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,2
* Instituto de Biología y Medicina Experimental-Consejo Nacional de Investigaciones Científicas y Técnicas; and
Departmento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, Vuelta de Obligado 2490, (1428) Buenos Aires, Argentina
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Abstract |
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Key Words: luteoma • immunosuppression • cyclosporin A • dexamethasone
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Introduction |
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The experimental intrasplenic ovarian tumor, selected for the following experiments, was developed initially by Biskind and Biskind (3), with later modifications, including those of our laboratory (4). It is a typical benign endocrine tumor that secretes various hormones including estradiol, progesterone (5, 6), and inhibin (7), as well as growth factors such as insulin-like growth factor (IGF)-1 (8). The tumor develops when an ovary is grafted into the spleen of an adult bilaterally castrated female rat. In this new anatomic location, the steroids produced by the ovary reach the hepatic portal vein and are metabolized in the liver. In this way, the negative feedback normally exerted by the steroids on the hypothalamic-pituitary unit is abolished, and hypergonadotropinemia is established. The grafted ovary turns into a highly luteinized tumor (luteoma) under the constant stimulation of gonadotropins, which induce growth and hormonal activity (5, 9, 10). The dependency of this experimental luteoma on gonadotropin hyperstimulation for its growth is similar to the one observed in different ovarian pathologies as in certain ovarian carcinomas (11–13), granulosa-cell tumors after ovarian stimulation in treatments of infertility (14), or in the polycystic ovary syndrome (15, 16) among others.
The question concerning tumor localization from the immunological perspective is a very interesting point. The site of the ovarian graft was selected to ensure steroid metabolism by the liver and, as a consequence, tumor development under hypergonadotropinemia, as described above.
From the immunological point of view, this is a critical site of antigen presentation, taking into account the hypothesis proposed by Zinkernagel et al. (17), which postulates that antigen localization, dose, and time kinetics form a three-dimensional integral that determines the immunological response. Thus, the localization of the antigen (luteoma) in a secondary lymphoid organ (spleen) is a decisive component in this paradigm.
In view of this, and due to the lack of information regarding the compromise of the immune system in the development of luteoma, the study of tumor progress under immune suppression was an attractive approach.
To establish if immunosuppression could alter the development of this experimental tumor, the effects of cyclosporin A (CsA) and dexamethasone (Dex) were evaluated in tumor-bearing animals. These drugs exert their actions on the immune system by different mechanisms. CsA is a microbial product with potent immunosuppressing properties that result from a selective inhibition of T lymphocyte activation. CsA interacts with the intracellular signaling triggered by the activation of the T cell receptor, forming a complex with cyclophilin and calcineurin, which blocks the translocation of the transcription factor NF-AT to the nucleus, inhibiting the synthesis of many cytokines. This selectively suppresses the adaptive response of T lymphocytes as well as their de novo development in the thymus (18). Because of its capacity of interfering with T cell activation, this drug is used as a potent immunotherapeutic agent in autoimmune diseases and also to prevent transplanted organ rejection (19). On the other hand, Dex is a synthetic glucocorticoid 10- to 20-fold more active than cortisol and corticosterone. It is a potent anti-inflammatory and immunosuppressor that works by inhibiting transcription factors as activation protein-1 (AP-1) and NF-
B, which in turn suppress cytokine, receptor, and adhesion molecule induction involved in the activation, migration, and cell recruiting. As a consequence, an inhibition of peripheral T lymphocyte proliferation is produced, accompanied by an inhibition of cell migration to inflammation sites and control of leukocyte recirculation (20–22). Furthermore, it induces CD4+CD8+ thymocyte apoptosis, inducing thymus atrophy (23, 24).
In sum, our aim was to analyze the effect of immunosuppressor drugs, CsA and Dex, on the development of a hormone-dependent ovarian tumor grafted into the spleen after 7 weeks of treatment in comparison with vehicle-treated animals. For this purpose, various endocrine and immune parameters were evaluated as well as survival rates, body weight, and tumor volume and histology at the end of the experimental procedures.
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Methods |
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Animals.
Adult female virgin Sprague-Dawley rats (200–250 g) from the Instituto de Biología y Medicina Experimental colony were housed in groups in an air-conditioned room, with lights on from 0700 to 1900 hr. They were given free access to laboratory chow and tap water. To obtain tumor-bearing animals, surgical procedures were performed as previously described (4–6). Briefly, animals were anesthetized with ketamine (100 mg/kg body wt ip), both ovaries were removed, and one gonad was cleared from the adherent fat and oviduct and was inserted into the spleen. Sham animals had a piece of abdominal muscle inserted into the spleen after being bilaterally ovariectomized.
