of monoclonal antibodies against the TSH receptor are blocked in part by the cyclooxygenase inhibitor indometh- acin (1). AA and eicosanoid formation have ...
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Cyclooxygenase-Dependent Thyroid Cell Proliferation Induced by Immunoglobulins from Patients with Graves’ Disease* ROSA DI PAOLA, CLAUDIA MENZAGHI, VITO DE FILIPPIS, DANIELA CORDA, AND ALFREDO DI CERBO Division of Endocrinology (R.D.P., C.M., V.D.F., A.D.C.), Istituto di Ricovero e Cura a Carattere Scientifico “Casa Sollievo della Sofferenza” General Hospital, San Giovanni Rotondo (Foggia); and the Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche “Mario Negri,” Consorzio Mario Negri Sud (D.C.), S. Maria Imbaro (Chieti), Italy ABSTRACT IgG associated with Graves’ disease bind to the TSH receptor and alter thyroid growth and function, mainly through the stimulation of adenylyl cyclase. In addition, Graves’ IgG are able to interact with the phospholipase C (PLC)/Ca21 and phospholipase A2 (PLA2)/arachidonic acid (AA) cascades. The activation of this latter pathway leads to thyroid cell growth in vitro. The elucidation of additional mechanisms of action of Graves’ IgG has made possible the identification of four subgroups of patients, characterized by IgG with different biochemical activities (extent of cAMP and AA release stimulation in in vitro assays). On the basis of these results, a novel therapeutic approach could be proposed based on the inhibition of PLA2 and AA
metabolism. To test this hypothesis, the ability of IgG from 56 Graves’ patients to stimulate [3H]thymidine incorporation in FRTL5 thyroid cells in the presence and absence of the cyclooxygenase inhibitor indomethacin (2.5 3 1026 mol/L) was measured. A significant reduction in [3H]thymidine incorporation was found (33% inhibition; P , 0.0001) upon pretreatment with indomethacin, suggesting that in vitro thyroid cell growth is regulated by cyclooxygenase metabolites. This strengthens the argument for involvement of the PLA2/AA cascade in the pathophysiology of Graves’ disease and the proposal for novel selective pharmacological treatments of these patients. (J Clin Endocrinol Metab 82: 670 – 673, 1997)
T
of AA in inducing thyroid growth led us to propose a novel therapeutic approach in Graves’ disease based on the inhibition of PLA2 and AA metabolism. The regulation of growth has been well characterized in FRTL5 cells and appears related to multiple transduction pathways including cAMP- and Ca21-dependent mechanisms activated by TSH, a kinase-dependent pathway stimulated by insulin-like growth factor I, and an AA-dependent pathway that produces PGE2 (6 – 8). PG synthesis in FRTL-5 thyroid cells is under multihormonal control, as insulin, insulin-like growth factor I, and serum are required for the TSH-dependent activation of cyclooxygenase (9, 10). Furthermore, DNA synthesis induced by TSH and the activity of monoclonal antibodies against the TSH receptor are blocked in part by the cyclooxygenase inhibitor indomethacin (1). AA and eicosanoid formation have been shown to be involved in the control of cell growth in several cell systems (11–14). Similarly, AA and its metabolites might play a role in the regulation of thyroid growth in Graves’ disease. We have analyzed this possibility using the cyclooxygenase inhibitor indomethacin in FRTL5 thyroid cells stimulated to proliferate by IgG from Graves’ patients.
SH RECEPTOR- stimulating Igs play a pathogenetic role in Graves’ disease. They are believed to act via the TSH receptor to increase the cellular levels of cAMP and other second messengers and thus alter thyroid growth and function (1–3). We have previously demonstrated that the Igs (IgG) obtained from Graves’ patients stimulate not only adenylyl cyclase (AC), but also phospholipase A2 (PLA2) (4). This latter effect is easily distinguished from the known effect on AC, as a subpopulation of Graves’ IgG could be identified that induces arachidonic acid (AA) release without affecting cAMP (5). Moreover, four subgroups of Graves’ patients have been characterized on the basis of the different biochemical activities of their IgG (extent of cAMP production and AA release in in vitro assays) (5). Interestingly, we have shown that the IgG able to stimulate both AC and PLA2 induced the highest levels of [3H]thymidine incorporation in in vitro assays, and the corresponding patients were those with more severe manifestations of the disease (5). The role Received June 27, 1996. Revision received September 16, 1996. Accepted October 25, 1996. Address all correspondence and requests for reprints to: Dr. Alfredo Di Cerbo, Division of Endocrinology, Istituto di Ricovero e Cura a Carattere Scientifico “Casa Sollievo della Sofferenza,” 71013 San Giovanni Rotondo (Foggia), Italy. * Part of these data has been presented as an abstract at the 22nd International Congress of Endocrinology, San Francisco, CA, June 1996 (Abstract P3-162). This work was supported in part by the Italian Association for Cancer Research, the Italian National Research Council (no. 95.00558.PF39-ACRO and Convenzione CNR-Consorzio Mario Negri Sud), and the Italian Ministry of Health (EDRF 9201).
