Augumented interleukin 6 production by rat thyrocytes (FRTL5): Effect of interleukin 1β and thyroid-stimulating hormone

AUGUMENTED INTERLEUKIN 6 PRODUCTION BY RAT THYROCYTES (FRTL5): EFFECT OF INTERLEUKIN lp AND THYROID-STIMULATING HORMONE Masahiro Iwamoto,’ Toshiko Sakihama,’ Nobutoshi Kimura,3 Kachio Tasaka,’ Toshimasa Onaya” This study shows that rat thyroid follicular (FRTW) cells can produce interleukin 6 (IL-6) spontaneously and that IL-ll3 enhanced the production of IL-6 dose-dependently. In addition, 10 ng/mL of IL-ll3 in combination with 10 to 1,000 pU/mL of thyroid-stimulating hormone (TSH) synergistically enhanced IL-6 production. It is suggested that IL-6 released from thyroid follicular cells may be a costimulator of autoreactive B and T cells in autoimmune thyroid disease and may constitute another link between the immune and endocrine systems. Copyright o 1991 by W.B. Saunders Company

The thyroid gland is known to be one of the main target organs involved in autoimmune disorders.’ Thyroid follicular cells may participate directly in local immune responses by expressing class II antigens,’ by presenting antigens,3 or by their phagocytic activity.4 Interleukin 6 (IL-6) manifests a broad spectrum of biological activities, including the promotion of B-cell growth5 and differentiation,’ T-cell activation,’ a role in the acute phase reaction,* and other effects. Various types of cells can produce IL-6 including monocytes,93”’ fibroblasts, “J endothelial cells,‘3,‘4keratinocytes,” chondrocytes,” and osteoblasts.” Some endocrine organs, e.g., the p cells of the pancreatic islets’* and the anterior pituitary cells,‘” can also produce IL-6. Recently, thyroid cells were found to produce IL-6 constitutively.2”*2’ It has been reported that elevated levels of IL-6 were detected both in the synovial effusions of patients with rheumatoid arthritisz2.” and in the urine of patients with mesangioproliferative glomerulonephritis,24 emphasizing the possible importance of IL-6 in the pathogenesis of immunoinflammatory disorders. Cytokines produced by thyroid follicular cells may cause the amplification or propagation of autoimmune

From

the Third Department of Internal Medicine,’ Department of Parasitology & Immunology,* and Department of Urology,3 University of Yamanashi Medical School, Yamanashi 409-38, Japan. *To whom reprint requests should be addressed. Copyright o 1991 by W.B. Saunders Company 1043-4666/91/0304-0006$5.00/O KEY WORDS: thyrocyteiTSH

CYTOKINE,

autoimmunityiinterleukin

Vol.

3, No. 4 (July),

1Biinterleukin

1991: pp 345-349

61

thyroid disease. In the present study, we determined whether IL-ll3 and thyroid-stimulating hormone (TSH) alone or in combination could stimulate the production of IL-6 by a rat thyroid follicular cell line (FRTLS).

RESULTS Dose-Dependent Interleukin I p

Induction

of Interleukin

6 by

To examine whether rat thyroid follicular cells could produce IL-6, quiescent FRTLS cells were cultured in 5 H medium with or without IL-ll3 for 12 h, and the IL-6 activity in the culture supernatants was measured by its effect on hybridoma growth. As shown in Fig. 1, FRTL.5 cells produced IL-6 activity even without any stimulation, whereas supernatants from cultures stimulated with IL-ll3 stimulated the growth of IL-6-dependent 7TDl cells in a dose-dependent manner. Kinetics of Induction Interleukin l/3

of Znterleukin

6 by

Quiescent FRTLS cells were cultured in 5 H medium with 10 ng/mL of IL-l@ Supernatants were harvested at 2,4,6,8, or 12 h and tested for their ability to induce 7TDl cell proliferation. IL-6 activity was observed within 2 h, increased in a time-dependent manner, and reached a plateau level after 8 h (Fig. 2). Enhancement

of Interleukin

6 Production

The addition of 1,000 lo,U/mL of TSH also caused an increase in IL-6 production, whereas the combina345

346

I Iwamoto

et al.

CYTOKINE,

50

TABLE

1.

Enhancement

Vol. 3, No. 4 (July

1991: 345-349)

of IL-6 production.

I

Additive” TSH

IL-1p

wJ/w

WmL) 0

0 0 10 10 10 10

1,000 0 10 100 1,000

IL-6 productionh

(UlmL

per 10’ cells)

1.5 15.7 19.7 71.7 145.8 202.4

k k f k k k

1.7 3.1 1.8 6.6 18.8 11.0

“FRTL5 cells were cultured with IL-lp (10 ng/mL) and/or TSH (10 to 1,000 @/mL) for 12 h. hResults are expressed as the mean + SD of three separate cultures.

tion in FRTL.5 cells, and these combinations also did not stimulate IL-6 production. Moreover, IL-lp treated with anti-IL-1P antiserum did not stimulate IL-6 production by FRTLS cells (Table 2).