Treatments.
Tumors were left to develop for 1 month. Thereafter, tumor-bearing animals were randomly distributed into the different groups that were treated subcutaneously with CsA (25 mg/kg, following the protocols of Murphy et al. [25] and Hojo et al. [26]), Dex (0.1 mg/kg), or vehicle (75:25 castor oil:ethanol) in the morning on alternate days for 50 days. Body weight was determined once a week to adjust the dose. Each group comprised seven to 12 animals.
Sampling.
After finishing the treatments, a jugular vein blood sample was taken with heparin under ether anesthesia. Thereafter, animals were sacrificed by cervical dislocation according to protocols for animal use approved by the Institutional Animal Care and Use Committee, which follows National Institutes of Health guidelines. Autopsies of animals were performed, searching for tumor-induced disorders or metastatic propagation. A macroscopic observation of several organs and tissues, including lungs, heart, liver, kidneys, adrenal glands, intestines, uterus, spleen, adipose tissue, and peritoneum was performed to establish the presence of any anomalies.
Thymi were dissected and weighed. Tumors were measured in two dimensions with a caliper, and tumor volumes were calculated based on an ellipsoid tumor shape (V: 4/3
. r12. r2, where r1 is the minor radius (10, 27). Some tumors of each group were placed in formaline for histological studies.
Hematological Parameters.
To determine the immunological status of the animals, a leukocyte differential count was performed, and the number of lymphocytes, segmented neutrophils, and monocytes in heparin-treated blood was quantified with a cell counter. In addition, red blood cell number and hemoglobin content were also determined.
Hormonal Determinations.
Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) were determined by radioimmunoassay (RIA) using kits provided by the NIDDK. Results were expressed in terms of RP3 rat LH, FSH, and PRL standards. Assay sensitivities were 0.015 ng/ml for LH and 0.1175 ng/ml for FSH. Intra- and interassay coefficients of variation for LH were 7.2% and 11.4%, respectively, 8.0% and 13.2%, respectively, for FSH, and 8.1% and 11.4%, respectively, for PRL.
Serum estradiol was also quantified by RIA using a specific antiserum kindly provided by Dr. G. D. Niswender. Labeled estradiol was purchased from New England Nuclear (Boston, MA). Assay sensitivity was 1.7 pg and intra- and interassay coefficients of variation were 9.3% and 11.4%, respectively.
Histology.
Tumors from CsA-, Dex-, or vehicle-treated animals were kept in 10% formaline, embedded in paraffin wax, and sectioned at 4 µm using a microtome, as in previous studies (7). For light microscopic examination, sections were stained with hematoxylin and eosin using standard procedures. Three or four different tumors in each experimental group were subjected to blind evaluation by an independent pathologist. Several slides were examined from each tumor.
Statistical Analysis.
Results are shown as the mean ± SEM. Differences in means among groups were analyzed with Statistica v 5.0 software by multiple variance analysis (two-way ANOVA), followed by Newman-Keuls test for interaction factors, when the interaction was significant (P < 0.05), or for the main effects, when the interaction was nonsignificant (28). Differences between percentages were evaluated by test of comparison of two percentages (29). P < 0.05 was considered significant.
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Results |
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). Deaths occurred 6–7 weeks after treatments were started.
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).
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In CsA-treated tumor-bearing animals, body weight increased from the 3rd week of treatment and continued to increase until the end of the experiment, similar to vehicle-treated animals (P < 0.05). In CsA-treated sham animals, body weight increased already in the 2nd week (P < 0.001), continued to increase up to the 5th week, and then abruptly fell (P < 0.001), reaching initial levels.
Immunological Parameters.
With the purpose of assessing the immunosuppressor effect of the treatments (CsA and Dex), some immunological indicators were determined. Dex induced a significant decrease in thymus weight in both tumor-bearing (P < 0.005) and sham (P < 0.001) animals when compared with vehicle-treated animals (Fig. 2). CsA induced thymus weight loss only in sham animals (P < 0.005), and no effect was observed in tumor-bearing rats. The same results were observed when thymus weight was expressed relative to body weight (data not shown).
