INTERLEUKIN-1 RECEPTORS ON HUMAN THYROID CELLS AND ON THE RAT THYROID CELL LINE FRTL-5 M. Svenson,l
L. Kayser,2’3
M.B. Hansen,’ K. Bendtzen4,*
A. Krogh Rasmusseq3
Cellular binding of interleukin-1 (IL-l) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human ‘%-IL-lo. Scatchard plots of data obtained at 4°C indicated the presence of a single population of receptors with a & of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-l. The IL-1 receptors on these cells had I(d values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-lo and rIL-1B inhibited lz51-rIL-lo binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of ‘*%rIL-la to long-term cultivated human thyroid cells or to the FRTL-5 cells. We conclude that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.
Copyright o 1991 by W.B. Saunders Company
Interleukin-1 (IL-l) is a small family of pleiotropic cytokines produced primarily by monocytes and macrophages.‘,’ IL-l acts as a costimulator of T and B lymphocytes,‘,’ and it stimulates endothelial cells to produce inflammatory factors.3 It thus appears to be centrally involved in infectious and immunoinflammatory reactions. Furthermore, IL-l has been found to be cytotoxic to islets of Langerhans: and it affects the function of thyrocytes’ and pituitary cells6 IL-l may therefore play a role in the regulation of endocrine cell functions and, possibly, in the development of autoimmune and endocrine diseases. Two distinct molecules with IL-l activity have been identified, termed IL-la and IL-lB.7,8 The spectrum of biological activities shared by these two mole-
cules seems to be mediated by binding of IL-la and IL-@ to the same receptor, even though the receptor for IL-l may not be identical on different cell types.9^12 Recently, differential effects of IL-la and IL-lp on human thyroid cells in secondary culture and on the rat thyroid cell line FRTL-5 have been described.13 In the present study, we examined the presence of IL-l receptors on these cells and, in addition, the specific binding of ‘251-IL-l~ to long-term cultures of human thyroid cells. Only human thyroid cells in secondary culture expressed detectable specific receptors for rIL-la. RESULTS Binding of ‘251-rIL-1 a to Human Tlzyrocytes in Secondary Culture and to Murine T-Cell Lines
‘Department of Infectious Diseases M, Rigshospitalet University Hospital. ‘Department of Anatomy, The Panum Institute, University of Copenhagen. 3Medical Department E, Frederiksberg Hospital. “Deoartment of Medicine TTA. Rieshosuitalet Universitv ~ Hospital, _ DK-2200 Copenhagen N, Denm&k. A *Correspondence: K. Bendtzen, MD, Lab. Med. Immunol. 7544, Rigshospitalet, 20 Tagensvej, DK-2200 Copenhagen N, Denmark. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0302-0006$05.00/O KEY
WORDS:
CYTOKINE,
interleukin-1
receptors/thyroid
Vol. 3, No. 2 (March),
cells/FRTL-S/EL-4
1991: pp 125-130
Figure 1 shows the kinetics of binding of ‘251-rIL-lo to human thyrocytes in secondary culture and to the murine T-cell line NOB-l. On both cell types, maximum saturable binding was reached after about 800 min of incubation at 4°C. At room temperature, maximal binding of lz51-rIL-lor was seen after 240 min (data not shown). The specific lZI-rIL-la binding to NOB-l and EL-4 cells was saturated by 1 nM rIL-la (Fig. 2A). However, 10 times higher concentrations were needed 125
126 I Svenson
CYTOKINE,
et al.
Binding of “‘I-IL-1 LXto the Rat Thyroid Cell Line FRTL-5 and to Long-Term Culturesof Human Thyrocytes
BOUNO (cpml
: vu &em o-..'-. 0
Vol. 3, No. 2 (March 1991: 125-130)
200
'. 400
I 600
. ..l....,....,....,. BOO 1000
. 1200
.
1400
TIME (min) Figure 1. Kinetics to human tbyrocytes
of binding ‘Z51-rIL-la in secondary culture
to NOB-1 @I#).
cells
(0,O)
and
Duplicate cultures were incubated at 4°C with 0.2 @ml of ‘251-rIL-la alone (0,O) or in the presence of excess (SO @ml) unlabeled rIL-la (.,V
to saturate the binding to human thyroid cells (Fig. 2B). Scatchard analyses of the binding data at 4°C indicate the presence of a single class of IL-lo receptors on these cells (Fig. 2, A and B). Using thyroid cells, the equilibrium dissociation constant (Kd) was almost 10 times higher than on the murine T-cell lines. However, the number of receptors on the thyroid cells was comparable to that on the murine T-cell lines (Fig. 2 and Table 1). When binding was measured at room temperature, increased amounts of 1251-rIL-la associated with the human thyrocytes (Fig. 3). Scatchard analyses of data obtained in experiments at 4°C and at 22°C resulted in parallel lines. This indicates that the cells expressed increased numbers of receptors at room temperature. Internalization of the ‘251-rIL-10-receptor complex and recycling of the receptor would contribute to an apparent increase in receptor numbers. Therefore, the effect of acid treatment on the cell-associated radioactivity was determined. As shown in Table 2, a substantial amount of the cell-associated tracer was nonextractable by acid treatment at room temperature. Thus, internalization of the receptor-ligand complex and recycling or increased synthesis of the receptor are likely causes of the increased binding of ‘=I-rIL-la to the thyrocytes at room temperature. At 4°C both human rIL-la and rIL-ll3 competed with ?-rIL-lcx binding to EL-4 cells and to human thyrocytes in secondary culture. Using EL-4 cells, the competition by rIL-lo was stronger than by rIL-ll3, but the reverse was found when using thyrocytes (Fig. 4).
