JOURNAI. OF BONE A N D MINERAL RESEARCH Volume 6. Numher I , 1991 Mary Ann Liehert, Inc., Publishers

Interleukin-6 Does Not Stimulate Bone Resorption in Neonatal Mouse Calvariae ABDULKARIM AL-HUMXDAN, STUART H. RALSTON,* DAVID E. HUGHES, KAREN CHAPMAN, LUCIEN AARDEN,? R . GRAHAM G . RUSSELL, and MAXINE GOWEN

ABSTRACT Recombinant human interleukin-6 (1L-6) was assessed for its ability to stimulate bone resorption in prelabeled mouse calvariae in vitro. IL-6 had no effect on bone resorption at concentrations ranging from 300 to 10,000 U/ml (3-1000 pg/ml). Neither the presence of indomethacin nor prolonged incubation periods (96 h) affected this result. IL-6 did not affect resorption stimulated by human recombinant IL-ltu (rIL-la) but inhibited resorption stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D, [1,25(OH),D1]. rIL-la, PTH, and 1,25-(OH),D, induced IL-6 release by calvariae. We conclude from these studies that IL-6 does not stimulate bone resorption in neonatal mouse calvariae. However, it may act as a locally produced inhibitor and therefore a paracrine regulator of bone resorption induced by osteotropic hormones. IL-6 could also function as a long-range stimulator of systemic reactions and acute-phase responses to local inflammatory and neoplastic lesions in bone.

INTRODUCTION

fect. Finally, IL-6 has been identified in the synovial fluid and serum of patients with rheumatoid arthritis"" in whom periarticular bone resorption is a feature of the disease.

NTERLEUKIN-6 is a pieiotropic cytokine with activities described by its previous names: hepatocyte stimulating factor,'" B cell growth factor 2,l') hybridoma and plasmacytoma growth factor,(" and interferon-&.(*) I t has a number of activities in common with interleukin-l (ILparticularly those associated with the organism's response to infection or injury. IL-I is also known to be a potent stimulator of bone resorption,I6 '' and to have complex effects on osteoblast function,(R-lO1 and is a powerful inducer of IL-6 production.'"' It has been suggested that in a number of systems IL-6 may therefore mediate the actions of IL-1, including its bone-resorptive capacity. An additional stimulus for this study was the description of 1L-6 as an autocrine growth regulator of myeloma cells.'") Myeloma is associated with bone loss driven by tumor-derived factors, and we suggested that IL-ti, if capable of stimulating bone resorption, could contribute to this ef-

I

MATERIALS AND METHODS Recombinant human IL-6 was cloned and expressed in Escherichia coli as described previously. ( I " The specific activity of the purified 1L-6 was 10' U/mg using the B9 assay as described previously"' (see also later discussion). Sheep antihuman IL-6 antiserum was prepared using pure recombinant hIL-6, and has no cross-reactivity with other cytokines. Recombinant human IL-ICYwas kindly supplied by Dr. P. Lomedico, Hoffmann-LaRoche (Nutley, NJ), specific activity 3 x 10RU/mg (D10 assay). The 11BI I antimouse IL-4 monoclonal antibody was generously provided by DNAX."" This had no cross-reactivity with other cytokines.

Department of Human Metabolism and Clinical Biochemistry, Sheffield University Medical School, UK. *Current address: Rheumatic Diseases Unit, Northern General Hospital, Edinburgh, U K . ?Current address: Central Laboratory for the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands.

3

AL-HUMIDAN ET AL.

4

Antirat IL-6 antiserum was generously supplied by Dr.

J . Gauldie, McMaster University, Canada. The antiserum did not cross-react with human IL-6 but did neutralize mouse IL-6. Antiserum was dialyzed against culture medium before use. Bone resorption was assessed using the prelabeled mouse calvarial assay as described with the addition of M indomethacin during the 24 h preculture period as described by L e r r ~ e r . “ ~Bones ) were usually cultured for 48 h with test substances, although some experiments were extended to 96 h. Endotoxin was undetectable by the Limulus amebocyte lysate assay, sensitivity 0.001 ng/ml, in any of the media and reagents at the concentrations used. IL-6 release was measured using a specific bioassay utilizing an 1L-6 dependent hybridoma cell line B9.‘” Proliferation was quantitated using the uptake and metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).‘”’ This gave results equivalent to the incorporation of [’H)thymidine (results not shown), although it actually measures the overall metabolic activity of the cells, rather than DNA synthesis. Briefly, cells were seeded into microtiter wells at 5000 cells per well and cultured for 72 h with samples containing IL-6 or known concentrations of recombinant IL-6. Phosphate buffered saline (PBS, 100 pl) containing 50 rng/rnl of MTT was added to each well 4 h before the end of the culture period. Medium was then removed and replaced with 100 pI propan-201 and 0.04 M HCI, which solubilized the metabolized dye. Samples were then measured spectrophotometrically in a Dynatech micro-ELISA reader using 570 nM as the test and 630 as standard wavelengths. OD,,,,.,,,, was plotted against standard concentrations to generate a standard curve. Sample OD,,,,.,,,, could then be used to quantitate