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). Neither CsA nor Dex altered red blood cell number (not shown). Segmented neutrophil number/mm3 did not vary with treatment, but was higher in sham animals than in tumor-bearing animals (P < 0.05; Fig. 3B). Lymphocyte number/mm3 was significantly decreased in Dex-treated rats with regard to vehicle (P < 0.005) and to CsA (P < 0.001) injected animals (Fig. 3C). CsA had no effect on lymphocyte number. CsA, on the other hand, induced an increase monocytes/mm3 only in sham animals (P < 0.005), whereas Dex had no effect (Fig. 3D).
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), and no differences due to drug treatments were apparent. With regard to PRL, CsA induced a significant increase only in sham animals (P < 0.05; Fig. 4C); the different treatments did not alter PRL secretion in tumor-bearing rats. No variations in estradiol levels under any treatment or in any group were observed (Fig. 4D).
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). In addition, no visible metastases or alterations in other organs were observed.
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). No signs of corpora lutea involution were observed, in agreement with lack of TUNEL-positive cell detection in these tumors (30). Immunosuppressor treatments did not alter tumor histology, and no signs of malign transformation were observed. A collagen interphase was observed in three of 10 tumors with a focalized pattern.
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Discussion |
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With regard to survival rates, only CsA induced a significant decrease in this parameter both in sham and tumor-bearing animals, suggesting a noxious effect of this drug at the present protocol of administration.
In sham vehicle-treated rats, the expected postcastration weight increase was observed. Tumor presence delayed this weight increase in both vehicle- or CsA-injected animals. This may imply that tumors produce some factor(s) that is inhibitory on weight increase or that the postcastration state is not established in tumor-bearing animals to the same degree as in sham animals (in agreement with lower gonadotropin levels, see below). The CsA-treated tumor-bearing animals that survived until the end of the experiment showed a mean body weight similar to vehicle-treated animals, whereas in sham animals, CsA induced an important loss of weight after 6 weeks of treatment, suggesting a protective effect in tumor-bearing animals with regard to CsA action. On the other hand, Dex induced considerable body weight loss starting in the 2nd week of treatment in both sham and tumor-bearing animals, in agreement with the catabolic effect extensively described for this drug (31).
The effects of CsA on survival rates and body weight could be due to side effects of the drug. In this context, Whiting and colleagues (32) described that rats treated for 7 weeks with CsA presented structural and functional renal anomalies as well as liver damage.
When analyzing thymus weight, Dex induced the expected reduction (approximately 50%) in both sham and tumor-bearing animals (23). On the other hand, CsA induced a 30% decrease in sham animals, in agreement with previous data (33), whereas no effect was observed in tumor-bearing rats. This once again points to the presence of some factor secreted by the tumor that protects the thymus from CsA action. Previous studies from our laboratory have shown that these tumors secrete inhibin (7, 8). Hedger and colleagues (34) have shown that inhibin stimulated [H3]thymidine incorporation in adult, male rat thymocyte nonstimulated or mitogen-stimulated cultures. Therefore, a proliferative action of inhibin could oppose the antiproliferative effect of CsA, preventing thymus weight loss in tumor-bearing animals.
To establish if immunosuppressor-treated tumor-bearing animals were still able to produce inhibins, mRNA for inhibin subunits was studied by Northern blot. The results indicated that tumors still had the capacity to synthesize inhibins in vehicle-treated and immunosuppressed animals, though ß subunits were expressed in low amounts (unpublished results from this laboratory). In contrast to the results from Hedger and colleagues (34), inhibin could not counteract the CsA-induced inhibition of proliferation in ovariectomized female rat thymocyte cultures stimulated with phytohemagglutinin in our experimental conditions (unpublished observations from this laboratory).
Therefore, under our conditions, inhibin does not seem to be responsible for counteracting CsA actions in tumor-bearing animals. Various other proteins not inactivated by the liver, such as IGF-I, which is produced by these tumors (8) and has been shown to induce proliferation of thymocytes (35), may be involved in these effects. Moreover, IGF-I has been shown to accelerate reconstitution of the rat thymus after CsA-induced involution (36).
When analyzing other immunosuppression parameters, a Dex-induced decrease in lymphocyte number was observed, as expected (20), whereas CsA had no effect. The lack of CsA effect on lymphocyte number may be due to insufficient dosage, which is improbable because with the dose of CsA used, animals showed side effects. This response may also be conditioned to the strain of animals used. With regard to monocytes, in sham animals, CsA induced an increase monocyte number, in agreement with data from Whiting et al. (32). Similarly to what occurs with thymus and body weight, some factor produced by the tumor also blocked CsA action on monocyte number in tumor-bearing animals. Though red blood cell numbers were not modified by any treatment, a low hemoglobin concentration was observed in CsA-treated animals, also in agreement with previous data (32); this parameter did not seem to be altered by the putative tumor-secreted factor, as it occurred in both sham and tumor-bearing animals.
