Estrogen Induction of the Granzyme B Inhibitor, Proteinase Inhibitor 9, Protects Cells against Apoptosis Mediated by Cytotoxic T L
http://www.100md.com
《内分泌学杂志》
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
Abstract
Exposure to estrogens is associated with an increased risk of developing breast, cervical, and liver cancer. Estrogens strongly induce the human granzyme B inhibitor, proteinase inhibitor 9 (PI-9). Because cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the granzyme pathway to induce apoptosis of target cells, we tested the ability of activated CTLs and the human NK cell line, YT cells, to lyse human liver cells. Estrogen induction of PI-9 protected the liver cells against CTL and NK cell-mediated, granzyme-dependent, apoptosis. Knockdown of PI-9 by RNA interference blocked the protective effect of estrogen. This work demonstrates that estrogens can act on target cells to control their destruction by immune system cells and shows that induction of PI-9 expression can inhibit both CTL and NK cell-mediated apoptosis. Estrogen induction of PI-9 may reduce the ability of cytolytic lymphocytes-mediated immune surveillance to destroy newly transformed cells, possibly providing a novel mechanism for an estrogen-mediated increase in tumor incidence.
Introduction
CYTOLYTIC LYMPHOCYTES, including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells can use the granule-mediated pathway or the Fas-Fas ligand-mediated pathway to induce apoptosis of neoplastic cells or cells infected with intracellular pathogens. The granule-mediated pathway is often the predominant pathway for CTL and NK cell-induced cell death. Fas-Fas ligand mediated apoptosis is important in lymphocyte homeostasis (1, 2, 3). In granule-mediated apoptosis, the major granzyme protease, granzyme B, and perforin enter target cells via endocytosis of a multiprotein complex. Perforin then facilitates release of granzyme B from the endosome. Intracellular granzyme B induces apoptosis by direct cleavage and activation of pro-caspase-3 and other effector caspases, by cleavage of BH3 interacting domain death agonist, which activates the mitochondrial apoptosis pathway, and direct cleavage of intracellular protein substrates (4, 5).
In vitro, proteinase inhibitor 9 (PI-9; SerpinB9) is an effective inhibitor of granzyme B, a reasonably good inhibitor of caspase-4 and a weak inhibitor of caspase-1 (6, 7). Consistent with a role for PI-9 in protecting cells against misdirected granzyme B released during immune responses, PI-9 is expressed at high levels in cytolytic lymphocytes and dendritic cells, protecting against CTL-induced apoptosis and prolonging the life span of memory cells (8, 9, 10). Misregulation of PI-9 expression in early stage human atherosclerotic plaques led to the suggestion that PI-9 has the potential to regulate immune and inflammatory responses in vivo (11). Elevated constitutive expression of PI-9 in some tumors and tumor-derived cell lines may enhance their ability to evade apoptosis mediated by CTLs and NK cells (12).
PI-9 levels are regulated by control of PI-9 gene expression by estrogen and modulators of inflammation. We showed that PI-9 is an estrogen-inducible gene in human liver biopsy specimens and estrogen receptor (ER)-positive HepG2ER7 human hepatoblastoma cells (13). Subsequent identification of a unique downstream estrogen response unit (ERU), which is responsible for estrogen induction of PI-9 (14), and strong estrogen-dependent binding of ER to the PI-9 ERU in intact cells demonstrated that PI-9 is a primary estrogen-inducible gene in humans (14, 15). Modulators of inflammation, such as IL-1 and lipopolysaccharide acting at PI-9’s nuclear factor (NF)-B and activator protein-1 sites, also induce PI-9 (16). The antiviral cytokine interferon- is an effective inducer of PI-9 in liver cells (17).
Estrogens exert important effects on immunity and immune responses (18, 19, 20, 21), and these effects are likely important in pathologies ranging from autoimmune diseases to cancer. However, most studies have focused on effects of estrogens on cells of the immune system. Much less is known about the ability of estrogens to influence the susceptibility of target cells to lysis by CTLs and NK cells. Because PI-9 is induced by estrogen and is a potent inhibitor of the granzyme B used by CTLs and NK cell to induce apoptosis of target cells, we hypothesized that induction of PI-9 might represent a new site at which estrogens act to modulate immune responses. To test the idea that estrogen induction of PI-9 in target cells might suppress the ability of CTLs and NK cells to induce apoptosis of target cells, we used estrogen to induce PI-9, and RNA interference (RNAi) to knockdown PI-9 expression.
Materials and Methods
Cell lines and cell culture
Although early passages of HepG2 cells contained ER (22), this cell line subsequently lost receptor and became ER negative. In HepG2ER7 cells, ER expression has been restored by stable transfection of ER (13). ER was used because mammalian liver cells normally contain significant levels of ER and negligible levels of ER (23, 24). HepG2ER7 cells were maintained in RPMI 1640 supplemented with 10% charcoal-dextran-treated fetal bovine serum (FBS) plus 50,000 IU penicillin and 50 mg streptomycin per liter. YT cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and penicillin-streptomycin. CTL clone 2C cells were derived from transgenic 2C T-cell receptor (TCR) mice that were crossed with RAG-1 knockout mice (TCR/RAG mice) (25). These mice express a monoclonal population of naive T cells. Activated 2C CTLs were generated by in vitro stimulation of TCR/recombination activating gene (RAG) splenocytes with 3 mM agonist peptide SIYRYYGL for 3 d before cytotoxicity assays (25). For studies of killing by the 2C CTLs, dithiothreitol-treated HepG2ER7 cells were incubated with SPDP (Pierce, Rockford, IL) cross-linked monoclonal antibody 1B2 for 30 min (26).
Western blot analysis
In HepG2 and other liver cells, the cytochrome P450 system rapidly degrades the prototypical estrogen, 17-estradiol (13). We therefore used the poorly metabolized potent estrogen, moxestrol (MOX). Chromatin immunoprecipitation assays showed that 10 nM MOX and high concentrations of estradiol are identical in their abilities to recruit ER to the PI-9 ERU (15). Cell extracts were prepared in radioimmunoprecipitation assay buffer containing protease inhibitors (27) and run on 10% sodium dodecyl sulfate gels. After transfer, PI-9 was visualized by incubation for 24 h with a 1:1000 dilution of monoclonal antibody to PI-9 (Alexis, San Diego, CA). Bands were visualized by phosphor imager analysis using ECL Plus (Amersham, Piscataway, NJ).
Quantitative real-time PCR
HepG2ER7 cells were grown in medium containing either ethanol vehicle or hormone for 24 and 48 h. The cells were then harvested using Trizol (Invitrogen, Carlsbad, CA) to isolate total RNA following the manufacturer’s protocol. RNA was purified using the RNeasy minikit (QIAGEN, Valencia, CA) following the manufacturer’s protocol. Total RNA (1 mg) was reverse transcribed with moloney murine leukemia virus reverse transcriptase (Invitrogen) with 5 μM random hexamer primers according to the manufacturer’s instructions. For quantitative real-time PCR, 10 μl of one to 10 diluted reverse transcription reactions was added to quantitative RT-PCR containing the following in a total volume of 25 μl: 12.5 μl of 2x SYBR Green master mix (Applied Biosystems, Foster City, CA) and 100 nM forward and reverse primers specific for the genes of interest. Detection and data analysis were carried out with the Bio-Rad iCycler system. Actin mRNA expression was used to normalize gene expression for sample-to-sample variation in input and reverse transcription efficiency. Primers were designed using Primer Express software (Applied Biosystems). To ensure specificity, primer sequences were searched against GenBankTM using BLAST and were found to be unique.
Flow cytometry
HepG2ER7 cells were incubated with 10 nM MOX or 10 nM MOX plus 200 ng/ml epithelial growth factor (EGF) (Biosource, Camarillo, CA) for 2 d. In one experiment, CH11 anti-Fas activating antibody (Upstate, Charlottesville, VA) was then added at 50 ng/ml. After 24 h, the cells were harvested, resuspended in 0.7 ml PBS, Dioc(6)3 was added to a final concentration of 20 nM, and the cell suspensions were incubated for 15 min at 37 C and analyzed by fluorescence-activated cell sorter as described (28).
DNA fragmentation assay
DNA fragmentation is a hallmark of apoptosis (29). To assay cytolytic lymphocyte-induced cytotoxicity, we used a DNA fragmentation assay (30), with some modifications. Briefly, HepG2ER7 cells were labeled with 3H-thymidine (ICN Biochemical, Irvine, CA) at 5 mCi/ml for 4 h and washed three times on the plate. The cells were then incubated in 0.5 ml of PBS containing 0.5 mM dithiothreitol per well for 30 min at room temperature, washed twice with PBS, and harvested with PBS containing 1 mM EDTA. For the experiments testing DNA fragmentation induced by the 2C CTLs, the cells were then cross-linked with the 1B2 antibody and washed twice with RPMI 1640 containing 10% FBS. HepG2ER7 cells (10,000 cells/well) were incubated at the indicated ratios of 2C CTLs to liver cells for 6 h in medium containing 1.5 μM unlabeled thymidine. DNA fragmentation was calculated as the percentage of released against total radioactivity, as described (12). The YT cell-mediated DNA fragmentation assay was similar to the assay for 2C CTL-induced DNA fragmentation except that there was no antibody cross-linking. We assayed Fas activating antibody induced DNA fragmentation as described (31).