Materials and Methods Reagents and cells Hormones used in the tissue culture media, Coon’s modified Ham’s F-12 medium, ethyleneglycol-bis-(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid, Tris(hydroxymethyl) aminomethane, and indomethacin were obtained from Sigma Chemical Co. (St. Louis, MO). Tissue culture materials were purchased from Life Technologies (Grand Island, NY).
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Protein A-Sepharose CL4B was obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden); glycine was purchased from Bio-Rad Laboratories (Richmond, CA); Minicon B15 concentrators were obtained from Amicon Division (W. R. Grace Co., Danvers, MA). [3H]Thymidine was purchased from New England Nuclear Corp. (Boston, MA). FRTL5 cells, a continuous line of functioning rat thyroid cells, were cultured as previously described in Coon’s modified Ham’s F-12 medium, 5% calf serum, and a six-hormone mixture (TSH, insulin, cortisol, transferrin, glycyl-l-histidyl-l-lysine acetate, and somatostatin; 6H medium) (15).
Patients Sera were obtained from 88 patients with active Graves’ disease randomly selected from the group of 104 enrolled for our previous study (5) and from 54 normal subjects. The characteristics of patients have been previously described (5).
IgG purification and [3H]thymidine incorporation IgG purification and [3H]thymidine incorporation were performed as previously described (4, 5). To evaluate the effect of the cyclooxygenase inhibition on [3H]thymidine incorporation, FRTL5 cells were preincubated with 2.5 3 1026 mol/L indomethacin for 30 min before adding IgG. All determinations were made in triplicate for each experimental point. The cut-off value discriminating between [3H]thymidine incorporation due to pathological and normal IgG was calculated using Receiver Operating Characteristic Curves analysis (16).
Statistical analysis For statistical analysis of the data, the Wilcoxon signed rank test was used. Differences were considered statistically significant for P , 0.05.
Results
IgG from 88 patients with Graves’ disease and 54 normal subjects were characterized for their ability to stimulate [3H]thymidine incorporation in FRTL5 thyroid cells. In agreement with previous studies (17, 18) [3H]thymidine incorporation was significantly higher in Graves’ patients (P , 0.0001; Fig. 1). On the basis of the cut-off value (equal to a 116% increase over the basal value), IgG from 88 patients with Graves’ disease were divided into 2 groups according to whether their IgG were able to stimulate [3H]thymidine incorporation in FRTL5 thyroid cells. Fifty-six of 88 (63.6%) IgG gave values higher than the cut-off (Fig. 1). [3H]Thymidine incorporation induced by these IgG was then assayed in the presence or absence of 2.5 3 1026 mol/L indomethacin, which is known to block cyclooxygenase, a key enzyme leading to the synthesis of PGE2 (19). In the entire group of patients, a significant reduction of [3H]thymidine incorporation was found (33% inhibition; P , 0.0001; range, 0 – 64% inhibition) when cells were pretreated with 2.5 3 1026 mol/L indomethacin, indicating the ability of this cyclooxygenase inhibitor to reduce thyroid cell growth in in vitro studies (Fig. 2). Moreover, [3H]thymidine incorporation was completely abolished after preincubation with 2.5 3 1026 mol/L indomethacin in 38 of 56 IgG with elevated [3H]thymidine incorporation in the absence of indomethacin (Fig. 3). To exclude a possible direct toxic effect of indomethacin on FRTL5 cells, we also measured [3H]thymidine incorporation induced by cholera toxin in the presence and absence of the drug. We found that [3H]thymidine incorporation is not modified by pretreatment with the drug (data not shown). These data as well as the finding that some IgG retained the
FIG. 1. Frequency distribution patterns of [3H]thymidine incorporation induced by IgG from 88 patients with active Graves’ disease (shaded bars) and 54 normal subjects (open bars). The vertical solid line indicates the sample cut-off value. See text for details.
