0021-972X/91/7206-1296$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society

Vol. 72, No. 6 Printed in U.S.A.

Effects of Recombinant Human Interleukin-2 and Tumor Necrosis Factor-a with or without Interferon-7 on Human Thyroid Tissues from Patients with Graves' Disease and from Normal Subjects Xenografted into Nude Mice* YOSHIO KASUGAt, SUNAO MATSUBAYASHIJ, FUMITO AKASUt, NAOMI MILLER, CHRISTOPHER JAMIESON, AND ROBERT VOLPE Endocrinology Research Laboratory and Departments of Medicine, Pathology (N.M.), and Surgery (C.J.), Wellesley Hospital, University of Toronto, Toronto, Ontario, Canada

ABSTRACT. We have compared the effects of interleukin-2 (IL-2) or tumor necrosis factor-a (TNFa) administration with or without interferon-7 (IFN7) on Graves' and normal thyroid tissue xenografts in the nude mouse (in the absence of an intact immune system) in terms of possible functional, immunological, or histological changes. The dosages of recombinant human IL2, TNFa, and IFN7 given to each mouse were 250, 800, and 4000 U, respectively; they were injected ip daily for 6 consecutive weeks. The parameters measured included the free T4 index, thyroid autoantibodies, and mouse TSH during the course of the study. Thyroid epithelial cell (TEC) HLA-DR expression was measured in thyroid tissue before xenotransplantation and at death; in addition, light microscopic studies were carried out at those times. There were no significant differences in thyroid function between the results in unstimulated (control) animals and those obtained with cytokine administration in either group of tissues, with the exception of the group receiving TNFa together with IFN7; in this latter group, the free T4 index declined significantly 4-6 weeks after commencement of treatment in the animals with normal thyroid tissue xenografts. The reduction of thyroid function induced by the combination of IFN7 and TNFa observed in normal thyroid tissue may be due

R

ECENTLY, a number of reports have appeared describing the development of autoimmune thyroid disease (AITD) or hypothyroidism in humans or experimental animals after the administration of interferons or interleukin-2 (IL-2) (1-9), whereas tumor necrosis factor-a (TNFa) has been reported to reduce thyroid function in experimental animals (10). Received June 28,1990. Address all correspondence and requests for reprints to: Dr. Robert Volpe, 160 Wellesley Street East, Room 112D Jones Building, Toronto, Ontario, M4Y 1J3 Canada. * This work was supported by a grant from the Medical Research Council of Canada (MT859). t Fellow, Wellesley Hospital Research Foundation. $ Fellow, Angus Foundation, Wellesley Hospital.

to inhibition of thyroperoxidase and thyroglobulin gene transcription. However, there was no such effect on the Graves' thyroid tissue xenografts, perhaps because of down-regulation of this tissue in response to cytokines, after having been released from long term in vivo immune stimulation. On the other hand, TNFa plus IFN7 induced TEC HLA-DR expression on both types of thyroid xenografts at death, although IL-2 alone did not induce HLA-DR expression, and IFN7 induced TEC significantly only on normal thyroid xenografts (but not on Graves' xenografts). In light microscopic examination, Graves' thyroid xenografts treated with IL-2 alone or TNFa plus IFN7 appeared normal at death. In addition, normal thyroid xenografts treated with the same cytokines did not show discernible differences compared to those at human surgery or when the xenografts were untreated at death. We conclude that Graves' TEC did not differ from normal TEC in any significant fashion at the time of death, aside from a reduced responsiveness to the stimuli applied. These observations are again consistent with the previous proposition that Graves' TEC are not unique or abnormal, and may be mere passive captives to immunological events in relation to the pathogenesis of autoimmune thyroid disease. (J Clin Endocrinol Metab 72: 1296-1301, 1991)