RNA interference
The sequence of the PI-9 small interfering RNA (siRNA) is 5'-CAG UUC CUC UCA ACG UUU AdTdT-3'. This sequence is located in the PI-9 open reading frame starting at nucleotide 379. This sequence was BLAST searched against the human genome and showed no exact or near exact matches to any other sequences. The 21-nt sequence of pGL3 luciferase siRNA was previously described (32). Cells were seeded into a 24-well plate at 40,000 cells/well, maintained for 24 h, and then transfected with oligofectamine (Invitrogen) (see Fig. 5, C and D) or DF4 (Dharmacon, Lafayette, CO) (Fig. 5, A and B) with 100 nM PI9 siRNA following the manufacturer’s protocol. The medium was changed after 24 h and MOX or MOX + EGF was added. After an additional 48 h, the cells were harvested for the cytotoxicity assay.
Statistical analysis
Results were expressed as mean ± SEM of three independent experiments. Student’s t test was used for comparison of the means between two groups and one-way ANOVA for comparison between multiple groups. Tukey’s honestly significant difference was used for the posttest of ANOVA using SAS (Cary, NC). P < 0.05 was considered significant.
Results
EGF enhances estrogen induction of PI-9
To analyze the effect of estrogen induction of PI-9 on CTL and NK cell-mediated apoptosis, it was important to identify conditions that result in a broad range of PI-9 levels. Estrogen-ER complex activates PI-9 gene transcription (13, 14, 15). Because ER-mediated transcription is often stimulated by phosphorylation of ER induced by activation of the ERK signaling pathway, we used quantitative real-time PCR to test whether EGF activation of the ERK pathway potentiates the estrogen induction of PI-9 mRNA. The poorly metabolized potent estrogen, MOX, induced PI-9 mRNA by approximately 5-fold after 24 h and approximately 11-fold after 48 h. EGF alone did not induce PI-9 mRNA. Addition of EGF to the medium enhanced the MOX induction of PI-9 mRNA from 5- to 11-fold after 24 h and from 11- to 15-fold after 48 h (Fig. 1A). To determine whether the synergistic effect of MOX and EGF treatment on PI-9 mRNA levels was reflected in a corresponding increase in the level of PI-9 protein, we carried out Western blot analysis. EGF did not induce PI-9 protein. MOX + EGF induced PI-9 protein by 11-fold. Because MOX alone induced PI-9 protein by 5.5-fold and addition of MOX + EGF and the ERK inhibitor PD98059 produced a 5.4-fold induction, activation of the ERK pathway by EGF plays a role in the ability of EGF to potentiate induction of PI-9 by MOX (Fig. 1B). These studies established two levels of induction of PI-9 protein for subsequent studies, 5- to 6-fold by MOX and 10- to 12-fold by MOX + EGF.
CTL clone 2C uses the granzyme-mediated pathway to induce apoptosis of target cells
The assay for CTL and NK cell-mediated killing of target cells must quantitate damage to the target cells in the presence of an excess of CTLs or NK cells. Because a detailed study concluded that for studies involving granzyme B-mediated apoptosis, assays based on DNA fragmentation, as measured by release of 3H-thymidine, provided a much better approximation of the in vivo situation than classical chromium release assays (33), we used an assay based on DNA fragmentation. We prelabeled the liver cell’s DNA with 3H-thymidine and measured DNA fragmentation using a modified thymidine release assay (30).
To determine whether treating the cells with MOX or MOX + EGF induces a general antiapoptotic state, we analyzed their effect on apoptosis induced by FAS antibody. We used the same thymidine release assay to measure FAS antibody-induced apoptosis that we used to measure CTL and NK cell-induced apoptosis. Inducing PI-9 with MOX or MOX + EGF did not protect the HepG2ER7 cells from apoptosis induced by Fas activating antibody (Fig. 2A). To evaluate whether the DNA fragmentation assay produced the same outcome as an assay for apoptosis based on mitochondrial dysfunction, we also quantitated apoptosis using an assay for mitochondrial damage based on exclusion of the dye, Dioc(6)3, and flow cytometry (28). The results were quite similar (Fig. 2B), validating the use of the thymidine release assay.
Although the granzyme pathway is usually the predominant pathway (2, 3, 4), CTLs can use the granyzme-mediated pathway or the Fas-Fas ligand-mediated pathway to induce apoptosis of target cells. To determine whether the Fas pathway contributes to apoptosis of the target cells induced by the 2C CTL clone used in these studies, we used the specific inhibitor of the granzyme-mediated pathway, concanamycin A (CMA) (Sigma, St. Louis, MO) (34). To assay CTL-induced apoptosis, we prelabeled the liver cell’s DNA with 3H-thymidine and measured DNA fragmentation induced by different levels of CTLs using the thymidine release assay. Inhibiting the granyzme pathway with CMA completely blocked the ability of the 2C CTLs to induce cell death and DNA fragmentation in the target liver cells (Fig. 2C). Therefore, the granzyme-mediated pathway is essential for 2C CTL-induced apoptosis of human liver cells.
Estrogen protects cells against CTL-mediated apoptosis
We first tested the effect of MOX treatment on CTL-mediated apoptosis. MOX, which induced PI-9 5- to 6-fold (Fig. 1B), reproducibly reduced CTL-induced DNA fragmentation by approximately 30% at all of the ratios of CTLs to liver cells that we tested (2:1, 6:1, and 12:1). The difference between control and MOX treatment at each ratio of CTLs to target cells is significant at P < 0.05 (Fig. 3A). Consistent with the enhanced induction of PI-9, MOX + EGF reduced CTL-induced apoptosis by nearly 50% (P < 0.05) (Fig. 3B). Because EGF does not induce PI-9 (Fig. 1) and EGF is a mitogen, we tested whether EGF alone could protect the target cells from CTL-induced apoptosis. EGF did not protect the target cells from CTL-induced DNA fragmentation (Fig. 3C).
Estrogen protects against human NK cell-mediated apoptosis
The studies shown in Fig. 3 used a CTL clone to induce apoptosis of the liver cells. NK cells represent another major class of cytolytic lymphocyte. NK cells play important roles in liver immunobiology, including disposal of infected or malignant cells (35, 36). The granzyme B pathway is the major pathway for NK cell-mediated lysis of target cells (37, 38). YT cells, a human NK cell line, have been widely used as effector NK cells (39, 40). We therefore evaluated the ability of YT cells to induce apoptosis of HepG2ER7 cells. Initially we determined the contribution of the Fas-Fas ligand pathway to YT cell-mediated apoptosis. Inhibiting the granzyme pathway by treating the cells with CMA greatly reduced the ability of YT cells to induce DNA fragmentation in the target liver cells (Fig. 4A). Because a 12:1 ratio of YT cells to HepG2ER7 cells produced substantial DNA fragmentation, we used a 12:1 ratio of YT cells/liver cells in subsequent experiments. Then we tested whether MOX and MOX + EGF, which induce different levels of PI-9 (Fig. 1), could protect the HepG2ER7 cells from YT cell-induced apoptosis. Consistent with the data, we obtained with the 2C CTLs (Fig. 3C), EGF alone did not protect the liver cells from YT cell-induced apoptosis (Fig. 4B). MOX treatment reduced YT cell-mediated DNA fragmentation of the target HepG2ER7 cells by approximately 50%. Incubating the cells in MOX + EGF, which induces PI-9 more effectively than MOX alone, further protected the target liver cells, reducing YT cell-mediated DNA fragmentation by approximately 70% (Fig. 4B).
RNAi knockdown of PI-9 abolishes estrogen protection against CTL-induced apoptosis
Whereas the data in Figs. 1, 3, and 4 showed that the estrogen, MOX, induces the granzyme B inhibitor, PI-9, and that estrogen treatment protects the liver cells against CTL and NK cell-induced apoptosis mediated through the granzyme system, these experiments did not explicitly show that induction of PI-9 was responsible for the protective effect of estrogen. Because neither MOX nor MOX + EGF protected liver cells against Fas-mediated apoptosis (Fig. 2, A and B), it was clear that MOX and MOX + EGF were not simply inducing a progrowth, antiapoptotic state. To directly determine whether the estrogen induction of PI-9 was responsible for the ability of estrogen to protect the liver cells against CTL-induced apoptosis, we used RNAi to selectively knock down PI-9 expression.