Assay conditions similar to those described above were used to detect 1251-rIL-la binding to FRTL5 cells and to cultures of human thyrocytes passaged 8 times during a 5-month cultivation period. Morphologically, the latter had changed to small cells without detectable thyroid-specific functions such as thyroglobulin production and TSH-induced CAMP production. At 4°C neither of these two thyroid cell populations showed changes in the bound/free (B/F) ‘=I-rIL-la values using free ligand concentrations between 5 x 10-l’ M and 6 x 10m9M (Table 3). To increase the ability to detect receptors, which may internalize the ligand, binding to FRTL5 cells was also performed at 37°C. This increased the binding of lZI-rIL-la, but the binding was proportional to the free ligand concentration (Table 3). When kept in secondary culture for 3 months, human thyroid cells still possessed detectable IL-la receptors. Human thyroid cells passaged in parallel exhibited unchanged receptor binding up to the second passage. After the third passage, the cells had lost all IL-lo receptors (see Table 1). These cells did not show any morphological changes.
DISCUSSION The number of receptors for IL-l and their binding properties vary considerably depending upon the target cell and, most likely, the experimental conditions. Thus, dissociation constants from 3 pM to 500 pM, binding sites from 100 to more than 10,000 per cell, and differences in the relative binding affinities of the two forms of IL-l have been reported.9’14-20Generally, T cells, in contrast to B cells, seem to bind rIL-la more strongly than rIL-l~.9~‘5~20Judged from the present experiments, using rIL-la and rIL-1B competition with lZI-rIL-la binding, human thyroid cells in secondary culture bind rIL-1B more strongly than rIL-la. In contrast, and in agreement with other studies,‘4,15,21,22 preferential binding of rIL-la was found using the murine T-cell lines EL-4 and NOB-l. Thus, the pattern of binding of IL-l to human thyroid cells in secondary culture resembles that to B cells. Using EL-4 and NOB-l cells, the K values of 8 to 17 pM for rIL-lo agree with those reported by Lowenthal and McDonald,‘* but they are 10 to 30 times lower than those reported by others.14~15~21~22 In contrast to Lowenthal and McDonald, we and others failed to obtain data indicating the presence of both high- and low-affinity receptors on the EL-4 cells.‘* Using thyroid cells in secondary culture, the Scat-
Interleukin-1 receptors on thyroid cells / 127
0.10
0.05
-
0.00 10 -13
IO -12
10 -1'
IO -'Cl
10-g
10-B
mol/ I
6
B/F
Figure 2. Binding of ‘%-IL-k to NOB-l, EL-4 cells (A) and to human thyrocytes in secondary culture (B) at 4°C.
The results are shown as total bound ‘Z51-rIL-la to 5 x 10’ cells free ligand (B/F) as a function of the free ligand concentration. The inserts show the Scatchard plot of the same binding data after subtraction of unspecific binding. Unspecific binding was calculated on the basis of binding obtained at lo-’ M of the ligand. The x-axes show the bound ‘Z51-rIL-1a in pg/5 x lo5 cells. (A) (0) NOB-l cells, K, = 10 pM and 2,400 receptors/cell, (+) EL-4 cells, K, = 9 pM and 2,000 receptors/cell. (B) Human thyrocytes in secondary culture: K, = 170 pM, 6,000 receptors/cell.
chard plots of the binding data showed single slopes, indicating a single population of 1251-rIL-10 binding sites. Single slopes were also obtained with murine T-cell lines. The calculated K value of 80 pM for ‘251-rIL-la binding to human thyroid cells is similar to that found on human rheumatoid synovial cells’7 and lung fibroblasts.16 A high-affinity rIL-1B receptor on porcine thyroid cells has recently been reported by Kasai et a1.23The Kd value was similar to that of the rIL-lol receptor on human thyroid cells. In contrast to our observation, Kasai et al. found identical cross-reactivity when using unlabeled rIL-la and rIL-1B. Also, the human thyroid cells expressed 4,500 IL-1 binding sites per cell (this study), and this is more than ten times the number of
mol/l IL-l receptors found on porcine thyroid cells.” These differences between IL-1 binding to human and porcine thyrocytes may be explained by variations in the experimental conditions, but species differences cannot be excluded. Biological responses to IL-l may be obtained at extremely low concentrations of the cytokine (lo-l4 to lo-l*M). In some cell systems, this potency of IL-l seems to require less than one receptor occupancy for biological activation of a single ce11,24,25 indicating the presence of IL-l receptors of higher affinity than those presently characterized. Occupation of only a few receptors per cell has been shown to activate IL-lresponsive target cells.16’17EL-4 and NOB-l cells respond to both species of IL-l at 10-14M (references 26
128 I Svenson
CYTOKINE,
et al,
Table 1. IL-k receptors on various cell lines and human thyrocytes. Direct binding was performed at 4°C as described in Materials and Methods. The I& values and receptor numbers were calculated from Scatchard plots. Receptors/cell
UPM) Cells
Range
Murine T-cell lines EL4 NOB-l Rat thyrocyte cell line FRTL-5 Human thyrocytes Secondary culture Long term2
Mean
Range
Mean
Table 2. Effect of pH 3 wash of human thyrocytes on cell-associated ‘z51-rIL-lcy, Human thyrocytes in secondary culture were incubated at 22°C for 6 h with ‘ZSI-rIL-lcu alone or with excess unlabeled rIL-loi. The cultures were washed two times in the incubation buffer followed by 3 min incubation at pH 7.4 or pH 3 (made by adding 6.25 ml of 1 M HCl to 100 ml of the pH 7.4 incubation buffer). The cells were then washed and treated as described in Materials and Methods.
n rIL-la (34 @ml)
Wash pH3
+
-
-
+ + +
+ +
+ +
‘J%rIL-1OI (0.16 ngiml)
8-10 8-17
9 12
1,200-3,000 2,500-6,000
nd’ 30-170 (80)
2,100 3,600
3 3
nd 80
2
2,000-6,000
Vol. 3, No. 2 (March 1991: 125-130)
4,500
cpm/loI cells (mean t SD; n = 3)
648 177 491 180
f f k f
2.5 8 12 5
6 3
ww
‘Not detectable. ‘Obtained with one culture of cells passaged three times and with two cultures passaged more than three times. The latter cells did not show specific binding of ‘xI-rIL-loi.