We have previously reported that human recombinant interferon-7 (IFN7) could not enhance, sustain, or induce AITD in xenografted thyroid tissue from patients with Graves' disease or normal subjects (actually paranodular tissue) in a nude athymic mouse model, which lacks a T-lymphocyte immune system (11). Our aim has been to determine whether Graves' thyroid tissue reacts in any way differently from normal thyroid tissue in response to various cytokines. In this context, we decided to determine whether IL-2 or TNFa with or without IFN7 has any different functional or histological effects on Graves' tissue xenografts as opposed to normal thyroid tissue. Possible effects on thyrocyte HLA-DR expression were also studied. It should be noted that these thyroid xenografts cannot be compared directly to

1296

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EFFECTS OF IL-2 AND TNFa ON GRAVES' THYROID TISSUE the intact human or animal situation, in which the immune system is either normal or minimally affected; the effect of cytokines under some circumstances might exacerbate an established low level autoimmune response.

Materials and Methods Male BALB/c nude (athymic) mice of an outbred strain (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were used in this study. They were 4-6 weeks old, with an initial body weight of approximately 20 g. The thyroid gland of each mouse was completely ablated with 0.2 mCi 131I (Merck Frosst Canada, Inc., Kirkland Lake, Canada). The mice were shown to be hypothyroid by routine laboratory tests, including low T4 and high TSH values (12). Xenografting was performed 3-4 weeks after the 131I ablation at a time when hypothyroidism was clearly demonstrable by the above laboratory tests. The xenograft procedure was carried out by the methods of Smeds et al. (13) and Usadel et al. (14). Briefly, human thyroid tissue was obtained at surgery from patients with Graves' disease, whereas paranodular (normal) tissue was obtained at the time of removal of discrete thyroid nodules. The latter tissue was histologically normal at the time of human surgery. Surgical specimens selected for xenografting were cut into fragments measuring about 2 x 2 x 3 mm. About five fragments were xenografted sc into each groin of the mouse within 2 h after the human surgery. Survival of the xenograft was clearly indicated by the return of thyroid function to normal in the animals that were xenografted, whereas there was no return to normal in those animals undergoing 131I ablation but no xenograft, which were used as controls (12). Approximately 4 weeks after the xenografts, recombinant human IL-2, TNFa, and IFN7 (Genzyme Co., Boston, MA) were injected ip into the mice on a daily basis for 6 consecutive weeks. The dosages of recombinant human IL-2, TNFa, and IFN7 given to each mouse were 250, 800, and 4000 U, respectively, which were considered analogous to dosages employed for anticancer therapy (5-10). Blood was taken from the tail veins of the mice every 2 weeks until death. In this study Graves' thyroid tissue was xenografted into 20 mice, and normal human thyroid tissue into 14 mice. The animals survived the procedure well.

1297

cyte HLA-DR expression, the thyroid tissue was cultured as previously described (17, 18). Briefly, the finely minced tissue was digested with 2 mg/mL collagenase (type II, Sigma, St. Louis, MO) at 37 C for 60 min. The pellet obtained after 10 min of centrifugation at 600 x g was washed twice with phosphate-buffered saline (PBS) and once with complete medium [RPMI (Gibco, Burlington, Ontario, Canada) containing 20% fetal bovine serum substitute (Sigma), 1% antibiotic-antimycotic solution, and 2 nmol/L glutamine (Gibco)], plated in a petri dish, and incubated at 37 C in a humidified atmosphere of 5% CO2. The confluent cells after 1 day of culture were trypsinized (0.5% trypsin in 0.02% EDTA; Gibco), washed with PBS and medium, and transferred into the wells (40,000 cells/ well) of a Linbro 96-well tissue culture plate (Flow Laboratories, Inc., McLean, VA). Cell viability by trypan blue exclusion was greater than 90%. The thyrocyte HLA-DR expression was measured by an enzyme-linked immunosorbent assay technique, which has been reported previously (19-21). This was performed on thyroid tissue before xenografting (i.e. immediately after the human surgery) and on the same tissues after death of the nude mouse. HLA-DR expression in the thyrocytes was calculated as follows: experimental optical density (OD) with anti-HLADR antibody - optical density of control without anti-HLADR antibody. Light microscopic studies The initial thyroid tissue before xenografting was submitted to the Department of Pathology for routine light microscopic studies for the purpose of comparison with the subsequent appearance of the same tissues. After death, the xenografted thyroid tissues were subjected to the same studies. These tissues were fixed in neutral formalin and embedded in paraffin for routine light microscopic examination. The staining was carried out using hematoxylin-eosin. Statistical analysis Results are given as the mean ± SD. Statistical analysis of data was performed by Student's t test and analysis of variance (Anova) (see Fig. 5); P < 0.05 was considered significant.