IL-1 p (rig/ml) Figure

1.

Dose-dependent

induction

FRTLS cells were incubated 12 h. Results are expressed

of IL-6

by IL-IS.

with various concentrations as the mean + SD of three

of IL-lp cultures.

for

tion of 10 ng/mL of IL-lp with 10 pU/mL to 1 mU/mL of TSH induced greater IL-6 production than either IL-lp (10 ng/mL) or TSH (1 mU/mL) alone (Table 1). The combination of 10 ng/mL of IL-lfi with 1 mU/mL of TSH enhanced IL-6 production lo-fold compared with each stimulus alone. Exclusion of Contamination

Northern Blot Analysis In an effort to demonstrate further evidence that IL-lp induces IL-6 production by thyroid follicular cells, we investigated the expression of IL-6 mRNA in IL-lp-stimulated thyroid follicular cells by Northern blot analysis. As shown in Fig. 3, two faint but discrete bands were identified in RNA isolated from IL-lpstimulated thyroid follicular cells, which were comparable with the bands shown by IL&producing P815 cells.

by Endotoxin

To exclude the possibility of contamination by endotoxin, IL-lp and TSH preparations were treated at 100°C for 20 min and subsequently tested for their effects on IL-6 production. Both heat-treated IL-lp and heat-treated TSH did not enhance IL-6 produc-

DISCUSSION Our study showed that the functioning rat thyroid follicular cell line, FRTL.5, could produce IL-6 even in an unstimulated state. IL-lp stimulated IL-6 production by thyroid follicular cells in a dose-dependent manner, and significantly greater quantities of IL-6 TABLE Experiment

11

8 2

I 12

4

Incubatk Figure

2.

Supernatants incubation. cultures.

Kinetics

of induction

of IL6

were assayed for IL-6 Results are expressed

Tim:

(h)

by IL-l@

(10 ng/mL).

activity after different periods of as the mean 4 SD of three

2.

Exclusion

of contamination Treatment

None (control) IL-lp + normal rabbit serum IL-l!3 + anti-IL-lp None (control) IL-1p IL-1p (100°C) None (control) IL-1p TSH IL-lp + TSH TSH (100°C) IL-lp (100°C) + TSH (100°C)

by endotoxin. IL-6 production” (UlmL per 10s cells) 12.7 25.2 13.5 2.8 12.0 2.7 2.3 14.3 18.2 113.5 2.5 1.9

2 f -+ k k -+ + e ” f k f

0.7 3.5 2.0d 0.1 0.1 O.gd 0.6 2.4 2.4 26.0 0.5d 0.3d

“Results are expressed as the mean + SD of three separate cultures. ‘FRTL5 cells were cultured with or without IL-l@ (OS ng/mL) for 12 h. IL-lp was neutralized by the addition of a rabbit anti-IL-lp antiserum (1:SOO). ‘FRTL5 cells were culmred with or without IL-lp (10 ngimL) for 12 h. IL-lp and TSH were inactivated by treatment at 100°C for 20 min [IL-l6 (100°C); TSH (lWC)]. “No significant difference compared with the control.

Augumented

Lane

1234

28s -

5 .. .( 2-.:&i%,,’ ;::*:‘“.F. ,:,: : I

18S-

Lane

123

28S-

.

IL-6

production

by FRTLS

cells

/ 347

4

18S-

0B

Q9 Figure 3. in lane 5.

(A) Detection

of IL-6

mRNA

by Northern

blot analysis;

5 pg of RNA

in lanes

1 to 4, and 10 pg of RNA

Lanes 1 and 5, P815 cells as a positive control; lane 2, unstimulated FRTLS cells; lane 3, FRTLS cells stimulated with IL-lp (10 ng/mL) for 1 h; lane 4, FRTLS cells stimulated with IL-l@ (10 ng/mL) for 2 h. Autoradiograph exposed for 5 days. (B) Same RNA preparations hybridized with p-actin probe; 5 kg of RNA in all lanes. Lanes to-4 same as in (A). Autoradiograph exposed for 16 h.