and 27, and our own data). However, EL-4 cells require a second signal such as a calcium ionophore in order to respond to IL-Lz6 whereas the NOB-l cells respond with interleukin-2 production if exposed to IL-lo//p a1one.27 Judged by the Scatchard plot analysis of binding at 4°C 1 to 6 receptors per cell would be occupied on the murine T cells at lo-l4 M rIL-lo. In secondary cultures of human thyrocytes, the secretion of thyroglobulin and CAMP is suppressed by a minimum of lo-l3 M human IL-~cx/B.~~‘~At this concentration, an average of 2 to 6 receptors per cell would be occupied by rIL-la. These findings, therefore, support the notion that high-affinity receptors are involved in mediating the
biological effects of IL-1 on the murine T-cell lines (Kd I 10 PM) and on human thyroid cells (Kd I 100 PM) in secondary culture. In contrast to human thyroid cells in secondary culture, the growth of the rat thyroid cell line FRTL5, but not its secretion of CAMP, has been found to be inhibited by rIL-1B but not rIL-10.13 The lack of effect of rIL-la on this cell line is in agreement with the observed absence of specific ‘2SI-rIL-la binding to the cells when assayed at 4°C as well as 37°C. Similar findings have been reported in the case of another peptide mediator, epidermal growth factor.% Though present on primary cultures of sheep thyroid cells, the receptors for this factor were absent on FRTL5 cells.** This, and the observed loss of IL-1 receptors during passage of human thyroid cells, indicates that dediffer% INH
B/F
0.02
0.01
0 IO -2
,
. .Jd....l 10-I
. “I,,,,’ 100
“I,,.,’ 10 1
’ 102
w/ml 0.00
0
IO
20
30
40
50 6
Figure 3. Scatchard plots cytes in secondary culture.
of ““I-rIL-la
binding
to human
thyro-
Assay at 4°C (W) and at 22°C (0). The experiments were performed in parallel.
Figure 4. Ability of unlabeled binding of ‘LsI-rIL-lcy.
rIL-lru
or rIL-lp
to compete
with
the
‘251-rIL-la, 180 pgiml, was incubated at 4°C with EL-4 cells (0,O) in the presence of the indicated amounts of unlabeled rIL-lu (open symbols) and rIL-1B (closed symbols). The same experiment was carried out in parallel with human thyroid cells in secondary culture (0,m) using 280 p&ml of rz51-rIL-la. The y-axis shows percent reduction of ‘“I-rIL-lo binding.
Interleukin-1 Table 3. Binding of ‘zSI-rIL-lcu to FRTLd thyrocytes passaged eight times.
cells and human
Results are given as means of duplicates. (A) human thyrocytes passaged 8 times during a 5-month cultivation period. The cell density was 6 x 106/well. (B) FRTL-5 (2.5 x 106/well).
Cont.
of ‘2SI-rIL-l~
(M)
5 x lo-l2 lo-” 3 x 1om9 6 x 1O-9
3TC3
A
B
Ei
0.0049 0.0048 0.0050
0.0019 0.0019 0.0018 0.0017
0.009 0.008 0.009 0.008
‘cpm boundicpmiml supernatant. ‘incubated for 22 h. ‘incubated for 5 h.
entiation during transformation of thyroid cells results in marked changes in the expression of receptors for peptide hormones.
MATERIALS
AND METHODS
Cells Human thyroid cells were obtained from paraadenomatous tissue during strumectomies. They were isolated and grown in Hams F12 medium as modified by Coon and supplemented with 5% fetal calf serum and six growth factors, as previously described.5 The FRTL5 cells were kindly provided by P. Ealey (University College Hospital, London, UK). The cells were grown as described by AmbesiImpiombato et al.29in the same medium as above. The mouse thymocyte cell lines EL-4 and NOB-l were obtained from the American Type Culture Collection (Rockville, MD, USA) and the European Collection of Animal Cell Cultures (Wilts, England), respectively. The cells were maintained in RPMI-1640 with 25 mM Hepes (Gibco Biocult, Paisley, UK) containing 2.5 km 2-mercaptoethanol, 2.5 mM L-glutamine (Sigma, St. Louis, MO, USA), 25 p,l/ml gentamycin (Flow Laboratories, Scotland), and 5% fetal calf serum (Gibco). Cultures were grown at concentration between 1 x 10’ and 10 x 10’ cells/ml.
Procedures After 1 to 2 weeks, the thyroid cells were passaged by seeding 5 x lo5 cells into 6-well culture plates (9.6 cm’/well, Greiner, Frickenhausen, Germany). The cells were then grown for 3 to 5 daysbefore the binding assaywas performed. To evaluate the stability of the expression of IL-la receptors of human thyroid cells in long-term culture, 2 experiments were performed: 1. 2.
passage. Every third week, 4 to 6 days after passage, one plate with passaged cells was investigated, along with one plate with cells kept in secondary passage from the same original cell culture.
Interleukin-1
Em’ 4”CZ
receptors on thyroid cells / 129
Cells were kept in culture and passaged every second to third week for 5 months and evaluated in the eighth passage. Cells were kept in secondary culture for 3 months, with parallel passages every third week until the fourth
Human rIL-la and rIL-1P were generous gifts from T. Tsuboi (Dainippon Pharmaceutical Company, Osaka, Japan) and C.A. Dinarello (Tufts New England Medical Center, Boston, MA, USA). The biological activities of rIL-lol and rIL-1P were detected by the murine cell lines EL-4 and CTLL-2, as described.3oThe specific biological activity of rIL-la was lo* U/mg, as determined by a mouse thymocyte costimulatory assaycalibrated with an interim international rIL-la reference preparation (NIBC, Potters Bar, UK).
Radioiodination
of rIL-1 cy
Human rIL-lol was radioiodinated using chloramine T, and the bioactivity of 1251-rIL-1awas determined using EL-4 and CTLL-2, as previously described”’ More than 96% of the lZ51in the rIL-101 fraction could be precipitated with 5% trichloroacetic acid. The specific activity of the labeled rIL-101 was 3 X 10’ to 4 X 10’ cpm/ng.