FIG. 2. [3H]Thymidine incorporation, as measured before (closed circles) and after (open circles) treatment with 2.5 3 1026 mol/L indomethacin in 56 patients with active Graves’ disease. Results are expressed as a percentage of basal [3H]thymidine incorporation (Hanks’ Balanced Salt Solution-BSA only). Individual data points represent the means of triplicate determinations. The difference between the two groups of data is statistically significant (P , 0.0001).
ability to stimulate [3H]thymidine incorporation in FRTL5 cells pretreated with indomethacin (see Fig. 2) rule out a possible toxic effect of the drug. In conclusion, these data suggest that the AA/PGE2 pathway may contribute to thyroid growth in some patients with Graves’ disease. More-
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growth may also depend on activation of the PLA2/AA/ PGE2 pathway. We have previously shown that a subgroup of Graves’ IgG acts mainly via the PLA2/AA cascade and that Graves’ patients whose IgG are able to stimulate both AC/ cAMP and PLA2/AA pathways have a more severe form of the disease (5). These data and the results of the present study could represent the molecular basis for a clinical trial using inhibitors of AA metabolism, such as indomethacin and acetylsalicylic acid, in association with the thionamide antithyroid agents to improve management of some Graves’ patients. Acknowledgments We thank Dr. C. Checchia de Ambrosio for IgG purification, and Dr. B. Dallapiccola for critical reading of the manuscript. We acknowledge the gift of purified human TSH from the National Hormone and Pituitary Program (NIH, Bethesda, MD).
References
FIG. 3. Frequency distribution patterns of [3H]thymidine incorporation induced by IgG from 56 patients with active Graves’ disease, measured before (shaded bars) and after (open bars) treatment with 2.5 3 1026 mol/L indomethacin. The vertical solid line indicates the sample cut-off value. See text for details.
over, our findings confirm the existence of subgroups of Graves’ IgG able to activate both cyclooxygenase- and noncyclooxygenase-dependent pathways. Discussion
IgG produced by B lymphocytes infiltrating the thyroid are believed to play a major role in the development of thyrotoxicosis and goiter in Graves’ disease (20, 21). IgG bind the TSH receptor and stimulate cAMP production in several thyroid systems, including human thyroid slices and membranes (2, 3). Activation of the AC/cAMP pathway is related to thyroid growth in Graves’ disease (6, 7, 22). Recently, it has also been shown that the PLC (23–26) and PLA2 (4, 5, 26) pathways are involved in the action of Graves’ IgG. This effect and previous data showing that indomethacin blocks the mitogenic effect of several monoclonal antibodies derived from Graves’ disease patients (1) suggest a role for AA metabolites, particularly cyclooxygenase products, in the regulation of thyroid growth in Graves’ disease. Indeed, we found that indomethacin inhibits the mitogenic activity of IgG from Graves’ patients in FRTL5 thyroid cells. The molecular mechanism involved in the stimulation of the PLA2/AA/PGE2 pathway has not been elucidated. It has been reported that different regions of the TSH receptor are involved in the activation of AC and PLC mediated by Graves’ IgG (24). Likewise, Graves’ IgG that release AA could bind to specific regions of the TSH receptor and, via activation of the PLA2/AA/PGE2 pathway, regulate thyroid cell growth. The present results strengthen the argument that thyroid
1. Kohn LD, Aloj SM, Tombaccini D, et al. 1985 The thyrotropin receptor. In: Litwack G, ed. Biochemical action of hormones. New York: Academic Press; vol 12:457–512. 2. Zakarija M, McKenzie JM. 1987 The spectrum and significance of autoantibodies reacting with the thyrotropin receptor. Endocrinol Metab Clin North Am. 16:343–363. 3. Rees Smith B, McLachlan SM, Furmaniak J. 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev. 9:106 –121. 4. Di Cerbo A, Di Girolamo M, Guardabasso V, De Filippis V, Corda D. 1992 Immunoglobulins from Graves’ patients stimulate phospholipase A2 in FRTL5 thyroid cells. J Clin Endocrinol Metab. 74:585–592. 