HepG2ER7 cells were treated with MOX + EGF to induce PI-9 and transfected with a PI-9 siRNA or a control pGL3 luciferase siRNA. Transfection with the specific PI-9 siRNA nearly blocked the induction of PI-9 by MOX + EGF. In contrast, treatment with MOX + EGF strongly induced PI-9 in the cells transfected with the control pGL3 siRNA (Fig. 5A). We then determined whether the reduction in PI-9 levels by transfection with the PI-9-specific siRNA was associated with loss of estrogen protection of the liver cells against CTL-induced apoptosis. We used a 12:1 ratio of CTLs to liver cells to test the effect of RNAi knockdown of PI-9 on the ability of MOX + EGF to protect the target liver cells against CTL-mediated apoptosis. The level of CTL-induced DNA fragmentation was similar in mock-transfected target cells that were not treated with MOX + EGF and in target cells treated with MOX + EGF and transfected with the PI-9-specific siRNA (Fig. 5B). In contrast, transfecting the cells with the control pGL3 luciferase siRNA did not interfere with the ability of MOX + EGF treatment to protect the target cells from CTL-induced lysis (Fig. 5B). We also tested the effect of RNAi-mediated knockdown of PI-9 on the ability of MOX to protect the target cells against CTL-induced apoptosis across a broader set of ratios, CTLs to target cells (2:1, 6:1, and 12:1). Transfection with the PI-9-specific siRNA nearly abolished the ability of MOX + EGF to protect the target liver cells against CTL-induced apoptosis (Fig. 5C). In contrast, transfection with the control pGL3 siRNA had no effect on estrogen protection of the HepG2ER7 cells against CTL-mediated apoptosis (Fig. 5D). These data demonstrate that estrogen induction of the granzyme B inhibitor, PI-9, is responsible for estrogen’s ability to protect the target cells against CTL-induced apoptosis.
Discussion
Estrogens exert diverse effects on development and regulation of the immune system. Estrogens are thought to play a role in enhancing immune responses and increasing the incidence and severity of some autoimmune diseases (41). Exposure to estrogens stimulates antibody production, but decreases T cell-mediated delayed-type hypersensitivity (42, 43). Estrogen delays neutrophil apoptosis (44) and modulates cytokine and chemokine expression in human monocyte-derived dendritic cells (45). Estrogen can affect chemotaxis of T cells through regulation of CC-chemokine receptor gene expression (46) and drive expansion of the regulatory T cell compartment, which is important in autoimmunity (47). Most of the many studies of estrogen as an immunomodulator have focused on estrogen modulation of immune system cells, not target cells (20, 21).
Our work provides the first demonstration that estrogens can control the potential of target cells to be destroyed by cells of the immune system. However, induction of PI-9 by the estrogen-ER complex does not completely block CTL and NK cell-induced cell death. This finding is reasonable in terms of the biology of the system. If estrogen induction of PI-9 completely blocked CTL and NK cell-mediated apoptosis through the granzyme B system, this could be highly deleterious to the organism because it would be much more difficult for the immune system to destroy transformed or infected cells. Whereas the granzyme B pathway that is inhibited by PI-9 is thought to be the most important pathway in CTL and NK cell-mediated apoptosis, granzymes A, M, and K are present in granules and may play a role in CTL and NK cell-mediated apoptosis (48). It is not presently known whether PI-9 is an effective inhibitor of these granzymes.
A recent report shows that ER and ER elicit different effects on p53 activity and TNF-induced apoptosis in MCF-7 cells (49). Although the ER used in this study is ER, in transient transfections using PI-9 promoter-luciferase reporter gene constructs, MOX-ER and MOX-ER exhibit similar abilities to transactivate the reporter (Krieg, S., and D. Shapiro, unpublished observations). This study looks at apoptosis of target cells induced by the proteolytic activity of granzymes produced by cytolytic lymphocytes, not endogenous apoptosis. It is the induction of PI-9 by estrogen that is responsible for estrogens ability to protect the target cells from cytolytic lymphocytes-mediated, granzyme-dependent apoptosis (Fig. 5). It is therefore likely that it will be the level to which estrogens can induce PI-9 in a particular cell context, rather than the presence of a specific ER subtype, that will be critical for estrogen protection of target cells against cytolytic lymphocyte-mediated apoptosis.
Recent studies report a correlation between high constitutive levels of PI-9 and its closest mouse ortholog, serine proteinase inhibitor (SPI)-6, and either a poor therapeutic prognosis for tumors or the resistance of tumor cells to immune system-mediated killing. Overexpression of PI-9 correlated with a poor prognosis and lower rate of patient survival in anaplastic large cell lymphomas (50). In a more direct study, tumor cells expressing the highest levels of PI-9/SPI-6 were resistant to CTL-induced apoptosis (12). However, that study did not directly demonstrate that it was the level of PI-9 that was responsible for the inhibition of CTL-induced apoptosis. Our demonstration that knockdown of PI-9 by a PI-9 siRNA blocks estrogen protection against CTL-induced apoptosis provides a direct demonstration that it is the level of PI-9 that controls the susceptibility of the target liver cancer cells to CTL-induced apoptosis. In contrast to studies in which a limited subset of a large number of tested tumors and tumor cell lines exhibit dramatically elevated constitutive levels of PI-9 and are therefore protected from CTL-mediated killing, we demonstrate that induction of PI-9 by the naturally occurring hormone, estrogen, can protect cancer cells from cytolytic lymphocytes-mediated lysis.
A direct test of our hypothesis that estrogen induction of PI-9 may play an in vivo role in protecting tumor cells against immune system mediated apoptosis will be quite difficult. Although estrogen plays an important role in the development of breast cancer, it is not known whether estrogen induction of PI-9 exerts a protective role in some of these tumors. In widely studied ER-positive MCF-7, human breast cancer cells, PI-9 is constitutively expressed at a high level and is not further induced by estrogens or any of the four inflammatory modulators that induce PI-9 in liver and immune system cells (Krieg, A., and D. Shapiro, unpublished observations). In another ER-positive breast cancer cell line, ZR-75, PI-9 also could not be induced by estrogen. A direct test of the proposed roles of estrogens based on induction or knockout of PI-9/SPI6 in mouse models will be complicated by the substantial differences between the families of protease inhibitors in humans and mice. The human granzyme B inhibitor, PI-9, appears to be a unique protein with no very closely related human proteins. In contrast, its closest mouse ortholog, SPI-6, is a member of a large family of closely related protease inhibitors (12). The size and complexity of this family of protease inhibitor genes makes studies using knockout mouse models quite complex. There have been no reports of functional studies using knockouts of SPI-6. We therefore focused on functional studies of PI-9 in cultured human cells using estrogens to up-regulate PI-9 levels and RNAi to knock down PI-9 levels.
In lieu of animal studies, large clinical studies of human populations may provide insights into the effects of estrogen on liver cancer. The estrogens in oral contraceptives expose the liver to significant levels of estrogen. Several clinical studies report that in populations that did not have a high incidence of hepatitis-B infection and chronic liver disease, long-term use of estrogen-containing combined oral contraceptives increases the risk of developing hepatocellular carcinoma (51, 52, 53, 54). A recent consensus report of the World Health Organization International Agency for Research on Cancer concluded that long-term use of estrogen-containing combined oral contraceptives increases the risk of developing breast, cervical, and liver cancer (55, 56). Our demonstration that estrogen induction of PI-9 protects liver cancer cells against CTL and NK cell-induced apoptosis suggests that in transformed cells estrogen induction of PI-9 might reduce cytolytic lymphocytes-mediated immune surveillance of the transformed cells and thereby increase tumor incidence. Of course, the direct and indirect effects of estrogens in the intact organism are highly complex, and the induction of PI-9 will be only one of the several systems involved in the estrogen-mediated sparing of tumor cells from cytolytic lymphocytes-induced damage.
These studies extend our earlier finding that estrogens induce the human serpin PI-9 (13, 14, 15). We describe a new role for estrogens: regulating the susceptibility of target cells to the induction of apoptosis by cells of the immune system.
Acknowledgments
We are grateful to Dr. C. J. Froelich for providing YT cells.
Footnotes
This work was supported by National Institutes of Health Grants HD16720 and DK071909 (to D.J.S.) and GM55767 (to D.M.K.).
The authors have no conflict of interest.
First Published Online November 23, 2005
Abbreviations: CMA, Concanamycin A; CTL, cytotoxic T lymphocyte; EGF, epithelial growth factor; ER, estrogen receptor; ERU, estrogen-responsive unit; FBS, fetal bovine serum; MOX, moxestrol; NK, natural killer; PI-9, proteinase inhibitor 9; RAG, recombination gene activating; RNAi, RNA interference; siRNA, small interfering RNA; SPI, serine proteinase inhibitor; TCR, T-cell receptor.
Accepted for publication November 14, 2005.
References
Goping IS, Barry M, Liston P, Sawchuk T, Constantinescu G, Michalak KM, Shostak I, Roberts DL, Hunter AM, Korneluk R, Bleackley RC 2003 Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition. Immunity 18:355–365
Russell JH, Ley TJ 2002 Lymphocyte-mediated cytotoxicity. Annu Rev Immunol 20:323–370
Lieberman J 2003 The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol 3:361–370.