IL-l CYBinding Assay FRTL5 and human thyroid cells were cultured to confluence in 6-well culture plates as described above. Before assay,the medium was removed from each well and replaced with 1 ml of RPMI-1640 (Gibco) plus 0.5% bovine serum albumin (BSA) (Sigma) containing ‘251-rIL-lol with or without rIL-la and rIL-1P. Nonspecific binding of lZI-rIL-la was determined in the presence of 100 to 200 @ml of unlabeled rIL-l&. The plates were incubated under gentle agitation at 4°C for 20 to 22 h, at 22°C for 6 h, or at 37°C for 5 h. After incubation, the supernatants were removed for counting of lZ51activity and the plates were washed by dipping them rapidly three times in 4 L of ice-cold, phosphatebuffered saline (20 mM phosphate buffer, 0.125 mM NaCl, pH 7.4). Bound *251-rIL-la was then solubilized in two times 0.5 ml of 2 M KOH and the samples were counted with an error of 4% or less in a gamma counter (1272 CliniGamma, LKB, Wallac OY, Finland). One chamber was used on each plate for the detection of cell numbers after the washing procedure. The cell number was determined by DNA measurement31 using an index of 15 pg DNA/lo6 cells.32EL-4 and NOB-l cells, 1 x 106 to 2 x 106/ml, were incubated under continuous agitation with lZ51-rIL-la in medium RPMI-1640 containing 0.5% BSA. After 20 h, 200 ~1 of the cell suspensions were added to Microtubes EET-23 (Struers, Glostrup, Denmark) containing dibutyl phthalate:bis( -2ethylhexyl) phthalate 3:2 (Merck, Darmstadt, Germany). The cells were then centrifuged at S,OOOgfor 2 min in a Biofuge B centrifuge (Hereus-Christ, Osterode, Germany). The medium and pellet were separated by cutting through the oil layer and lz51was counted with an error of less than 2%. All procedures were carried out at 4°C.
130 / Svenson et al.
Acknowledgments
We thank The Copenhagen Thyroid Cell Group (Ulla Feldt-Rasmussen, Karine Beth, and Hans Perrild) for valuable help and Susanne Meldgird for excellent technical assistance. The study was supported by the Danish Cancer Society, the Danish Hospital Foundation for Medical Research, Region of Copenhagen, the Faroe Islands, and Greenland, the Danish Medical Research Council, the Novo Foundation, the Willumsen Foundation, and the Danish Biotechnology Program.
REFERENCES 1. Bendtzen K (1988) Interleukin 1, interleukin 6 and tumor necrosis factor in infection, inflammation and immunity. Immunol Lett 19:183-192. 2. Dinarello CA (1989) Interleukin-1 and its biologically related cytokines. Adv Immunol44:153-205. 3. Mantovani A, Dejana E (1989) Cytokines as communication signals between leukocytes and endothelial cells. Immunol Today 10:370-375. 4. Bendtzen K, Mandrup-Poulsen T, Nerup J, Nielsen JH, Dinarello CA, Svenson M (1986) Human p1 7 interleukin 1 is cytotoxic for pancreatic islets of Langerhans. Science 232~1545-1547. 5. Krogh Rasmussen A, Beth K, Feldt-Rasmussen U, Paulsen S. Holten I. Rvberlr M. Dinarello CA. Siersbcek-Nielsen K. Friis T. Bendtzen K (i9885 Interleukin-1 affects the function of ‘cultured human thyroid cells. Allergy 43:435-441. 6. Besedovsky H, de1 Rey A (1987) Neuroendocrine and metabolic responses induced by interleukin-1. J Neurosci Res 18:172-178. 7. Gray PW, Glaister D, Chen E, Goeddel DV, Pennica D (1986) Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin lp. J Immunol137:3644-3648. 8. March CJ, Mosley B, Larsen A, Cerretti DP, Braedt G, Price V, Gillis S, Henney CS, Kronheim SR, Grabstein K, Conlon PJ, Hopp TP, Cosman D (1985) Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs. Nature 315:641-647. 9. Dower SK, Kronheim SR, Hopp TP, Cantrell M, Deeley M, Gillis S, Henney CS, Urdal DL (1986) The cell surface receptors for interleukin-la and interleukin-1B are identical. Nature 324:266-268. 10. Dower SK, Cosman D, Gallis B, March CJ, Gillis S, Urdal DL, Sims JE (1989) Molecular structure of the interleukin-1 receptor. Lymph Ret Interact Colloque INSERM 179:17-24. 11. Solari R (1990) Identification and distribution of two forms of the interleukin 1 receptor. Cytokine 2:21-28. 12. Solari R, Smithers N, Page K, Bolton E, Champion BR (1990) Interleukin 1 responsiveness and receptor expression by murine Thl and Th2 clones. Cytokine 2:129-141. 13. Krogh Rasmussen A, Kayser L, Beth K, Feldt-Rasmussen U, Perrild H, Bendtzen K (1990) Differential effects of interleukin la and 1B on cultured human and rat thyroid epithelial cells. Acta Endocrinol122:520-526. 14. Bird TA, Gearing AJH, Saklatvala J (1988) Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin l-binding protein. J Biol Chem 263:12063-12069. 15. Savage N, Puren AJ, Orencole SF, Ikejima T, Clark BD, Dinarello CA (1989) Studies on IL-l receptors on DlOS T-helper
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cells: demonstration of two molecularly and antigenically distinct IL-l binding proteins. Cytokine 1:23-35. 16. Chin J, Cameron PM, Rupp E, Schmidt JA (1987) Identification of a high-affinity receptor for native human interleukin ll3 and interleukin 101 on normal human lung fibroblasts. J Exp Med 165:70-86. 17. Chin J, Rupp E, Cameron PM, MacNaul KL, Lotke PA, Tocci MJ, Schmidt JA, Bayne EK (1988) Identification of a highaffinity receptor for interleukin 1 alpha and interleukin 1 beta on cultured human rheumatoid synovial cells. J. Clin Invest 82:420-426. 18. Lowenthal JW, MacDonald HR (1986) Binding and internalization of interleukin 1 by T cells. Direct evidence for high- and low-affinity classes of interleukin 1 receptor. J Exp Med 164:10601074. 19. Bensimon C, Wakasugi N, Tagaya Y, Takakura K, Yodoi J, Tursz T. Wakasuei H (1989) Two distinct affinitv bindinn sites for IL-l on human ceil lines. J Immunol143:1168-1174. ” 20. Matsushima K, Akahoshi T, Yamada M, Furutani Y, Oppenheim JJ (1986) Properties of a specific interleukin 1 (IL 1) receptor on human Epstein Barr virus-transformed B lymphocytes: identity of the receptor for IL l-a and IL 1-B. J Immunol 136:44964502. 