TABLE1. EFFECTOF IL-6

ON

BONERESORPTION”

the amount of IL-6 in the conditioned medium. As the B9 cells are extremely sensitive to IL-6, samples were diluted in medium and assayed at a range of concentrations from 1:1000 to 1:100,000.

Statistics Results were analyzed using the nonpaired Student’s t-test.

1

FIG. 1. Effect of IL-6 on IL-1-stimulated bone resorption. Calvariae were cultured in three groups with either no IL-6 (light hatching) or 500 or 5000 U/ml of IL-6 (dark hatching). Paired half-calvariae were cultured with or without 10 U/ml of rIL-la (approximately 10 pM). The first bar therefore represents basal bone resorption. In each case the rlL- la-stimulated bone released significantly more “Ca than its paired control ( P < 0.001), and in no case did the IL-6 significantly modulate rlL-la-stimulated resorption. n = 5 . This is representative of three experiments.

TABLE2. EFFECTOF IL-6 ON PTH AND 1 ,25-(OH),DI-STIMULATED RESORPTION~

Yo Release treated/control

Treatment (U/mI) Control IL-1 IL-6 I L-6 IL-6 IL-6 IL-6 IL-6

Mean

SEM

1.OO

10 300 500 1,000 3,000 5,000 10,000

1.64b 1.16 1.20 0.96 1.12 I .08 I .03

Ratio of % release of V a

n 65

0.07 0.11 0.12 0.04 0.11 0.09 0.01

T

40 5

18 26 5

17 12

“Data shown are from 10 separate experiments. Bone resorption was measured as the percentage release of ‘”Ca from individual calvariae, and treated/control ratios were calculated to facilitate comparisons between experiments. The only consistent and statistically significant stimulation was obtained using rIL-la. n = number of bones studied. Wgnificantly greater than 1, p < 0.001.

Treatments 1,25-(OH),D,/control 1,25-(OH),D, + IL-6/control IL-6/control PTH/control PTH + IL-6/control IL-6/control

Mean

SEM

1.65 1.29 1.04 1.64 1.17 I .01

0. lob 0.llC 0.03 0.09b 0.08d 0.03

al,ZS-(OH),-vitamin D , [I,25-(OH),Dr] was present at lo-* M , P T H at 10.” M, and 1L-6 at 500 U/ml. A total of five and four experiments were performed with 1,25(OH),D, and P T H , respectively, and the values shown are mean and SEM of all experiments. Within each experiment each treatment was assessed using between four and eight half-calvaria. Wgnificantly greater than 1 .OO, p < 0.01. CSignificantly lower than 1.25-D alone, p < 0.05. dSignificantly lower than P T H alone, p < 0.01.

5

IL-6 AND BONE RESORPTION TABLE3. NEUTRALIZATION OF IL-6 EFFECTBY ANTIHUMAN IL-6 ANTISERUM^ ?lo Release

of "'Ca Addirion to bone

None PTH IL-6 IL-6Ab IL-6 PTH + IL-6 IL-6 + IL-6Ab PTH None 1,25-(OH),Di 1L-6 IL-6 + IL-6Ab 1,25-(OH),Dt + IL-6 1,25-(OH),Di + IL-6 + IL-6Ab