Barry M, Bleackley RC 2002 Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol 2:401–409
Thomas DA, Du C, Xu M, Wang X, Ley TJ 2000 DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity 12:621–632
Bird CH, Sutton VR, Sun J, Hirst CE, Novak A, Kumar S, Trapani JA, Bird PI 1998 Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol Cell Biol 18:6387–6398
Annand RR, Dahlen JR, Sprecher CA, De Dreu P, Foster DC, Mankovich JA, Talanian RV, Kisiel W, Giegel DA 1999 Caspase-1 (interleukin-1-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9. Biochem J 342(Pt 3):655–665
Bladergroen BA, Strik MC, Bovenschen N, van Berkum O, Scheffer GL, Meijer CJ, Hack CE, Kummer JA 2001 The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites. J Immunol 166:3218–3225
Medema JP, Schuurhuis DH, Rea D, van Tongeren J, de Jong J, Bres SA, Laban S, Toes RE, Toebes M, Schumacher TN, Bladergroen BA, Ossendorp F, Kummer JA, Melief CJ, Offringa R 2001 Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells. J Exp Med 194:657–667
Phillips T, Opferman JT, Shah R, Liu N, Froelich CJ, Ashton-Rickardt PG 2004 A role for the granzyme B inhibitor serine protease inhibitor 6 in CD8+ memory cell homeostasis. J Immunol 173:3801–3809
Young JL, Sukhova GK, Foster D, Kisiel W, Libby P, Schonbeck U 2000 The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1-converting enzyme (caspase-1) activity in human vascular smooth muscle cells. J Exp Med 191:1535–1544
Medema JP, de Jong J, Peltenburg LT, Verdegaal EM, Gorter A, Bres SA, Franken KL, Hahne M, Albar JP, Melief CJ, Offringa R 2001 Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors. Proc Natl Acad Sci USA 98:11515–11520
Kanamori H, Krieg S, Mao C, Di Pippo VA, Wang S, Zajchowski DA, Shapiro DJ 2000 Proteinase inhibitor 9, an inhibitor of granzyme B-mediated apoptosis, is a primary estrogen-inducible gene in human liver cells. J Biol Chem 275:5867–5873
Krieg SA, Krieg AJ, Shapiro DJ 2001 A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1-converting enzyme (caspase 1). Mol Endocrinol 15:1971–1982
Krieg AJ, Krieg SA, Ahn BS, Shapiro DJ 2004 Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. J Biol Chem 279:5025–5034
Kannan-Thulasiraman P, Shapiro DJ 2002 Modulators of inflammation use nuclear factor-B and activator protein-1 sites to induce the caspase-1 and granzyme B inhibitor, proteinase inhibitor 9. J Biol Chem 277:41230–41239
Barrie MB, Stout HW, Abougergi MS, Miller BC, Thiele DL 2004 Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6. J Immunol 172:6453–6459
Jansson L, Holmdahl R 1998 Estrogen-mediated immunosuppression in autoimmune diseases. Inflamm Res 47:290–301
Vegeto E, Belcredito S, Etteri S, Ghisletti S, Brusadelli A, Meda C, Krust A, Dupont S, Ciana P, Chambon P, Maggi A 2003 Estrogen receptor- mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci USA 100:9614–9619
Lang TJ 2004 Estrogen as an immunomodulator. Clin Immunol 113:224–230
Bouman A, Heineman MJ, Faas MM 2005 Sex hormones and the immune response in humans. Hum Reprod Update 11:411–423
Archer TK, Tam SP, Deugau KV, Deeley RG 1985 Apolipoprotein C-II mRNA levels in primate liver. Induction by estrogen in the human hepatocarcinoma cell line, HepG2. J Biol Chem 260:1676–1681
Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors and . Endocrinology 138:863–870
Taylor AH, Al-Azzawi F 2000 Immunolocalisation of oestrogen receptor in human tissues. J Mol Endocrinol 24:145–155
Manning TC, Rund LA, Gruber MM, Fallarino F, Gajewski TF, Kranz DM 1997 Antigen recognition and allogeneic tumor rejection in CD8+ TCR transgenic/RAG(–/–) mice. J Immunol 159:4665–4675
Kranz DM, Eisen HN 1987 Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T lymphocytes. Proc Natl Acad Sci USA 84:3375–3379
Goolsby KM, Shapiro DJ 2003 RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis. Nucleic Acids Res 31:5644–5653
Obrero M, Yu DV, Shapiro DJ 2002 Estrogen receptor-dependent and estrogen receptor-independent pathways for tamoxifen and 4-hydroxytamoxifen-induced programmed cell death. J Biol Chem 277:45695–45703
Nagata S 2000 Apoptotic DNA fragmentation. Exp Cell Res 256:12–18
Bierer B, Coligan JE, Margulies DH, Shevach EM, Strober W, Kruisbeek A 2003 Curr Protoc Immunol 3.11.8–3.11.20
Shen WH, Zhou JH, Broussard SR, Freund GG, Dantzer R, Kelley KW 2002 Proinflammatory cytokines block growth of breast cancer cells by impairing signals from a growth factor receptor. Cancer Res 62:4746–4756
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T 2001 Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498
Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, Barry M, Shostak I, Sawchuk T, Holmes CF, Gauldie J, Bleackley RC 2000 Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell 103:491–500
Kataoka T, Shinohara N, Takayama H, Takaku K, Kondo S, Yonehara S, Nagai K 1996 Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J Immunol 156:3678–3686
Wiltrout RH 2000 Regulation and antimetastatic functions of liver-associated natural killer cells. Immunol Rev 174:63–76
Mackay IR 2002 Hepatoimmunology: a perspective. Immunol Cell Biol 80:36–44
Vermijlen D, Luo D, Froelich CJ, Medema JP, Kummer JA, Willems E, Braet F, Wisse E 2002 Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway. J Leukoc Biol 72:668–676
Mahrus S, Craik CS 2005 Selective chemical functional probes of granzymes a and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem Biol 12:567–577
Chuang SS, Kumaresan PR, Mathew PA 2001 2B4 (CD244)-mediated activation of cytotoxicity and IFN- release in human NK cells involves distinct pathways. J Immunol 167:6210–6216
Miyagawa S, Kubo T, Matsunami K, Kusama T, Beppu K, Nozaki H, Moritan T, Ahn C, Kim JY, Fukuta D, Shirakura R 2004 -Short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis. J Immunol 173:3945–3952
Cutolo M, Sulli A, Capellino S, Villaggio B, Montagna P, Seriolo B, Straub RH 2004 Sex hormones influence on the immune system: basic and clinical aspects in autoimmunity. Lupus 13:635–638
Carlsten H, Holmdahl R, Tarkowski A, Nilsson LA 1989 Oestradiol- and testosterone-mediated effects on the immune system in normal and autoimmune mice are genetically linked and inherited as dominant traits. Immunology 68:209–214
Carlsten H, Holmdahl R, Tarkowski A, Nilsson LA 1989 Oestradiol suppression of delayed-type hypersensitivity in autoimmune (NZB/NZW)F1 mice is a trait inherited from the healthy NZW parental strain. Immunology 67:205–209
Molloy EJ, O’Neill AJ, Grantham JJ, Sheridan-Pereira M, Fitzpatrick JM, Webb DW, Watson RW 2003 Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone. Blood 102:2653–2659
Bengtsson AK, Ryan EJ, Giordano D, Magaletti DM, Clark EA 2004 17-Estradiol (E2) modulates cytokine and chemokine expression in human monocyte-derived dendritic cells. Blood 104:1404–1410
Mo R, Chen J, Grolleau-Julius A, Murphy HS, Richardson BC, Yung RL 2005 Estrogen regulates CCR gene expression and function in T lymphocytes. J Immunol 174:6023–6029
Polanczyk MJ, Carson BD, Subramanian S, Afentoulis M, Vandenbark AA, Ziegler SF, Offner H 2004 Cutting edge: estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment. J Immunol 173:2227–2230
Bade B, Boettcher HE, Lohrmann J, Hink-Schauer C, Bratke K, Jenne DE, Virchow Jr JC, Luttmann W 2005 Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes. Int Immunol 17:1419–1428
Lewandowski SA, Thiery J, Jalil A, Leclercq G, Szczylik C, Chouaib S 2005 Opposite effects of estrogen receptors and on MCF-7 sensitivity to the cytotoxic action of TNF and p53 activity. Oncogene 24:4789–4798
ten Berge RL, Oudejans JJ, Ossenkoppele GJ, Meijer CJ 2003 ALK-negative systemic anaplastic large cell lymphoma: differential diagnostic and prognostic aspects—a review. J Pathol 200:4–15
Forman D, Vincent TJ, Doll R 1986 Cancer of the liver and the use of oral contraceptives. Br Med J (Clin Res Ed) 292:1357–1361
Yu MC, Tong MJ, Govindarajan S, Henderson BE 1991 Nonviral risk factors for hepatocellular carcinoma in a low-risk population, the non-Asians of Los Angeles County, California. J Natl Cancer Inst 83:1820–1826
Tavani A, Negri E, Parazzini F, Franceschi S, La Vecchia C 1993 Female hormone utilisation and risk of hepatocellular carcinoma. Br J Cancer 67:635–637
Chen CJ, Yu MW, Liaw YF 1997 Epidemiological characteristics and risk factors of hepatocellular carcinoma. J Gastroenterol Hepatol 12:S294–S308
Vincent Cogliano, Yann Grosse, Robert Baan, Kurt Straif, Ghissassi BSFE 2005 Carcinogenicity of combined oestrogen-progestogen contraceptives and menopausal treatment. Lancet Oncol 6:552–553
La Vecchia C, Autier P, Rosano G, Stafford R, Vatten L, Shapiro S, Fernandez E, Beral V, Peto J, Anderson G, Bosland M, Haslam S, Junghans T, Kaufman D, Molinolos A, Olin S, Newcomb P, Parl F, Roy D, Stanczyk F, Thomas D, Combined estrogen-progestogen contraceptives and combined estrogen-progestogen menopausal therapy. IARC Monogr Eval Carcinog Risks Hum, in press(Xinguo Jiang, Brent A. Orr, David M. Kra)
Abstract
Exposure to estrogens is associated with an increased risk of developing breast, cervical, and liver cancer. Estrogens strongly induce the human granzyme B inhibitor, proteinase inhibitor 9 (PI-9). Because cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the granzyme pathway to induce apoptosis of target cells, we tested the ability of activated CTLs and the human NK cell line, YT cells, to lyse human liver cells. Estrogen induction of PI-9 protected the liver cells against CTL and NK cell-mediated, granzyme-dependent, apoptosis. Knockdown of PI-9 by RNA interference blocked the protective effect of estrogen. This work demonstrates that estrogens can act on target cells to control their destruction by immune system cells and shows that induction of PI-9 expression can inhibit both CTL and NK cell-mediated apoptosis. Estrogen induction of PI-9 may reduce the ability of cytolytic lymphocytes-mediated immune surveillance to destroy newly transformed cells, possibly providing a novel mechanism for an estrogen-mediated increase in tumor incidence.