21. Sims JE, March CJ, Cosman D, Widmer MB, MacDonald HR, McMahan CJ, Grubin CE, Wignall JM, Jackson JL, Call SM, Friend D, Alpert AR, Gillis S, Urdal DL, Dower SK (1988) cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily. Science 241:585-589. 22. Urdal DL, Call SM, Jackson JL, Dower SK (1987) Affinity purification and chemical analysis of the interleukin-1 receptor. J Biol Chem 263:2870-2877. 23. Kasai K, Hiraiwa M, Emoto T, Kuroda H, Hattori Y, Mochizuki Y, Nakamura T, Shimoda S (1990) Presence of high affinity receptor for interleukin-1 (IL-l) on cultured porcine thyroid cells. Horm Metab Res 22:75-79. 24. Dower SK, Kronheim SR, March CJ, Conlon PJ, Hopp TP, Gillis S. Urdal DL (1985) Detection and characterization of high affinity plasma membrane’receptors for human interleukin 1. J Eip Med 162:501-515. 25. Dower SK, Call SM, Gillis S, Urdal DL (1986) Similarity between the interleukin 1 receptors on a murine T-lymphoma cell line and on a murine fibroblast cell line. Proc Nat1 Acad Sci USA 83:1060-1064. 26. Lowenthal JW, Cerottini J-C, MacDonald HR (1986) Interleukin l-dependent induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells. J Immunol 137:1226-1231. 27. Gearing AJH, Bird CR, Bristow A, Poole S, Thorpe R (1987) A simple sensitive bioassay for interleukin-1 which is unresponsive to 10’ U/ml of interleukin-2. J Immunol Meth 99:7-11. 28. Eggo MC, Mak WW, Bachrach LK, Errick LE, Burrow GN (1985) Cultured thyroids-Is immortality the answer? In Eggo MC, Burrow GN (eds) Thyroglobulin-The Prothyroid Hormone, Raven Press, New York, pp 201-210. 29. Ambesi-Impiombato FS, Picone R, Tramontano D (1982) Influence of hormones and serum on growth and differentiation of the thyroid cell strain FRTL. Growth of cells in hormonally-defined media. In Sato GH, Pardee A, Sirbasku DA (eds) Cold Spring Harbor Conference on Cell Proliferation, pp 483-492. 30. Svenson M, Poulsen LK, Fomsgaard A, Bendtzen K (1989) IgG autoantibodies against interleukin la in sera of normal individuals. Stand J Immunol29:489-492. 31. Giles KW, Myers A (1965) An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 4979:93. 32. Weiss SJ, Philp NJ, Grollman EF (1984) Iodide transport in a continuous line of cultured cells from rat thyroid. Endocrinology 114:1090-1098.
L. Kayser,2’3
M.B. Hansen,’ K. Bendtzen4,*
A. Krogh Rasmusseq3
Cellular binding of interleukin-1 (IL-l) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human ‘%-IL-lo. Scatchard plots of data obtained at 4°C indicated the presence of a single population of receptors with a & of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-l. The IL-1 receptors on these cells had I(d values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-lo and rIL-1B inhibited lz51-rIL-lo binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of ‘*%rIL-la to long-term cultivated human thyroid cells or to the FRTL-5 cells. We conclude that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.
Copyright o 1991 by W.B. Saunders Company
Interleukin-1 (IL-l) is a small family of pleiotropic cytokines produced primarily by monocytes and macrophages.‘,’ IL-l acts as a costimulator of T and B lymphocytes,‘,’ and it stimulates endothelial cells to produce inflammatory factors.3 It thus appears to be centrally involved in infectious and immunoinflammatory reactions. Furthermore, IL-l has been found to be cytotoxic to islets of Langerhans: and it affects the function of thyrocytes’ and pituitary cells6 IL-l may therefore play a role in the regulation of endocrine cell functions and, possibly, in the development of autoimmune and endocrine diseases. Two distinct molecules with IL-l activity have been identified, termed IL-la and IL-lB.7,8 The spectrum of biological activities shared by these two mole-
cules seems to be mediated by binding of IL-la and IL-@ to the same receptor, even though the receptor for IL-l may not be identical on different cell types.9^12 Recently, differential effects of IL-la and IL-lp on human thyroid cells in secondary culture and on the rat thyroid cell line FRTL-5 have been described.13 In the present study, we examined the presence of IL-l receptors on these cells and, in addition, the specific binding of ‘251-IL-l~ to long-term cultures of human thyroid cells. Only human thyroid cells in secondary culture expressed detectable specific receptors for rIL-la. RESULTS Binding of ‘251-rIL-1 a to Human Tlzyrocytes in Secondary Culture and to Murine T-Cell Lines
‘Department of Infectious Diseases M, Rigshospitalet University Hospital. ‘Department of Anatomy, The Panum Institute, University of Copenhagen. 3Medical Department E, Frederiksberg Hospital. “Deoartment of Medicine TTA. Rieshosuitalet Universitv ~ Hospital, _ DK-2200 Copenhagen N, Denm&k. A *Correspondence: K. Bendtzen, MD, Lab. Med. Immunol. 7544, Rigshospitalet, 20 Tagensvej, DK-2200 Copenhagen N, Denmark. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0302-0006$05.00/O KEY
WORDS:
CYTOKINE,
interleukin-1
receptors/thyroid
Vol. 3, No. 2 (March),
cells/FRTL-S/EL-4
1991: pp 125-130
Figure 1 shows the kinetics of binding of ‘251-rIL-lo to human thyrocytes in secondary culture and to the murine T-cell line NOB-l. On both cell types, maximum saturable binding was reached after about 800 min of incubation at 4°C. At room temperature, maximal binding of lz51-rIL-lor was seen after 240 min (data not shown). The specific lZI-rIL-la binding to NOB-l and EL-4 cells was saturated by 1 nM rIL-la (Fig. 2A). However, 10 times higher concentrations were needed 125
126 I Svenson
CYTOKINE,
et al.