+

+

~~~

Mean

SEM

No. of bones

7.3 13.3 8.1 8.3 9.7 13.0 9.2 18.9 9.9 10.4 14.3 18.3

0.36 1.09b 0.65 0.81 0.7Y 2.41d 0.97 1.45b 1.50 0.34

8

1.29

2.09d

5

4 4 4

5 6 4 4 2 4 4

~

aAgents were present at the concentrations shown in Table 2. Goat antihuman 1L-6 (1:1OOO dilution) was preincubated with 1L-6 for 1 h at 37°C before addition to the assay. Normal sheep serum (Glaxo Group Research, UK) was without effect in this assay. Wgnificantly different from control, p < 0.001. 'Significantly different from PTH alone, p < 0.05. dsignificantly different from control, p < 0.01. CSignificantly different from I ,25-(OH),D, alone, p < 0.05.

TABLE 4. STIMULATION OF RELEASIOF 1L-6 FROM CALVARIAE BY BONE-RESORBING AGENTS~

IL-6 rekase, frea[ed/control ratio Treat men i

Mean

SEM

Control IL-1, 10 U/ml M PTH, 1,25-(OH),Di lo-" M

100-760 22.5 7.5 1.25

U/ml 0.72b 0.99 0.04b

"IL-6 release was measured in medium conditioned by bones stimulated to resorb with the agents shown. Comparable resorption was achieved for each resorbing agent. The ratios were determined from four experiments, qexcluding that shown in Table 5. hSignificantly different from control, p < 0.001.

RESULTS IL-6 did not affect bone resorption at concentrations of 300-10,OOO U/ml (1 U is equivalent to 1 pg; Table I ) . rlLl a was used as a positive control. Neither addition of indomethacin to the culture medium nor exi.ension of the culture period to 96 h affected this result (data not shown). To investigate the possibility that IL-6 could enhance resorption effected by other agents we added IL-6 in combi-

nation with IL-I, 1,25-(OH),D,, and PTH. IL-6 had no effect on bone resorption stimulated by IL-1 at a range of concentrations (Fig. 1). However, IL-6 unexpectedly inhibited the resorption induced by 1,25-dihydroxyvitamin D, [ 1,25-(OH),D,] and parathyroid hormone (PTH; Table 2). To verify that this effect was due to 1L-6 and no other contaminating factor similar experiments were carried out after preincubation of IL-6 for 1 h at 37°C with goat antihuman 1L-6 antiserum prepared using pure recombinant human IL-6 as immunogen. Neutralizing antiserum ablated the inhibitory effect of IL-6 (Table 3). Since many cell types are known to synthesize 1L-6 we investigated the possibility that IL-6 is produced at high levels by the calvarial cells and hence the calvariae may respond differently to the exogenously added IL-6. The calvariae were found to release some IL-6 under basal conditions (100-800 U/ml), but this was negligible compared to the concentrations of IL-6 added (up to 10,OOO U/ml). Upon further investigation the calvariae were found to release IL-6 in response to rIL-la and PTH at the concentrations used to stimulate bone resorption. 1,25-(OH),D, significantly stimulated IL-6 release but to a much smaller extent (Table 4). This activity was inhibitable using a neutralizing antirat 1L-6 antiserum (Table 5 ) but unaffected by an antimouse IL-4 antiserum (data not shown). Murine IL-4 is the only cytokine other than 1L-6 known to affect this bioassay. We further investigated the possibility that endogenous production of IL-6 could mediate rlL-la-stimulated resorption by inhibiting rIL-la-induced IL-6 release with hydrocortisone (Table 6). IL-6 production, stimulated 100fold by rlL-la, was inhibited to basal levels by M hy-

AL-HUMIDAN ET AL.

6

TABLE 5 . NHJTRALIZATION OF IL-6 ACTIVITY RELEASEDBY CULTURED MOUSEC A L V A R I A E ~

OD

Treatnient

Control IL-I, 10 U/ml PTH, lo-" M 1,25-(OH),D, lo-" M

After incubation with anti-lL-6 antibody

U/ml After correction for dilution factor

0.132 0.257 0.202 0.167

480 11,010 1,555 650

OD

U/nd

0.003 0.005 0.003 0.003

0 0 0 0

aMedium conditioned by calvariae 5timulated with the agents shown were incubated f o r 1 h at 37°C in the presence of a 1:2O dilution of rabbit antirat IL-6 after dialysis of the antiserum versus culture medium or nonimmune rabbit serum before assay using BV cells. Samples were diluted appropriately for assay 50 that readings were obtained on thc linear part of the dose-response curve.

TABLE 6. EFFECTOF HYDROCORTISONE ON IL-6-STIMULATED RESORPTIONAND 11-6 RELEASE^ Bone resorption (% "Ca release) Treatinenf

IL-6 release (U/Ill I)

Control Hydrocortisone, 10." M IL-I, 10 U/ml IL-1 + hydrocortisone IL-6, 5000 U/ml IL-6 + hydrocortisone

380 900 49,000 1,000 5,500 4,600

Mean

SEM

12.1 12.2

0.43 0.48 0.98h 0.32h 0.43 0.26

17.1 16.0 11.8 11.9

aBones were cultured for 48 h in the presence of the agenis indicated. Medium was collected and assayed for 1L-6 after serial diluiions (1:lOO1:1OO,OOO) were made. 1L-6 concentrations were estimated by comparing activities o n the linear portion of each dilution curve, usually at half-maximal stimulation. defined as I U/ml. Thus the reciprocal dilution represented the concentration of 1L-6 (U/ml). An aliquot of the medium was also used to measure "Ca content. n = 6 for control and n = 4 for treatment groups. This experiment was repeated three times, with similar result?. Wgnificantly different from control, p < 0.001.