Introduction
CYTOLYTIC LYMPHOCYTES, including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells can use the granule-mediated pathway or the Fas-Fas ligand-mediated pathway to induce apoptosis of neoplastic cells or cells infected with intracellular pathogens. The granule-mediated pathway is often the predominant pathway for CTL and NK cell-induced cell death. Fas-Fas ligand mediated apoptosis is important in lymphocyte homeostasis (1, 2, 3). In granule-mediated apoptosis, the major granzyme protease, granzyme B, and perforin enter target cells via endocytosis of a multiprotein complex. Perforin then facilitates release of granzyme B from the endosome. Intracellular granzyme B induces apoptosis by direct cleavage and activation of pro-caspase-3 and other effector caspases, by cleavage of BH3 interacting domain death agonist, which activates the mitochondrial apoptosis pathway, and direct cleavage of intracellular protein substrates (4, 5).
In vitro, proteinase inhibitor 9 (PI-9; SerpinB9) is an effective inhibitor of granzyme B, a reasonably good inhibitor of caspase-4 and a weak inhibitor of caspase-1 (6, 7). Consistent with a role for PI-9 in protecting cells against misdirected granzyme B released during immune responses, PI-9 is expressed at high levels in cytolytic lymphocytes and dendritic cells, protecting against CTL-induced apoptosis and prolonging the life span of memory cells (8, 9, 10). Misregulation of PI-9 expression in early stage human atherosclerotic plaques led to the suggestion that PI-9 has the potential to regulate immune and inflammatory responses in vivo (11). Elevated constitutive expression of PI-9 in some tumors and tumor-derived cell lines may enhance their ability to evade apoptosis mediated by CTLs and NK cells (12).
PI-9 levels are regulated by control of PI-9 gene expression by estrogen and modulators of inflammation. We showed that PI-9 is an estrogen-inducible gene in human liver biopsy specimens and estrogen receptor (ER)-positive HepG2ER7 human hepatoblastoma cells (13). Subsequent identification of a unique downstream estrogen response unit (ERU), which is responsible for estrogen induction of PI-9 (14), and strong estrogen-dependent binding of ER to the PI-9 ERU in intact cells demonstrated that PI-9 is a primary estrogen-inducible gene in humans (14, 15). Modulators of inflammation, such as IL-1 and lipopolysaccharide acting at PI-9’s nuclear factor (NF)-B and activator protein-1 sites, also induce PI-9 (16). The antiviral cytokine interferon- is an effective inducer of PI-9 in liver cells (17).
Estrogens exert important effects on immunity and immune responses (18, 19, 20, 21), and these effects are likely important in pathologies ranging from autoimmune diseases to cancer. However, most studies have focused on effects of estrogens on cells of the immune system. Much less is known about the ability of estrogens to influence the susceptibility of target cells to lysis by CTLs and NK cells. Because PI-9 is induced by estrogen and is a potent inhibitor of the granzyme B used by CTLs and NK cell to induce apoptosis of target cells, we hypothesized that induction of PI-9 might represent a new site at which estrogens act to modulate immune responses. To test the idea that estrogen induction of PI-9 in target cells might suppress the ability of CTLs and NK cells to induce apoptosis of target cells, we used estrogen to induce PI-9, and RNA interference (RNAi) to knockdown PI-9 expression.
Materials and Methods
Cell lines and cell culture
Although early passages of HepG2 cells contained ER (22), this cell line subsequently lost receptor and became ER negative. In HepG2ER7 cells, ER expression has been restored by stable transfection of ER (13). ER was used because mammalian liver cells normally contain significant levels of ER and negligible levels of ER (23, 24). HepG2ER7 cells were maintained in RPMI 1640 supplemented with 10% charcoal-dextran-treated fetal bovine serum (FBS) plus 50,000 IU penicillin and 50 mg streptomycin per liter. YT cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and penicillin-streptomycin. CTL clone 2C cells were derived from transgenic 2C T-cell receptor (TCR) mice that were crossed with RAG-1 knockout mice (TCR/RAG mice) (25). These mice express a monoclonal population of naive T cells. Activated 2C CTLs were generated by in vitro stimulation of TCR/recombination activating gene (RAG) splenocytes with 3 mM agonist peptide SIYRYYGL for 3 d before cytotoxicity assays (25). For studies of killing by the 2C CTLs, dithiothreitol-treated HepG2ER7 cells were incubated with SPDP (Pierce, Rockford, IL) cross-linked monoclonal antibody 1B2 for 30 min (26).
Western blot analysis
In HepG2 and other liver cells, the cytochrome P450 system rapidly degrades the prototypical estrogen, 17-estradiol (13). We therefore used the poorly metabolized potent estrogen, moxestrol (MOX). Chromatin immunoprecipitation assays showed that 10 nM MOX and high concentrations of estradiol are identical in their abilities to recruit ER to the PI-9 ERU (15). Cell extracts were prepared in radioimmunoprecipitation assay buffer containing protease inhibitors (27) and run on 10% sodium dodecyl sulfate gels. After transfer, PI-9 was visualized by incubation for 24 h with a 1:1000 dilution of monoclonal antibody to PI-9 (Alexis, San Diego, CA). Bands were visualized by phosphor imager analysis using ECL Plus (Amersham, Piscataway, NJ).
Quantitative real-time PCR
HepG2ER7 cells were grown in medium containing either ethanol vehicle or hormone for 24 and 48 h. The cells were then harvested using Trizol (Invitrogen, Carlsbad, CA) to isolate total RNA following the manufacturer’s protocol. RNA was purified using the RNeasy minikit (QIAGEN, Valencia, CA) following the manufacturer’s protocol. Total RNA (1 mg) was reverse transcribed with moloney murine leukemia virus reverse transcriptase (Invitrogen) with 5 μM random hexamer primers according to the manufacturer’s instructions. For quantitative real-time PCR, 10 μl of one to 10 diluted reverse transcription reactions was added to quantitative RT-PCR containing the following in a total volume of 25 μl: 12.5 μl of 2x SYBR Green master mix (Applied Biosystems, Foster City, CA) and 100 nM forward and reverse primers specific for the genes of interest. Detection and data analysis were carried out with the Bio-Rad iCycler system. Actin mRNA expression was used to normalize gene expression for sample-to-sample variation in input and reverse transcription efficiency. Primers were designed using Primer Express software (Applied Biosystems). To ensure specificity, primer sequences were searched against GenBankTM using BLAST and were found to be unique.
Flow cytometry
HepG2ER7 cells were incubated with 10 nM MOX or 10 nM MOX plus 200 ng/ml epithelial growth factor (EGF) (Biosource, Camarillo, CA) for 2 d. In one experiment, CH11 anti-Fas activating antibody (Upstate, Charlottesville, VA) was then added at 50 ng/ml. After 24 h, the cells were harvested, resuspended in 0.7 ml PBS, Dioc(6)3 was added to a final concentration of 20 nM, and the cell suspensions were incubated for 15 min at 37 C and analyzed by fluorescence-activated cell sorter as described (28).
DNA fragmentation assay
DNA fragmentation is a hallmark of apoptosis (29). To assay cytolytic lymphocyte-induced cytotoxicity, we used a DNA fragmentation assay (30), with some modifications. Briefly, HepG2ER7 cells were labeled with 3H-thymidine (ICN Biochemical, Irvine, CA) at 5 mCi/ml for 4 h and washed three times on the plate. The cells were then incubated in 0.5 ml of PBS containing 0.5 mM dithiothreitol per well for 30 min at room temperature, washed twice with PBS, and harvested with PBS containing 1 mM EDTA. For the experiments testing DNA fragmentation induced by the 2C CTLs, the cells were then cross-linked with the 1B2 antibody and washed twice with RPMI 1640 containing 10% FBS. HepG2ER7 cells (10,000 cells/well) were incubated at the indicated ratios of 2C CTLs to liver cells for 6 h in medium containing 1.5 μM unlabeled thymidine. DNA fragmentation was calculated as the percentage of released against total radioactivity, as described (12). The YT cell-mediated DNA fragmentation assay was similar to the assay for 2C CTL-induced DNA fragmentation except that there was no antibody cross-linking. We assayed Fas activating antibody induced DNA fragmentation as described (31).