Binding of “‘I-IL-1 LXto the Rat Thyroid Cell Line FRTL-5 and to Long-Term Culturesof Human Thyrocytes
BOUNO (cpml
: vu &em o-..'-. 0
Vol. 3, No. 2 (March 1991: 125-130)
200
'. 400
I 600
. ..l....,....,....,. BOO 1000
. 1200
.
1400
TIME (min) Figure 1. Kinetics to human tbyrocytes
of binding ‘Z51-rIL-la in secondary culture
to NOB-1 @I#).
cells
(0,O)
and
Duplicate cultures were incubated at 4°C with 0.2 @ml of ‘251-rIL-la alone (0,O) or in the presence of excess (SO @ml) unlabeled rIL-la (.,V
to saturate the binding to human thyroid cells (Fig. 2B). Scatchard analyses of the binding data at 4°C indicate the presence of a single class of IL-lo receptors on these cells (Fig. 2, A and B). Using thyroid cells, the equilibrium dissociation constant (Kd) was almost 10 times higher than on the murine T-cell lines. However, the number of receptors on the thyroid cells was comparable to that on the murine T-cell lines (Fig. 2 and Table 1). When binding was measured at room temperature, increased amounts of 1251-rIL-la associated with the human thyrocytes (Fig. 3). Scatchard analyses of data obtained in experiments at 4°C and at 22°C resulted in parallel lines. This indicates that the cells expressed increased numbers of receptors at room temperature. Internalization of the ‘251-rIL-10-receptor complex and recycling of the receptor would contribute to an apparent increase in receptor numbers. Therefore, the effect of acid treatment on the cell-associated radioactivity was determined. As shown in Table 2, a substantial amount of the cell-associated tracer was nonextractable by acid treatment at room temperature. Thus, internalization of the receptor-ligand complex and recycling or increased synthesis of the receptor are likely causes of the increased binding of ‘=I-rIL-la to the thyrocytes at room temperature. At 4°C both human rIL-la and rIL-ll3 competed with ?-rIL-lcx binding to EL-4 cells and to human thyrocytes in secondary culture. Using EL-4 cells, the competition by rIL-lo was stronger than by rIL-ll3, but the reverse was found when using thyrocytes (Fig. 4).
Assay conditions similar to those described above were used to detect 1251-rIL-la binding to FRTL5 cells and to cultures of human thyrocytes passaged 8 times during a 5-month cultivation period. Morphologically, the latter had changed to small cells without detectable thyroid-specific functions such as thyroglobulin production and TSH-induced CAMP production. At 4°C neither of these two thyroid cell populations showed changes in the bound/free (B/F) ‘=I-rIL-la values using free ligand concentrations between 5 x 10-l’ M and 6 x 10m9M (Table 3). To increase the ability to detect receptors, which may internalize the ligand, binding to FRTL5 cells was also performed at 37°C. This increased the binding of lZI-rIL-la, but the binding was proportional to the free ligand concentration (Table 3). When kept in secondary culture for 3 months, human thyroid cells still possessed detectable IL-la receptors. Human thyroid cells passaged in parallel exhibited unchanged receptor binding up to the second passage. After the third passage, the cells had lost all IL-lo receptors (see Table 1). These cells did not show any morphological changes.
DISCUSSION The number of receptors for IL-l and their binding properties vary considerably depending upon the target cell and, most likely, the experimental conditions. Thus, dissociation constants from 3 pM to 500 pM, binding sites from 100 to more than 10,000 per cell, and differences in the relative binding affinities of the two forms of IL-l have been reported.9’14-20Generally, T cells, in contrast to B cells, seem to bind rIL-la more strongly than rIL-l~.9~‘5~20Judged from the present experiments, using rIL-la and rIL-1B competition with lZI-rIL-la binding, human thyroid cells in secondary culture bind rIL-1B more strongly than rIL-la. In contrast, and in agreement with other studies,‘4,15,21,22 preferential binding of rIL-la was found using the murine T-cell lines EL-4 and NOB-l. Thus, the pattern of binding of IL-l to human thyroid cells in secondary culture resembles that to B cells. Using EL-4 and NOB-l cells, the K values of 8 to 17 pM for rIL-lo agree with those reported by Lowenthal and McDonald,‘* but they are 10 to 30 times lower than those reported by others.14~15~21~22 In contrast to Lowenthal and McDonald, we and others failed to obtain data indicating the presence of both high- and low-affinity receptors on the EL-4 cells.‘* Using thyroid cells in secondary culture, the Scat-
Interleukin-1 receptors on thyroid cells / 127
0.10
0.05
-
0.00 10 -13
IO -12
10 -1'
IO -'Cl
10-g
10-B
mol/ I
6
B/F
Figure 2. Binding of ‘%-IL-k to NOB-l, EL-4 cells (A) and to human thyrocytes in secondary culture (B) at 4°C.