were produced by thyroid follicular cells stimulated with 10 ng/mL of IL-lp in combination with 10 to 1,000 p U/mL of TSH. Neutralizing antibodies to rat IL-6 were not available at the time that the experiment was performed. However, the IL-6-dependent 7TDl cells have been reported to respond specifically to IL-6.‘4,25 It has been reported that rat IL-6 mRNA can hybridize to mouse IL-6 cDNA.*~ Northern blot analysis showed that IL-6 mRNA was expressed by FRTLS cells stimulated with IL-lp. But we could not detect the expression of IL-6 mRNA in unstimulated FRTLS cells, indicating very low amounts of IL-6 mRNA level. Van Snick et al. reported that a single band of - 1,300 base pairs was observed when RNA of activated T cells was hybridized with the same probe.2h However, Horii et al. detected another band with a size of -20 S, and our result is comparable to their findingZ4 The significance of the other band is not yet certain. The present results showing IL-6 production by thyroid follicular cells complement recent studies by Grubeck-Loebenstein, et al.*” and Weetman, et al.*’ Weetman et al. have found that gamma interferon, tumor necrosis factor, and TSH enhance IL-6 release by human thyrocytes. However, they have not documented the effects of cytokines in combination with TSH. The data presented here demonstrate that thyroid follicular cells can produce IL-6 and that IL-lp and TSH synergistically and markedly enhance IL-6 production. IL-l, which can be produced in the inflammatory response, has multiple effects on thyroid follicular cells, including the inhibition of thyroglobulin and cyclic AMP productionZ7 and increasing the expression of c-myc mRNA.2” In a variety of cell types including T

and B cells, IL-6 has been shown to have a large number of properties,s-x,29”1 but the effects of IL-6 on thyroid follicular cells are largely unknown. In Graves’ disease and Hashimoto’s thyroiditis, the

1

thyroid-stimulating antibody and serum TSH levels may be respectively elevated. In such a situation, IL-6 production may be enhanced in the thyroid gland, and may influence the inflammatory response by its effects on local T and B cells. For example, it is possible that the production of antimicrosome, antithyroglobulin, and anti-TSH receptor antibodies is enhanced by IL-6 effects on B cell differentiation. It is also likely that sentitized T cells are further activated by IL-6, which may have a direct action on thyrocytes themselves. Thus, IL-6 production by thyroid follicular cells may contribute to the development of autoimmune thyroid disease. MATERIALS

AND METHODS

Culture Medium and Reagents Ham’s F-12K medium and RPM1 1640 medium were purchased from Flow Laboratories, Inc. (Irvine, Scotland) and GIBCO Laboratories (Grand Island, NY), respectively. Bovine TSH, insulin, transferrin, and 3-(4,5-dimethylthiazol2-yl)-2, 5-diphenyl-tetrazolium bromide were obtained from Sigma Chemical Co. (St. Louis, MO). Somatostatin was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Glycyl-L-histidyl -L-lysine acetate was obtained from Aldrich Chemical Co, Inc. (Milwaukee, WI) and hydrocortisone from Nikken Chemical Co. (Tokyo, Japan). Fetal calf serum and calf serum were purchased from Flow Laboratories, Inc. and GIBCO Laboratories, respectively. Human rIL-1P and rabbit anti-human IL-lp antisera were gifts from Otsuka Pharmaceutical Co. (Tokushima, Japan). Murine rIL-6 was kindly provided by Dr. Sudo (Biomaterial Research Institute Co., Ltd., Yokohama, Japan).

Induction

of Interleukin

6

The FRTLS cells maintain the functional characteristics of iodide uptake, thyroglobulin synthesis, and have a TSHresponsive adenylate cyclase system. The FRTL.5 cells were

348

I Iwamoto

grown in Ham’s F-12K medium supplemented with 5% calf serum and a 6-hormone mixture (10 p. g/mL of insulin, 10-8M hydrocortisone, 5 p. g/mL of transferrin, 10 ng/mL of glycyl-Lhistidyl-l;lysine, 10 p g/mL of somatostatin, and 5 mU/mL of TSH).32 For the studies, 100,000 cells of FRTLS were seeded in 24-well plates (Nunc, Roskilde, Denmark). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO,. After 24 h of incubation in the medium supplemented with 1 mU/mL of TSH, the cells were washed twice with phosphate-buffered saline without MgZ’ or Ca2+ and switched to the medium without TSH (5 H medium) for 4 or 5 days. The quiescent FRTLS cells thus obtained were used in the assays. Quiescent cells were incubated in fresh 5 H medium with or without the addition of various doses of rIL-1P or TSH. Each of these cultures was performed in triplicate. After various periods of incubation, supernatants were collected, filtered using a 0.22~km filter (Millipore Corp., Bedford, MA), and then tested for IL-6 activity. After the supernatants were collected, cells were detached using a mixture of trypsin, (Difco Laboratories, Detroit, MI), collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 0.04% EDTA. Then, cell numbers were counted by a particle counter (Erma Inc., Tokyo, Japan).