The hormonal environment in these animals was also determined. In agreement with previous data from our laboratory (5), in tumor-bearing rats, LH and FSH were markedly increased with regard to estrous animals (LHestrous: 1.3 ± 0.2 ng/ml; FSHestrous: 6.6 ± 0,4 ng/ml), but were significantly lower than in sham animals. Immunosuppressor drugs did not alter gonadotropin secretion either in tumor-bearing or in sham animals. In contrast, an CsA-induced increase in PRL levels was observed in sham animals, although not in tumor-bearing rats, an observation that endorses our hypothesis of a factor secreted by the tumor that impedes some of CsA actions. A serum PRL increase due to CsA administration had already been shown by Esquifino et al. (37). CsA-induced effects on monocytes and PRL secretion could be related events, as it has been shown that human monocytes and T and B lymphocytes express the PRL receptor mRNA and protein (38, 39). In addition, PRL enhances absolute numbers of splenic granulocyte-macrophage colony-forming units in immunosuppressed mice (40).
Histological analysis of tumor samples showed a high degree of luteinization, which was not modified by immunosuppressor drug treatment. It is interesting to note that the percentage of tumors with predominance of luteinized cells (corpora lutea or luteinized follicles) over granulosa cells (well-developed follicular structures) was similar to the one observed in tumors after 1 year of development (7).
Our main aim had been to evaluate if immunosuppressor drugs could alter the development of this ovarian tumor, specially taking into consideration that, because of its intrasplenic localization, there was a possibility that the graft could have been limited in its development by the immune system (17). Neither CsA nor Dex significantly affected tumor volume or histological parameters, and no visible metastases could be observed in the tissues analyzed. Neither drug, in the present experimental conditions, induced tumor exacerbation or malignization. This lack of exacerbation under immunosuppressive therapy could be due to tumors presenting differentiation antigens and thus inducing a very weak immune response; in this case, immunosuppression would not substantially affect their development. In addition, one mechanism of malignant transformation is the reduction of major histocompatibility complex (MHC) class I molecules expression. Van Niekerk et al. (41), studying the marker profile of normal human ovarian tissues and their derived tumors, have shown a loss of MHC class I molecules expression in areas of tumoral cells. As our tumor did not present malignant transformation, the tissue probably expressed normal levels of MHC class I molecules presenting no foreign antigens, and as a consequence, the immune system may not have been induced. Furthermore, in a recent work, Ochsenbein et al. (42) described that tumor cells injected directly into the spleen but separated from T cells by barriers including collagen or hemostasis factors did not induce priming of cytotoxic T cells and were thus not rejected. From histological observations, a collagen interphase seems to be formed around some intrasplenic ovarian tumors (three of 10 tumors analyzed, see Table II
), which may, in part, explain the lack of differences in tumor development between control and immunosuppressant-treated animals.
It is interesting to note that although Hojo et al. (26) have shown that CsA promotes cancer progression by a direct cellular mechanism inducing morphological alterations in tumor cells such as membrane ruffling and acquirement of exploratory pseudopodia, no cell alterations were observed in intrasplenic ovarian tumors under CsA treatment.
We conclude that immunosuppression induced by CsA or Dex, although effective on immunological parameters, did not affect luteoma development. In contrast to tumor-bearing animals, in sham animals, CsA induced several significant effects on body weight, thymus weight, monocyte number, and PRL levels, suggesting the presence of tumor-secreted factor(s) that impede or counteract some of CsA actions. Future experiments will be designed to verify this hypothesis.
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Acknowledgments |
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Footnotes |
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2 To whom requests for reprints should be addressed at Instituto de Biología y Medicina Experimental, V. de Obligado 2490, (1428) Buenos Aires, Argentina. E-mail: libertun{at}dna.uba.ar
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References |
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B-dependent transcription involves direct interference with transactivation. Proc Natl Acad Sci U S A 94:13504–13509, 1997.
HOP) on lymphocyte proliferation. Clin Expt Immun 86:311–314, 1991.
HOP) effect `in vivo`. Acta Physiol Pharmacol Latinoamer 39:181, 1989.
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