RNA interference
The sequence of the PI-9 small interfering RNA (siRNA) is 5'-CAG UUC CUC UCA ACG UUU AdTdT-3'. This sequence is located in the PI-9 open reading frame starting at nucleotide 379. This sequence was BLAST searched against the human genome and showed no exact or near exact matches to any other sequences. The 21-nt sequence of pGL3 luciferase siRNA was previously described (32). Cells were seeded into a 24-well plate at 40,000 cells/well, maintained for 24 h, and then transfected with oligofectamine (Invitrogen) (see Fig. 5, C and D) or DF4 (Dharmacon, Lafayette, CO) (Fig. 5, A and B) with 100 nM PI9 siRNA following the manufacturer’s protocol. The medium was changed after 24 h and MOX or MOX + EGF was added. After an additional 48 h, the cells were harvested for the cytotoxicity assay.
Statistical analysis
Results were expressed as mean ± SEM of three independent experiments. Student’s t test was used for comparison of the means between two groups and one-way ANOVA for comparison between multiple groups. Tukey’s honestly significant difference was used for the posttest of ANOVA using SAS (Cary, NC). P < 0.05 was considered significant.
Results
EGF enhances estrogen induction of PI-9
To analyze the effect of estrogen induction of PI-9 on CTL and NK cell-mediated apoptosis, it was important to identify conditions that result in a broad range of PI-9 levels. Estrogen-ER complex activates PI-9 gene transcription (13, 14, 15). Because ER-mediated transcription is often stimulated by phosphorylation of ER induced by activation of the ERK signaling pathway, we used quantitative real-time PCR to test whether EGF activation of the ERK pathway potentiates the estrogen induction of PI-9 mRNA. The poorly metabolized potent estrogen, MOX, induced PI-9 mRNA by approximately 5-fold after 24 h and approximately 11-fold after 48 h. EGF alone did not induce PI-9 mRNA. Addition of EGF to the medium enhanced the MOX induction of PI-9 mRNA from 5- to 11-fold after 24 h and from 11- to 15-fold after 48 h (Fig. 1A). To determine whether the synergistic effect of MOX and EGF treatment on PI-9 mRNA levels was reflected in a corresponding increase in the level of PI-9 protein, we carried out Western blot analysis. EGF did not induce PI-9 protein. MOX + EGF induced PI-9 protein by 11-fold. Because MOX alone induced PI-9 protein by 5.5-fold and addition of MOX + EGF and the ERK inhibitor PD98059 produced a 5.4-fold induction, activation of the ERK pathway by EGF plays a role in the ability of EGF to potentiate induction of PI-9 by MOX (Fig. 1B). These studies established two levels of induction of PI-9 protein for subsequent studies, 5- to 6-fold by MOX and 10- to 12-fold by MOX + EGF.
CTL clone 2C uses the granzyme-mediated pathway to induce apoptosis of target cells
The assay for CTL and NK cell-mediated killing of target cells must quantitate damage to the target cells in the presence of an excess of CTLs or NK cells. Because a detailed study concluded that for studies involving granzyme B-mediated apoptosis, assays based on DNA fragmentation, as measured by release of 3H-thymidine, provided a much better approximation of the in vivo situation than classical chromium release assays (33), we used an assay based on DNA fragmentation. We prelabeled the liver cell’s DNA with 3H-thymidine and measured DNA fragmentation using a modified thymidine release assay (30).
To determine whether treating the cells with MOX or MOX + EGF induces a general antiapoptotic state, we analyzed their effect on apoptosis induced by FAS antibody. We used the same thymidine release assay to measure FAS antibody-induced apoptosis that we used to measure CTL and NK cell-induced apoptosis. Inducing PI-9 with MOX or MOX + EGF did not protect the HepG2ER7 cells from apoptosis induced by Fas activating antibody (Fig. 2A). To evaluate whether the DNA fragmentation assay produced the same outcome as an assay for apoptosis based on mitochondrial dysfunction, we also quantitated apoptosis using an assay for mitochondrial damage based on exclusion of the dye, Dioc(6)3, and flow cytometry (28). The results were quite similar (Fig. 2B), validating the use of the thymidine release assay.
Although the granzyme pathway is usually the predominant pathway (2, 3, 4), CTLs can use the granyzme-mediated pathway or the Fas-Fas ligand-mediated pathway to induce apoptosis of target cells. To determine whether the Fas pathway contributes to apoptosis of the target cells induced by the 2C CTL clone used in these studies, we used the specific inhibitor of the granzyme-mediated pathway, concanamycin A (CMA) (Sigma, St. Louis, MO) (34). To assay CTL-induced apoptosis, we prelabeled the liver cell’s DNA with 3H-thymidine and measured DNA fragmentation induced by different levels of CTLs using the thymidine release assay. Inhibiting the granyzme pathway with CMA completely blocked the ability of the 2C CTLs to induce cell death and DNA fragmentation in the target liver cells (Fig. 2C). Therefore, the granzyme-mediated pathway is essential for 2C CTL-induced apoptosis of human liver cells.
Estrogen protects cells against CTL-mediated apoptosis
We first tested the effect of MOX treatment on CTL-mediated apoptosis. MOX, which induced PI-9 5- to 6-fold (Fig. 1B), reproducibly reduced CTL-induced DNA fragmentation by approximately 30% at all of the ratios of CTLs to liver cells that we tested (2:1, 6:1, and 12:1). The difference between control and MOX treatment at each ratio of CTLs to target cells is significant at P < 0.05 (Fig. 3A). Consistent with the enhanced induction of PI-9, MOX + EGF reduced CTL-induced apoptosis by nearly 50% (P < 0.05) (Fig. 3B). Because EGF does not induce PI-9 (Fig. 1) and EGF is a mitogen, we tested whether EGF alone could protect the target cells from CTL-induced apoptosis. EGF did not protect the target cells from CTL-induced DNA fragmentation (Fig. 3C).
Estrogen protects against human NK cell-mediated apoptosis
The studies shown in Fig. 3 used a CTL clone to induce apoptosis of the liver cells. NK cells represent another major class of cytolytic lymphocyte. NK cells play important roles in liver immunobiology, including disposal of infected or malignant cells (35, 36). The granzyme B pathway is the major pathway for NK cell-mediated lysis of target cells (37, 38). YT cells, a human NK cell line, have been widely used as effector NK cells (39, 40). We therefore evaluated the ability of YT cells to induce apoptosis of HepG2ER7 cells. Initially we determined the contribution of the Fas-Fas ligand pathway to YT cell-mediated apoptosis. Inhibiting the granzyme pathway by treating the cells with CMA greatly reduced the ability of YT cells to induce DNA fragmentation in the target liver cells (Fig. 4A). Because a 12:1 ratio of YT cells to HepG2ER7 cells produced substantial DNA fragmentation, we used a 12:1 ratio of YT cells/liver cells in subsequent experiments. Then we tested whether MOX and MOX + EGF, which induce different levels of PI-9 (Fig. 1), could protect the HepG2ER7 cells from YT cell-induced apoptosis. Consistent with the data, we obtained with the 2C CTLs (Fig. 3C), EGF alone did not protect the liver cells from YT cell-induced apoptosis (Fig. 4B). MOX treatment reduced YT cell-mediated DNA fragmentation of the target HepG2ER7 cells by approximately 50%. Incubating the cells in MOX + EGF, which induces PI-9 more effectively than MOX alone, further protected the target liver cells, reducing YT cell-mediated DNA fragmentation by approximately 70% (Fig. 4B).
RNAi knockdown of PI-9 abolishes estrogen protection against CTL-induced apoptosis
Whereas the data in Figs. 1, 3, and 4 showed that the estrogen, MOX, induces the granzyme B inhibitor, PI-9, and that estrogen treatment protects the liver cells against CTL and NK cell-induced apoptosis mediated through the granzyme system, these experiments did not explicitly show that induction of PI-9 was responsible for the protective effect of estrogen. Because neither MOX nor MOX + EGF protected liver cells against Fas-mediated apoptosis (Fig. 2, A and B), it was clear that MOX and MOX + EGF were not simply inducing a progrowth, antiapoptotic state. To directly determine whether the estrogen induction of PI-9 was responsible for the ability of estrogen to protect the liver cells against CTL-induced apoptosis, we used RNAi to selectively knock down PI-9 expression.
HepG2ER7 cells were treated with MOX + EGF to induce PI-9 and transfected with a PI-9 siRNA or a control pGL3 luciferase siRNA. Transfection with the specific PI-9 siRNA nearly blocked the induction of PI-9 by MOX + EGF. In contrast, treatment with MOX + EGF strongly induced PI-9 in the cells transfected with the control pGL3 siRNA (Fig. 5A). We then determined whether the reduction in PI-9 levels by transfection with the PI-9-specific siRNA was associated with loss of estrogen protection of the liver cells against CTL-induced apoptosis. We used a 12:1 ratio of CTLs to liver cells to test the effect of RNAi knockdown of PI-9 on the ability of MOX + EGF to protect the target liver cells against CTL-mediated apoptosis. The level of CTL-induced DNA fragmentation was similar in mock-transfected target cells that were not treated with MOX + EGF and in target cells treated with MOX + EGF and transfected with the PI-9-specific siRNA (Fig. 5B). In contrast, transfecting the cells with the control pGL3 luciferase siRNA did not interfere with the ability of MOX + EGF treatment to protect the target cells from CTL-induced lysis (Fig. 5B). We also tested the effect of RNAi-mediated knockdown of PI-9 on the ability of MOX to protect the target cells against CTL-induced apoptosis across a broader set of ratios, CTLs to target cells (2:1, 6:1, and 12:1). Transfection with the PI-9-specific siRNA nearly abolished the ability of MOX + EGF to protect the target liver cells against CTL-induced apoptosis (Fig. 5C). In contrast, transfection with the control pGL3 siRNA had no effect on estrogen protection of the HepG2ER7 cells against CTL-mediated apoptosis (Fig. 5D). These data demonstrate that estrogen induction of the granzyme B inhibitor, PI-9, is responsible for estrogen’s ability to protect the target cells against CTL-induced apoptosis.