Results Measurements performed Serum T4 concentrations were measured by a commercial RIA (Joldon Diagnostics, Scarborough, Ontario, Canada). T 3 resin uptake was measured by a similar commercial kit, and the free T4 index (FT4I) was calculated as serum T4 (in nanomoles per L) X T 3 resin uptake (15, 16). The mouse TSH was measured by RIA, using rat TSH and anti-TSH (rat), generous gifts of Dr. Salvatore Raiti, NIDDK (Bethesda, MD), and a mouse TSH standard generously provided by Dr. A. F. Parlow, UCLA (Los Angeles, CA), and the National Hormone and Pituitary Program, University of Maryland School of Medicine. Thyroid autoantibodies (antithyroglobulin and antimicrosomal antibody) were measured by hemagglutination kits (Wellcome Diagnostics, Dartford, England). For measurements of thyro-

Thyroid autoantibodies were not detected at any time in any of the study groups. Figures 1, A and B, show the changes in serum TSH in nude mice with xenografted human normal tissue or with Graves' thyroid tissue, respectively. The serum TSH values in both controls and the group receiving TNFa declined to almost the normal range 4 weeks after commencing injection in normal thyroid tissue. However, the group receiving TNFa with IFN7 showed no further fall toward normal in TSH values at the same time that F T J decreased (by Anova test, P < 0.01). This is in contrast to the continuing decline in TSH results in the other groups of normal thyroid xenografts. On the other hand,

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KASUGA ET AL.

1298 Changes of Serum Mouse TSH in Nude Mouse with Xenografted Human Normal Thyroid Tissue

Mean ± SD O Control (n=4) TNFa(n=4) A TNFa + IFNy(n=4)

, Belore 3 Weeks Alter 11 3(0) 131-1 Administration

5(2) 7(4) 9(6) , Weeks Alter Xenograll (alter commencing injections)

Changes of serum mouse TSH in nude mice with xenografted Graves'thyroid tissue

160 120

—O—Control (n=6) — • — T N F a (n=5)

1 so

A 40

20

TNFa + IFNy (n=5)

iA

mean ± SD

Vj

10

a

. Before 3 Weeks After ,3(0)

B

131-1 Administration / Xenograft

Detectable line

a

«

5(2)

7(4)

9(6)

Weeks after xenograft (after commencing injection)

FlG. 1. Changes in serum TSH in nude mouse with xenografted human normal thyroid tissue (A) or Graves' thyroid tissue (B). The normal range is less than 5 ng/mL.

in Graves' thyroid tissue all three treatment groups declined to close to the normal range 4 weeks after injection and did not alter further. Table 1 shows the serum F T J in response to IL-2 on the xenografted human normal thyroid tissue and Graves' thyroid tissue, respectively. There was no signif-