Interleukin

6 Assay

Cells of the hybridoma line, 7TDlF kindly provided by Dr. J. Van Snick (Ludwig Cancer Institute, Brussels, Belgium), were cultured in RPM1 1640 medium containing 2 mM glutamine, 100 p,,g/mL of kanamycin, and 10% heatinactivated fetal calf serum supplemented with 40 U/mL of rIL-6 (7TDl medium). Before assay, samples were diluted to various twofold dilutions using phosphate-buffered saline without Mg” or Ca2+ in 96-well flat-bottomed microtiter plates (Falcon, Becton-Dickinson and Co., Lincoln Park, NJ). 7TDl cells were washed twice with RPM1 1640 medium, adjusted to a density of 4 x lo4 cells/ml in 7TDl-medium without rIL-6, and then 50 p,L of this cell suspension was added to 50 p,L of the diluted samples. These cultures were then incubated for 3 days. Cell proliferation was measured calorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, as described by Mosmann.33 The cell proliferation assays were performed in duplicate. Neither IL-lp nor TSH influenced the proliferation of the IL&dependent 7TDl cells (data not shown). IL-6 activity was expressed as U/mL per 10’ cells, and was determined by comparison with a simultaneous assay using murine rIL-6 as a standard. One unit of IL-6 was defined as the amount of IL-6 that induced half-maximal proliferation of 7TDl cells.

Northern

CYTOKINE,

et al.

Blot Analysis

Expression of IL-6 mRNA was assessed by Northern blot analysis. Quiescent FRTLS cells were stimulated with 10 ng/mL of IL-l@. Total cellular RNA was isolated by lysing cells in 6 M guanidium isothiocyanate followed by recovery of the RNA by centrifugation through cesium chloride.34 Then, samples of RNA were subjected to electrophoresis on 1.0%

Vol. 3, No. 4 (July 1991: 345-349)

agarose-formaldehyde gel and transferred to nitrocellulose filters (Schleicher and Schuell, Dassel, Germany). Total RNA from P815 cells, which express IL-6 mRNA constitutively, was used as a positive control. Hybridization was performed at 68°C in a solution containing 6 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.01 M EDTA, 5 x Denhardt’s solution, and 0.5% sodium dodecyl sulfate. For hybridization, 100 kg/mL of salmon sperm DNA and the labeled probe was added to the solution. After hybridization, the membrane was washed three times for 20 min each using 0.2 x SSC and 0.1% sodium dodecyl sulfate at 65°C. Blots were then exposed to X-ray film at -70°C. The EcoRI-Bgl II fragment (650 base pairs) of murine IL-6 cDNA (pHPlB5) was a gift from Dr. J. Van Snick (Ludwig Cancer Institute, Brussels, Belgium). This was labeled using a random primer labeling kit (Takara Shuzo Co., Kyoto, Japan) and a-[32P]dCTP (ICN Biomedical Inc., Irvine, CA).

Statistical Analysis Tests of significance for differences between dent means were performed by Student’s t test.

indepen-

Acknowledgment We thank Dr. J. Van Snick for providing 7TDl cells and cDNA probe for IL-6, Dr. Sudo for providing rIL-6, and Otsuka Pharmaceutical Co. for providing rIL-lj3 and anti-IL-@ antiserum. Dr. Hashimoto (Department of Physiology, University of Yamanashi Medical School) and Dr. Okada (Department of Biochemistry, University of Yamanashi Medical School) are greatly acknowledged for their support. Tomoko Kawaguchi, Yukiko Sato, and Yuko Nakamura are greatly acknowledged for their secretarial work. REFERENCES 1. De Groot LJ, Quintans J (1989) The causes of autoimmune thyroid disease. Endocr Rev 10:537-562. 2. Pujol-Borrell R, Hanafusa T, Chiorato L, Bottazzo GF (1983) Lectin-induced expression of DR antigen on human cultured follicular thyroid cells. Nature 304:71-73. 3. Eguchi K, Otsubo T, Kawabe Y, Shimomura C, Ueki Y, Nakao H, Tezuka H, Matsunaga M, Fukuda T, Ishikawa N, Ito K, Nagataki S (1988) Synergy in antigen presentation by thyroid epithelial cells and monocytes from patients with Graves’ disease. Clin Exp Immuno172:84-90. 4. Takazawa K, Endo T, Onaya T (1988) Phagocytotic activities of latex beads by FRTL cell. Acta Endocrinol (Copenh) 117:198-204. 5. Tosato G, Seamon KB, Goldman ND, Sehgal PB, May LT, Washington GC. Jones KD. Pike SE (1988) Monocvte-derived human E-cell growth factor idkntified as inierfeion-p, (BSF-2, IL-6). Science 239:502-504. 6. Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K, Koyama K, Iwamatsu A, Tsunasawa S. Sakivama F. Matsui H. Takahara Y, Taniauchi T. Kishimoto T’ (1986) Com&ementary’ DNA for a’ novel- human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 324:73-76. 7. Garman RD, Jacobs KA, Clark SC, Raulet DH (1987) B-cell-stimulatory factor 2 (& interferon) functions as a second

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