Discussion
Estrogens exert diverse effects on development and regulation of the immune system. Estrogens are thought to play a role in enhancing immune responses and increasing the incidence and severity of some autoimmune diseases (41). Exposure to estrogens stimulates antibody production, but decreases T cell-mediated delayed-type hypersensitivity (42, 43). Estrogen delays neutrophil apoptosis (44) and modulates cytokine and chemokine expression in human monocyte-derived dendritic cells (45). Estrogen can affect chemotaxis of T cells through regulation of CC-chemokine receptor gene expression (46) and drive expansion of the regulatory T cell compartment, which is important in autoimmunity (47). Most of the many studies of estrogen as an immunomodulator have focused on estrogen modulation of immune system cells, not target cells (20, 21).
Our work provides the first demonstration that estrogens can control the potential of target cells to be destroyed by cells of the immune system. However, induction of PI-9 by the estrogen-ER complex does not completely block CTL and NK cell-induced cell death. This finding is reasonable in terms of the biology of the system. If estrogen induction of PI-9 completely blocked CTL and NK cell-mediated apoptosis through the granzyme B system, this could be highly deleterious to the organism because it would be much more difficult for the immune system to destroy transformed or infected cells. Whereas the granzyme B pathway that is inhibited by PI-9 is thought to be the most important pathway in CTL and NK cell-mediated apoptosis, granzymes A, M, and K are present in granules and may play a role in CTL and NK cell-mediated apoptosis (48). It is not presently known whether PI-9 is an effective inhibitor of these granzymes.
A recent report shows that ER and ER elicit different effects on p53 activity and TNF-induced apoptosis in MCF-7 cells (49). Although the ER used in this study is ER, in transient transfections using PI-9 promoter-luciferase reporter gene constructs, MOX-ER and MOX-ER exhibit similar abilities to transactivate the reporter (Krieg, S., and D. Shapiro, unpublished observations). This study looks at apoptosis of target cells induced by the proteolytic activity of granzymes produced by cytolytic lymphocytes, not endogenous apoptosis. It is the induction of PI-9 by estrogen that is responsible for estrogens ability to protect the target cells from cytolytic lymphocytes-mediated, granzyme-dependent apoptosis (Fig. 5). It is therefore likely that it will be the level to which estrogens can induce PI-9 in a particular cell context, rather than the presence of a specific ER subtype, that will be critical for estrogen protection of target cells against cytolytic lymphocyte-mediated apoptosis.
Recent studies report a correlation between high constitutive levels of PI-9 and its closest mouse ortholog, serine proteinase inhibitor (SPI)-6, and either a poor therapeutic prognosis for tumors or the resistance of tumor cells to immune system-mediated killing. Overexpression of PI-9 correlated with a poor prognosis and lower rate of patient survival in anaplastic large cell lymphomas (50). In a more direct study, tumor cells expressing the highest levels of PI-9/SPI-6 were resistant to CTL-induced apoptosis (12). However, that study did not directly demonstrate that it was the level of PI-9 that was responsible for the inhibition of CTL-induced apoptosis. Our demonstration that knockdown of PI-9 by a PI-9 siRNA blocks estrogen protection against CTL-induced apoptosis provides a direct demonstration that it is the level of PI-9 that controls the susceptibility of the target liver cancer cells to CTL-induced apoptosis. In contrast to studies in which a limited subset of a large number of tested tumors and tumor cell lines exhibit dramatically elevated constitutive levels of PI-9 and are therefore protected from CTL-mediated killing, we demonstrate that induction of PI-9 by the naturally occurring hormone, estrogen, can protect cancer cells from cytolytic lymphocytes-mediated lysis.
A direct test of our hypothesis that estrogen induction of PI-9 may play an in vivo role in protecting tumor cells against immune system mediated apoptosis will be quite difficult. Although estrogen plays an important role in the development of breast cancer, it is not known whether estrogen induction of PI-9 exerts a protective role in some of these tumors. In widely studied ER-positive MCF-7, human breast cancer cells, PI-9 is constitutively expressed at a high level and is not further induced by estrogens or any of the four inflammatory modulators that induce PI-9 in liver and immune system cells (Krieg, A., and D. Shapiro, unpublished observations). In another ER-positive breast cancer cell line, ZR-75, PI-9 also could not be induced by estrogen. A direct test of the proposed roles of estrogens based on induction or knockout of PI-9/SPI6 in mouse models will be complicated by the substantial differences between the families of protease inhibitors in humans and mice. The human granzyme B inhibitor, PI-9, appears to be a unique protein with no very closely related human proteins. In contrast, its closest mouse ortholog, SPI-6, is a member of a large family of closely related protease inhibitors (12). The size and complexity of this family of protease inhibitor genes makes studies using knockout mouse models quite complex. There have been no reports of functional studies using knockouts of SPI-6. We therefore focused on functional studies of PI-9 in cultured human cells using estrogens to up-regulate PI-9 levels and RNAi to knock down PI-9 levels.
In lieu of animal studies, large clinical studies of human populations may provide insights into the effects of estrogen on liver cancer. The estrogens in oral contraceptives expose the liver to significant levels of estrogen. Several clinical studies report that in populations that did not have a high incidence of hepatitis-B infection and chronic liver disease, long-term use of estrogen-containing combined oral contraceptives increases the risk of developing hepatocellular carcinoma (51, 52, 53, 54). A recent consensus report of the World Health Organization International Agency for Research on Cancer concluded that long-term use of estrogen-containing combined oral contraceptives increases the risk of developing breast, cervical, and liver cancer (55, 56). Our demonstration that estrogen induction of PI-9 protects liver cancer cells against CTL and NK cell-induced apoptosis suggests that in transformed cells estrogen induction of PI-9 might reduce cytolytic lymphocytes-mediated immune surveillance of the transformed cells and thereby increase tumor incidence. Of course, the direct and indirect effects of estrogens in the intact organism are highly complex, and the induction of PI-9 will be only one of the several systems involved in the estrogen-mediated sparing of tumor cells from cytolytic lymphocytes-induced damage.
These studies extend our earlier finding that estrogens induce the human serpin PI-9 (13, 14, 15). We describe a new role for estrogens: regulating the susceptibility of target cells to the induction of apoptosis by cells of the immune system.
Acknowledgments
We are grateful to Dr. C. J. Froelich for providing YT cells.
Footnotes
This work was supported by National Institutes of Health Grants HD16720 and DK071909 (to D.J.S.) and GM55767 (to D.M.K.).
The authors have no conflict of interest.
First Published Online November 23, 2005
Abbreviations: CMA, Concanamycin A; CTL, cytotoxic T lymphocyte; EGF, epithelial growth factor; ER, estrogen receptor; ERU, estrogen-responsive unit; FBS, fetal bovine serum; MOX, moxestrol; NK, natural killer; PI-9, proteinase inhibitor 9; RAG, recombination gene activating; RNAi, RNA interference; siRNA, small interfering RNA; SPI, serine proteinase inhibitor; TCR, T-cell receptor.
Accepted for publication November 14, 2005.
References
Goping IS, Barry M, Liston P, Sawchuk T, Constantinescu G, Michalak KM, Shostak I, Roberts DL, Hunter AM, Korneluk R, Bleackley RC 2003 Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition. Immunity 18:355–365
Russell JH, Ley TJ 2002 Lymphocyte-mediated cytotoxicity. Annu Rev Immunol 20:323–370
Lieberman J 2003 The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol 3:361–370.