JCE & M • 1991 Vol 72 • No 6

icant difference between the control results and those obtained with IL-2 in either group of tissues from the period before commencing injection until the time of death. (Moreover, thyroid autoantibodies were not detected at any time throughout the treatment period with IL-2 in either group or in the control group.) Figure 2, A and B, show the change in serum F T J in response to TNFa with or without IFN7 on the xenografted human normal tissue and Graves' thyroid tissue, respectively. In normal thyroid tissue, there was no significant difference between controls and the group receiving TNFa 4 and 6 weeks after commencing injection. However, F T J values in the group receiving TNFa with IFN7 showed a slight but significant decline 4 and 6 weeks after injection compared to controls or those receiving TNFa alone. (This decline was associated with the higher TSH values, as noted above, which confirmed the significance of the decline in FTJ.) On the other hand, in Graves' thyroid tissue, there was no significant difference among the groups of controls us. those receiving TNFa or TNFa with IFN7. (In particular, there was no decline in F T J with TNFa plus IFN7 administration, as was seen with the normal thyroid tissue.) For comparison purposes, it may be noted that our previous study with IFN7 alone showed no significant effects on thyroid function tests (i.e. F T J and TSH) in either normal or Graves' thyroid xenografts (11). Figure 3, A and B, show the thyroid epithelial cell (TEC) HLA-DR expression values, expressed as experimental OD with anti-HLA-DR antibody minus OD of control without anti-HLA-DR antibody. For normal TEC unstimulated by cytokines the value was 9.8 ± 8.2 (±SD) x 10~3, whereas for normal TEC treated with IL2, TNFa, or TNFa plus IFN7 the values were 16.8 ± 3.3, 9.3 ± 5.3, and 106.0 ± 18.0 X 10"3, respectively. IL-2 or TNFa alone did not induce TEC HLA-DR significantly compared with that in the controls at death. On the other hand, TEC HLA-DR in the group receiving TNFa with IFN7 was markedly elevated, significantly different (P < 0.01) from the control value as well as from the value in the group receiving IFN7 alone, as reported by us previously, i.e. 61.0 ± 9.0 x 10"3. TEC HLA-DR expression in thyroid tissue obtained immediately after the

TABLE 1. Effect of human IL-2 on human thyroid tissue xenografted into nude mice: FTJ results Normal thyroid xenografts

Graves xenografts FTJ° Before injection After injection 2 weeks 4 weeks 6 weeks

Control Graves' (n = 6)

IL-2 group (n = 4)

Control normal (n = 4)

IL-2 group (n = 4)

15.2 ±3.6

18.8 ±5.1

18.9 ± 4.8

15.2 ± 2.0

15.0 ±5.8 19.6 ±2.9 20.6 + 5.0

18.9 ±4.9 24.1 ±3.2 17.7 + 1.9

17.3 ± 2.1 22.8 ± 1.9 20.6 ± 2.2

17.2 ± 1.9 23.1 ± 5.2 21.1 ± 2.5

' There were no significant changes in FTJ in any of these groups.

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EFFECTS OF IL-2 AND TNFa ON GRAVES' THYROID TISSUE Effect of human TNFa with or without human IFNY on human normal thyroid tissue xenografted into nude mice : Free T4 index (FT 4 1)

40

O

HLA-DR expression by xenografted normal thyroid epithelial cells (by ELISA)

control (n-4)



TNFa (n-4)

A

TNFa + IFNY (n-4)

30

140

* « - PcO.01 vs control NS - Not Signilicant vs control

10

1

120

mean ± SO * - P < 0.05 vs control

20

1299

I

100

c

80

£

60

mean ± SD * * - P < 0.01 NS - Not Significant

40 before 2 weeks 4 weeks 6 weeks injection (sacrifice)

20

After Injection

A

control control IL-2 (n-4) (no treatment) alone (n-4) (n-4)

Effect of human TNFa with or without human IFNY on Graves'thyroid tissue xenografted into nude mice : Free T4 index (FT4 | )

TNFa alone (n-4)

TNFa +IFN7 (n-4)

IFNy alone (n-4)

at sacrifice

at human surgery 30 -O—Control (n=6)

NS

-•

20

fcf

TNFa (n=5)

HLA-DR Expression of Graves1 Thyroid Epithelial Cells by ELISA

- A — T N F u t- IFNy (n=5) N S : Not Significant vs control

10

120

Mean ± SD * =P< 0.05 **=P

Effects of recombinant human interleukin-2 and tumor necrosis factor-alpha with or without interferon-gamma on human thyroid tissues from patients with Graves' disease and from normal subjects xenografted into nude mice.

We have compared the effects of interleukin-2 (IL-2) or tumor necrosis factor-alpha (TNF alpha) administration with or without interferon-gamma (IFN g...
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