5. Di Cerbo A, Di Paola R, Bonati M, Zingrillo M, De Filippis V, Corda D. 1995 Subgroups of Graves’ patients identified on the basis of the biochemical activities of their immunoglobulins. J Clin Endocrinol Metab. 80:2785–2790. 6. Jin S, Hornicek FJ, Neylan D, Zakarija M, McKenzie JM. 1986 Evidence that adenosine 39,59-monophosphate mediates stimulation of thyroid growth in FRTL5 cells. Endocrinology. 119:802– 810. 7. Tramontano D, Cushing GW, Moses AC, Ingbar SH. 1986 Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergize the stimulation of DNA synthesis induced by TSH and Graves’-IgG. Endocrinology. 119:940 –942. 8. Burch RM, Luini A, Mais DE, et al. 1986 a1-Adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. J Biol Chem. 261:11236 –11241. 9. Tahara K, Saji M, Aloj SM, Kohn LD. 1989 The arachidonic acid signal system in the thyroid: regulation by thyrotropin and insulin/IGF-I. Adv Exp Med Biol. 261:295–326. 10. Tahara K, Grollman EF, Saji M, Kohn LD. 1991 Regulation of prostaglandin synthesis by thyrotropin, insulin or insulin-like growth factor-I, and serum in FRTL-5 rat thyroid cells. J Biol Chem. 266:440 – 448. 11. Fafeur V, Jiang ZP, Bohlen P. 1991 Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells. J Cell Physiol. 149:277–283. 12. Patte C, Blanquet PR. 1992 Possible involvement of arachidonic acid metabolites in the synergistic action of endothelial mitogenesis by fibroblast growth factor and phorbol ester. Cell Mol Biol. 38:429 – 436. 13. Spisni E, Manica F, Tomasi V. 1992 Involvement of prostanoids in the regulation of angiogenesis by polypeptide growth factors. Prostaglandins Leukotriene Essential Fatty Acids. 47:111–115. 14. Dethlefsen SM, Shepro D, D’Amore P. 1994 Arachidonic acid metabolites in bFGF-, PDGF-, and serum-stimulated vascular cell growth. Exp Cell Res. 212:262–273. 15. Ambesi-Impiombato FS, Parks LAM, Coon HG. 1980 Culture of hormonedependent functional epithelial cells from rat thyroids. Proc Natl Acad Sci USA. 77:3455–3459. 16. Sackett DL, Haynes RB, Tugwell P. 1986 Clinical epidemiology. Boston: Little, Brown. 17. Valente WA, Vitti P, Rotella CM, et al. 1983 Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies. N Engl J Med. 309:1028 –1034. 18. Drexhage HA, Bottazzo GF, Doniach D, Bitensky L, Chayen J. 1980 Evidence for thyroid-growth-stimulating immunoglobulins in some goitrous thyroid diseases. Lancet. 2:287–292. 19. Meade EA, Smith WL, DeWitt DL. 1993 Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs. J Biol Chem. 268:6610 – 6614.
INDOMETHACIN AND THYROID GROWTH IN GRAVES’ DISEASE 20. McLachlan SM, Pegg CAS, Atherton MC, Middleton SL, Clark F, Rees Smith B. 1986 TSH receptor antibody synthesis by thyroid lymphocytes. Clin Endocrinol (Oxf). 24:223–230. 21. Kendall-Taylor P, Knox AJ, Steel NR, Atkinson S. 1984 Evidence that thyroidstimulating antibody is produced in the thyroid gland. Lancet. 1:654 – 656. 22. Zakarija M, Jin S, McKenzie JM. 1988 Evidence supporting the identity in Graves’ disease of thyroid-stimulating antibody and thyroid growthpromoting immunoglobulin G as assayed in FRTL5 cells. J Clin Invest. 81:879 – 884. 23. Kosugi S, Okajima F, Ban T, Hidaka A, Shenker A, Kohn LD. 1992 Mutation of alanine 623 in the third cytoplasmic loop of the rat thyrotropin (TSH) receptor results in a loss in the phosphoinositide but not cAMP signal induced by TSH and receptor autoantibodies. J Biol Chem. 267:24153–24156.
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24. Kosugi S, Okajima F, Ban T, Hidaka A, Shenker A, Kohn LD. 1993 Substitutions of different regions of the third cytoplasmic loop of the thyrotropin (TSH) receptor have selective effects on constitutive, TSH-, and TSH receptor autoantibody-stimulated phosphoinositide and 39,59-cyclic adenosine monophosphate signal generation. Mol Endocrinol. 7:1009 –1020. 25. Hidaka A, Okajima F, Ban T, Kosugi S, Kondo Y, Kohn LD. 1993 Receptor cross-talk can optimize assays for autoantibodies to the thyrotropin receptor: effect of phenylisopropyladenosine on adenosine 39,59-monophosphate and inositol phosphate levels in rat FRTL-5 thyroid cells. J Clin Endocrinol Metab. 77:1164 –1169. 26. Smallridge RC, Atwa MA, Burch HB, et al. 1996 Immunoglobulins from Graves’ disease patients stimulate phospholipase A2 and C systems in FRTL-5 and human thyroid cells. J Endocrinol. 148(Suppl):OC27.