The results are shown as total bound ‘Z51-rIL-la to 5 x 10’ cells free ligand (B/F) as a function of the free ligand concentration. The inserts show the Scatchard plot of the same binding data after subtraction of unspecific binding. Unspecific binding was calculated on the basis of binding obtained at lo-’ M of the ligand. The x-axes show the bound ‘Z51-rIL-1a in pg/5 x lo5 cells. (A) (0) NOB-l cells, K, = 10 pM and 2,400 receptors/cell, (+) EL-4 cells, K, = 9 pM and 2,000 receptors/cell. (B) Human thyrocytes in secondary culture: K, = 170 pM, 6,000 receptors/cell.
chard plots of the binding data showed single slopes, indicating a single population of 1251-rIL-10 binding sites. Single slopes were also obtained with murine T-cell lines. The calculated K value of 80 pM for ‘251-rIL-la binding to human thyroid cells is similar to that found on human rheumatoid synovial cells’7 and lung fibroblasts.16 A high-affinity rIL-1B receptor on porcine thyroid cells has recently been reported by Kasai et a1.23The Kd value was similar to that of the rIL-lol receptor on human thyroid cells. In contrast to our observation, Kasai et al. found identical cross-reactivity when using unlabeled rIL-la and rIL-1B. Also, the human thyroid cells expressed 4,500 IL-1 binding sites per cell (this study), and this is more than ten times the number of
mol/l IL-l receptors found on porcine thyroid cells.” These differences between IL-1 binding to human and porcine thyrocytes may be explained by variations in the experimental conditions, but species differences cannot be excluded. Biological responses to IL-l may be obtained at extremely low concentrations of the cytokine (lo-l4 to lo-l*M). In some cell systems, this potency of IL-l seems to require less than one receptor occupancy for biological activation of a single ce11,24,25 indicating the presence of IL-l receptors of higher affinity than those presently characterized. Occupation of only a few receptors per cell has been shown to activate IL-lresponsive target cells.16’17EL-4 and NOB-l cells respond to both species of IL-l at 10-14M (references 26
128 I Svenson
CYTOKINE,
et al,
Table 1. IL-k receptors on various cell lines and human thyrocytes. Direct binding was performed at 4°C as described in Materials and Methods. The I& values and receptor numbers were calculated from Scatchard plots. Receptors/cell
UPM) Cells
Range
Murine T-cell lines EL4 NOB-l Rat thyrocyte cell line FRTL-5 Human thyrocytes Secondary culture Long term2
Mean
Range
Mean
Table 2. Effect of pH 3 wash of human thyrocytes on cell-associated ‘z51-rIL-lcy, Human thyrocytes in secondary culture were incubated at 22°C for 6 h with ‘ZSI-rIL-lcu alone or with excess unlabeled rIL-loi. The cultures were washed two times in the incubation buffer followed by 3 min incubation at pH 7.4 or pH 3 (made by adding 6.25 ml of 1 M HCl to 100 ml of the pH 7.4 incubation buffer). The cells were then washed and treated as described in Materials and Methods.
n rIL-la (34 @ml)
Wash pH3
+
-
-
+ + +
+ +
+ +
‘J%rIL-1OI (0.16 ngiml)
8-10 8-17
9 12
1,200-3,000 2,500-6,000
nd’ 30-170 (80)
2,100 3,600
3 3
nd 80
2
2,000-6,000
Vol. 3, No. 2 (March 1991: 125-130)
4,500
cpm/loI cells (mean t SD; n = 3)
648 177 491 180
f f k f
2.5 8 12 5
6 3
ww
‘Not detectable. ‘Obtained with one culture of cells passaged three times and with two cultures passaged more than three times. The latter cells did not show specific binding of ‘xI-rIL-loi.
and 27, and our own data). However, EL-4 cells require a second signal such as a calcium ionophore in order to respond to IL-Lz6 whereas the NOB-l cells respond with interleukin-2 production if exposed to IL-lo//p a1one.27 Judged by the Scatchard plot analysis of binding at 4°C 1 to 6 receptors per cell would be occupied on the murine T cells at lo-l4 M rIL-lo. In secondary cultures of human thyrocytes, the secretion of thyroglobulin and CAMP is suppressed by a minimum of lo-l3 M human IL-~cx/B.~~‘~At this concentration, an average of 2 to 6 receptors per cell would be occupied by rIL-la. These findings, therefore, support the notion that high-affinity receptors are involved in mediating the
biological effects of IL-1 on the murine T-cell lines (Kd I 10 PM) and on human thyroid cells (Kd I 100 PM) in secondary culture. In contrast to human thyroid cells in secondary culture, the growth of the rat thyroid cell line FRTL5, but not its secretion of CAMP, has been found to be inhibited by rIL-1B but not rIL-10.13 The lack of effect of rIL-la on this cell line is in agreement with the observed absence of specific ‘2SI-rIL-la binding to the cells when assayed at 4°C as well as 37°C. Similar findings have been reported in the case of another peptide mediator, epidermal growth factor.% Though present on primary cultures of sheep thyroid cells, the receptors for this factor were absent on FRTL5 cells.** This, and the observed loss of IL-1 receptors during passage of human thyroid cells, indicates that dediffer% INH
B/F
0.02
0.01
0 IO -2
,
. .Jd....l 10-I
. “I,,,,’ 100
“I,,.,’ 10 1
’ 102
w/ml 0.00
0
IO
20
30
40
50 6
Figure 3. Scatchard plots cytes in secondary culture.
of ““I-rIL-la
binding
to human
thyro-
Assay at 4°C (W) and at 22°C (0). The experiments were performed in parallel.
Figure 4. Ability of unlabeled binding of ‘LsI-rIL-lcy.
rIL-lru
or rIL-lp
to compete
with
the
‘251-rIL-la, 180 pgiml, was incubated at 4°C with EL-4 cells (0,O) in the presence of the indicated amounts of unlabeled rIL-lu (open symbols) and rIL-1B (closed symbols). The same experiment was carried out in parallel with human thyroid cells in secondary culture (0,m) using 280 p&ml of rz51-rIL-la. The y-axis shows percent reduction of ‘“I-rIL-lo binding.