Barry M, Bleackley RC 2002 Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol 2:401–409
Thomas DA, Du C, Xu M, Wang X, Ley TJ 2000 DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity 12:621–632
Bird CH, Sutton VR, Sun J, Hirst CE, Novak A, Kumar S, Trapani JA, Bird PI 1998 Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol Cell Biol 18:6387–6398
Annand RR, Dahlen JR, Sprecher CA, De Dreu P, Foster DC, Mankovich JA, Talanian RV, Kisiel W, Giegel DA 1999 Caspase-1 (interleukin-1-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9. Biochem J 342(Pt 3):655–665
Bladergroen BA, Strik MC, Bovenschen N, van Berkum O, Scheffer GL, Meijer CJ, Hack CE, Kummer JA 2001 The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites. J Immunol 166:3218–3225
Medema JP, Schuurhuis DH, Rea D, van Tongeren J, de Jong J, Bres SA, Laban S, Toes RE, Toebes M, Schumacher TN, Bladergroen BA, Ossendorp F, Kummer JA, Melief CJ, Offringa R 2001 Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells. J Exp Med 194:657–667
Phillips T, Opferman JT, Shah R, Liu N, Froelich CJ, Ashton-Rickardt PG 2004 A role for the granzyme B inhibitor serine protease inhibitor 6 in CD8+ memory cell homeostasis. J Immunol 173:3801–3809
Young JL, Sukhova GK, Foster D, Kisiel W, Libby P, Schonbeck U 2000 The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1-converting enzyme (caspase-1) activity in human vascular smooth muscle cells. J Exp Med 191:1535–1544
Medema JP, de Jong J, Peltenburg LT, Verdegaal EM, Gorter A, Bres SA, Franken KL, Hahne M, Albar JP, Melief CJ, Offringa R 2001 Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors. Proc Natl Acad Sci USA 98:11515–11520
Kanamori H, Krieg S, Mao C, Di Pippo VA, Wang S, Zajchowski DA, Shapiro DJ 2000 Proteinase inhibitor 9, an inhibitor of granzyme B-mediated apoptosis, is a primary estrogen-inducible gene in human liver cells. J Biol Chem 275:5867–5873
Krieg SA, Krieg AJ, Shapiro DJ 2001 A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1-converting enzyme (caspase 1). Mol Endocrinol 15:1971–1982
Krieg AJ, Krieg SA, Ahn BS, Shapiro DJ 2004 Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. J Biol Chem 279:5025–5034
Kannan-Thulasiraman P, Shapiro DJ 2002 Modulators of inflammation use nuclear factor-B and activator protein-1 sites to induce the caspase-1 and granzyme B inhibitor, proteinase inhibitor 9. J Biol Chem 277:41230–41239
Barrie MB, Stout HW, Abougergi MS, Miller BC, Thiele DL 2004 Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6. J Immunol 172:6453–6459
Jansson L, Holmdahl R 1998 Estrogen-mediated immunosuppression in autoimmune diseases. Inflamm Res 47:290–301
Vegeto E, Belcredito S, Etteri S, Ghisletti S, Brusadelli A, Meda C, Krust A, Dupont S, Ciana P, Chambon P, Maggi A 2003 Estrogen receptor- mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci USA 100:9614–9619
Lang TJ 2004 Estrogen as an immunomodulator. Clin Immunol 113:224–230
Bouman A, Heineman MJ, Faas MM 2005 Sex hormones and the immune response in humans. Hum Reprod Update 11:411–423
Archer TK, Tam SP, Deugau KV, Deeley RG 1985 Apolipoprotein C-II mRNA levels in primate liver. Induction by estrogen in the human hepatocarcinoma cell line, HepG2. J Biol Chem 260:1676–1681
Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors and . Endocrinology 138:863–870
Taylor AH, Al-Azzawi F 2000 Immunolocalisation of oestrogen receptor in human tissues. J Mol Endocrinol 24:145–155
Manning TC, Rund LA, Gruber MM, Fallarino F, Gajewski TF, Kranz DM 1997 Antigen recognition and allogeneic tumor rejection in CD8+ TCR transgenic/RAG(–/–) mice. J Immunol 159:4665–4675
Kranz DM, Eisen HN 1987 Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T lymphocytes. Proc Natl Acad Sci USA 84:3375–3379
Goolsby KM, Shapiro DJ 2003 RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis. Nucleic Acids Res 31:5644–5653
Obrero M, Yu DV, Shapiro DJ 2002 Estrogen receptor-dependent and estrogen receptor-independent pathways for tamoxifen and 4-hydroxytamoxifen-induced programmed cell death. J Biol Chem 277:45695–45703
Nagata S 2000 Apoptotic DNA fragmentation. Exp Cell Res 256:12–18
Bierer B, Coligan JE, Margulies DH, Shevach EM, Strober W, Kruisbeek A 2003 Curr Protoc Immunol 3.11.8–3.11.20
Shen WH, Zhou JH, Broussard SR, Freund GG, Dantzer R, Kelley KW 2002 Proinflammatory cytokines block growth of breast cancer cells by impairing signals from a growth factor receptor. Cancer Res 62:4746–4756
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T 2001 Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498
Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, Barry M, Shostak I, Sawchuk T, Holmes CF, Gauldie J, Bleackley RC 2000 Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell 103:491–500
Kataoka T, Shinohara N, Takayama H, Takaku K, Kondo S, Yonehara S, Nagai K 1996 Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J Immunol 156:3678–3686
Wiltrout RH 2000 Regulation and antimetastatic functions of liver-associated natural killer cells. Immunol Rev 174:63–76
Mackay IR 2002 Hepatoimmunology: a perspective. Immunol Cell Biol 80:36–44
Vermijlen D, Luo D, Froelich CJ, Medema JP, Kummer JA, Willems E, Braet F, Wisse E 2002 Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway. J Leukoc Biol 72:668–676
Mahrus S, Craik CS 2005 Selective chemical functional probes of granzymes a and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem Biol 12:567–577
Chuang SS, Kumaresan PR, Mathew PA 2001 2B4 (CD244)-mediated activation of cytotoxicity and IFN- release in human NK cells involves distinct pathways. J Immunol 167:6210–6216
Miyagawa S, Kubo T, Matsunami K, Kusama T, Beppu K, Nozaki H, Moritan T, Ahn C, Kim JY, Fukuta D, Shirakura R 2004 -Short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis. J Immunol 173:3945–3952
Cutolo M, Sulli A, Capellino S, Villaggio B, Montagna P, Seriolo B, Straub RH 2004 Sex hormones influence on the immune system: basic and clinical aspects in autoimmunity. Lupus 13:635–638
Carlsten H, Holmdahl R, Tarkowski A, Nilsson LA 1989 Oestradiol- and testosterone-mediated effects on the immune system in normal and autoimmune mice are genetically linked and inherited as dominant traits. Immunology 68:209–214
Carlsten H, Holmdahl R, Tarkowski A, Nilsson LA 1989 Oestradiol suppression of delayed-type hypersensitivity in autoimmune (NZB/NZW)F1 mice is a trait inherited from the healthy NZW parental strain. Immunology 67:205–209
Molloy EJ, O’Neill AJ, Grantham JJ, Sheridan-Pereira M, Fitzpatrick JM, Webb DW, Watson RW 2003 Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone. Blood 102:2653–2659
Bengtsson AK, Ryan EJ, Giordano D, Magaletti DM, Clark EA 2004 17-Estradiol (E2) modulates cytokine and chemokine expression in human monocyte-derived dendritic cells. Blood 104:1404–1410
Mo R, Chen J, Grolleau-Julius A, Murphy HS, Richardson BC, Yung RL 2005 Estrogen regulates CCR gene expression and function in T lymphocytes. J Immunol 174:6023–6029
Polanczyk MJ, Carson BD, Subramanian S, Afentoulis M, Vandenbark AA, Ziegler SF, Offner H 2004 Cutting edge: estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment. J Immunol 173:2227–2230
Bade B, Boettcher HE, Lohrmann J, Hink-Schauer C, Bratke K, Jenne DE, Virchow Jr JC, Luttmann W 2005 Differential expression of the granzymes A, K and M and perforin in human peripheral blood lymphocytes. Int Immunol 17:1419–1428
Lewandowski SA, Thiery J, Jalil A, Leclercq G, Szczylik C, Chouaib S 2005 Opposite effects of estrogen receptors and on MCF-7 sensitivity to the cytotoxic action of TNF and p53 activity. Oncogene 24:4789–4798
ten Berge RL, Oudejans JJ, Ossenkoppele GJ, Meijer CJ 2003 ALK-negative systemic anaplastic large cell lymphoma: differential diagnostic and prognostic aspects—a review. J Pathol 200:4–15
Forman D, Vincent TJ, Doll R 1986 Cancer of the liver and the use of oral contraceptives. Br Med J (Clin Res Ed) 292:1357–1361
Yu MC, Tong MJ, Govindarajan S, Henderson BE 1991 Nonviral risk factors for hepatocellular carcinoma in a low-risk population, the non-Asians of Los Angeles County, California. J Natl Cancer Inst 83:1820–1826
Tavani A, Negri E, Parazzini F, Franceschi S, La Vecchia C 1993 Female hormone utilisation and risk of hepatocellular carcinoma. Br J Cancer 67:635–637
Chen CJ, Yu MW, Liaw YF 1997 Epidemiological characteristics and risk factors of hepatocellular carcinoma. J Gastroenterol Hepatol 12:S294–S308
Vincent Cogliano, Yann Grosse, Robert Baan, Kurt Straif, Ghissassi BSFE 2005 Carcinogenicity of combined oestrogen-progestogen contraceptives and menopausal treatment. Lancet Oncol 6:552–553
La Vecchia C, Autier P, Rosano G, Stafford R, Vatten L, Shapiro S, Fernandez E, Beral V, Peto J, Anderson G, Bosland M, Haslam S, Junghans T, Kaufman D, Molinolos A, Olin S, Newcomb P, Parl F, Roy D, Stanczyk F, Thomas D, Combined estrogen-progestogen contraceptives and combined estrogen-progestogen menopausal therapy. IARC Monogr Eval Carcinog Risks Hum, in press(Xinguo Jiang, Brent A. Orr, David M. Kra)