Interleukin-1 Table 3. Binding of ‘zSI-rIL-lcu to FRTLd thyrocytes passaged eight times.
cells and human
Results are given as means of duplicates. (A) human thyrocytes passaged 8 times during a 5-month cultivation period. The cell density was 6 x 106/well. (B) FRTL-5 (2.5 x 106/well).
Cont.
of ‘2SI-rIL-l~
(M)
5 x lo-l2 lo-” 3 x 1om9 6 x 1O-9
3TC3
A
B
Ei
0.0049 0.0048 0.0050
0.0019 0.0019 0.0018 0.0017
0.009 0.008 0.009 0.008
‘cpm boundicpmiml supernatant. ‘incubated for 22 h. ‘incubated for 5 h.
entiation during transformation of thyroid cells results in marked changes in the expression of receptors for peptide hormones.
MATERIALS
AND METHODS
Cells Human thyroid cells were obtained from paraadenomatous tissue during strumectomies. They were isolated and grown in Hams F12 medium as modified by Coon and supplemented with 5% fetal calf serum and six growth factors, as previously described.5 The FRTL5 cells were kindly provided by P. Ealey (University College Hospital, London, UK). The cells were grown as described by AmbesiImpiombato et al.29in the same medium as above. The mouse thymocyte cell lines EL-4 and NOB-l were obtained from the American Type Culture Collection (Rockville, MD, USA) and the European Collection of Animal Cell Cultures (Wilts, England), respectively. The cells were maintained in RPMI-1640 with 25 mM Hepes (Gibco Biocult, Paisley, UK) containing 2.5 km 2-mercaptoethanol, 2.5 mM L-glutamine (Sigma, St. Louis, MO, USA), 25 p,l/ml gentamycin (Flow Laboratories, Scotland), and 5% fetal calf serum (Gibco). Cultures were grown at concentration between 1 x 10’ and 10 x 10’ cells/ml.
Procedures After 1 to 2 weeks, the thyroid cells were passaged by seeding 5 x lo5 cells into 6-well culture plates (9.6 cm’/well, Greiner, Frickenhausen, Germany). The cells were then grown for 3 to 5 daysbefore the binding assaywas performed. To evaluate the stability of the expression of IL-la receptors of human thyroid cells in long-term culture, 2 experiments were performed: 1. 2.
passage. Every third week, 4 to 6 days after passage, one plate with passaged cells was investigated, along with one plate with cells kept in secondary passage from the same original cell culture.
Interleukin-1
Em’ 4”CZ
receptors on thyroid cells / 129
Cells were kept in culture and passaged every second to third week for 5 months and evaluated in the eighth passage. Cells were kept in secondary culture for 3 months, with parallel passages every third week until the fourth
Human rIL-la and rIL-1P were generous gifts from T. Tsuboi (Dainippon Pharmaceutical Company, Osaka, Japan) and C.A. Dinarello (Tufts New England Medical Center, Boston, MA, USA). The biological activities of rIL-lol and rIL-1P were detected by the murine cell lines EL-4 and CTLL-2, as described.3oThe specific biological activity of rIL-la was lo* U/mg, as determined by a mouse thymocyte costimulatory assaycalibrated with an interim international rIL-la reference preparation (NIBC, Potters Bar, UK).
Radioiodination
of rIL-1 cy
Human rIL-lol was radioiodinated using chloramine T, and the bioactivity of 1251-rIL-1awas determined using EL-4 and CTLL-2, as previously described”’ More than 96% of the lZ51in the rIL-101 fraction could be precipitated with 5% trichloroacetic acid. The specific activity of the labeled rIL-101 was 3 X 10’ to 4 X 10’ cpm/ng.
IL-l CYBinding Assay FRTL5 and human thyroid cells were cultured to confluence in 6-well culture plates as described above. Before assay,the medium was removed from each well and replaced with 1 ml of RPMI-1640 (Gibco) plus 0.5% bovine serum albumin (BSA) (Sigma) containing ‘251-rIL-lol with or without rIL-la and rIL-1P. Nonspecific binding of lZI-rIL-la was determined in the presence of 100 to 200 @ml of unlabeled rIL-l&. The plates were incubated under gentle agitation at 4°C for 20 to 22 h, at 22°C for 6 h, or at 37°C for 5 h. After incubation, the supernatants were removed for counting of lZ51activity and the plates were washed by dipping them rapidly three times in 4 L of ice-cold, phosphatebuffered saline (20 mM phosphate buffer, 0.125 mM NaCl, pH 7.4). Bound *251-rIL-la was then solubilized in two times 0.5 ml of 2 M KOH and the samples were counted with an error of 4% or less in a gamma counter (1272 CliniGamma, LKB, Wallac OY, Finland). One chamber was used on each plate for the detection of cell numbers after the washing procedure. The cell number was determined by DNA measurement31 using an index of 15 pg DNA/lo6 cells.32EL-4 and NOB-l cells, 1 x 106 to 2 x 106/ml, were incubated under continuous agitation with lZ51-rIL-la in medium RPMI-1640 containing 0.5% BSA. After 20 h, 200 ~1 of the cell suspensions were added to Microtubes EET-23 (Struers, Glostrup, Denmark) containing dibutyl phthalate:bis( -2ethylhexyl) phthalate 3:2 (Merck, Darmstadt, Germany). The cells were then centrifuged at S,OOOgfor 2 min in a Biofuge B centrifuge (Hereus-Christ, Osterode, Germany). The medium and pellet were separated by cutting through the oil layer and lz51was counted with an error of less than 2%. All procedures were carried out at 4°C.
130 / Svenson et al.
Acknowledgments
We thank The Copenhagen Thyroid Cell Group (Ulla Feldt-Rasmussen, Karine Beth, and Hans Perrild) for valuable help and Susanne Meldgird for excellent technical assistance. The study was supported by the Danish Cancer Society, the Danish Hospital Foundation for Medical Research, Region of Copenhagen, the Faroe Islands, and Greenland, the Danish Medical Research Council, the Novo Foundation, the Willumsen Foundation, and the Danish Biotechnology Program.
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