drocortisone but rIL-la-stimulated bone resorption was unaffected. Hydrocortisone alone or in the presence of IL-6 or rlL-la had no effect on bone resorption, as shown previously.oR'

DISCUSSION In this model system IL-6 had no stimulatory effect either alone or in combination with other resorbing agents. We therefore concluded that it is unlikely to be involved in the resorption that occurs in patients with myeloma or around rheumatoid joints. However, since these studies were performed there have been two reports of a stimulatory effect of IL-6 in models that utilize developmentally more immature bone."9.z0' It has been shown previously

that IL-6 has effects on hematopoietic stem cells,"" and this may explain the differences in results obtained using the different experimental systems. If this is so, it raises the possibility that we may now be able to localize the target cell of certain cytokines in the osteoclast lineage. I t also calls into question the relevance of such model systems in the attempt to understand adult remodeling with reference to such diseases as osteoporosis. An unexpected inhibition of PTH- and 1,25-(OH),D,stimulated resorption was detected in combination with enhanced release of IL-6 from resorbing calvariae. The stimulation of IL-6 release from murine bone cells by parathyroid hormone confirms the findings of others."9.zz1Such findings suggest a possible mechanism of control in which the extent of the bone resorption phase stimulated by a particular agent is limited by the concurrent production of

IL-6 AND BONE RESORPTION

an inhibitory mediator. The response to low levels of 1L-6 when relatively high levels of production can be induced by PTH may simply reflect the time course of each response. Exogenously added IL-6 can act at any stage of the osteoclastic response to PTH, whereas over the 48 h incubation used in this assay many cellular events will have occurred in response to PTH before IL-6 production takes place. A selective inhibition of resorption stimulated by different classes of agent is reminiscent of the action of interferony, which inhibits cytokine-stimulated but not PTH- or 1 ,25-(OH),D1-stimulated resorption.‘zJ’ These results further highlight the likelihood that different agents induce resorption via different cellular targets and are therefore potentially subject to different mechanisms of control. It is possible that IL-6 may also have an endocrine function, for example to help mediate the acute-phase response to local infection or inflammation in bone. Certainly these studies have demonstrated that although IL-6 shares some of the actions of IL-I, these appear to be predominantly those associated with the host’s response 10 infection or injury.

ACKNOWLEDGMENTS This work was presented in part at the Joint ICCRH/ ASBMR meeting in Montreal, Canada, September 9-14, 1989. This work was supported by the Arthritis and Rheumatism Council, UK, and the Science and Engineering Research Council, UK. Maxine Gowen is a Royal Society University Research Fellow.

REFERENCES Gauldie J. Richards C , Harnish D, Lansdorp P , Baumann H 1987 Interferon beta,/B cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-sr imulating factor and regulates the major acute phase protein response in liver cells. Proc Nail Acad Sci USA 84:7251-7255. Hirano T, Yasukawa K , Harad H, Taga T, el al. 1986 Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 324~73-76. Aarden LA, De Groot ER, Shaap OL, Lansdorp PM 1987 Production of hybridoma growth factor by monocytes. Eur J Immunol 17:1411-1416. Van Damme J , Van Beeuman JV, Decock B, Van Snick J , De Ley M, Billiau A 1988 Separation and comparison of two monokines with lymphocyte-activating factor activity: IL-l beta and hybridoma growth factor (HGF). J Immunol 140: 1534- 1541. Van Damme J , Opdenakker G, Simpson RJ, Rubira MR, Cayphas S, Vink A, Billiau A, Van Snick J 1987 Identification of the human 26 kD protein, interferon beta,, as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor. J Exp Med 165914-919,

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6. Gowen M, Mundy GR 1986 Actions of recombinant interleukin-1, interleukin-2 and interferon gamma on bone resorption in vitro. J lmmunol 136:2478-2482. 7. Heath J , Saklatvala J , Meikle MC, Atkinson SJ, Reynolds J J 1985 Pig interleukin-1 (catabolin) is a potent stimulator of bone resorption. Calcif Tissue Int 37:95-97. 8. Gowen M, Wood DD, Russell RGG 1985 Stimulation of the proliferation of human bone cells in vitro by human monocyte products with interleukin-l activity. J Clin Invest 75: 1223- 1229. 9. Stashenko P, Dewhirst FE, Rooney ML, Desjardins L A , Heeley J D 1987 Interleukin-l beta is a potent inhibitor of bone formation in vitro. J Bone Min Res 2:559-566. 10. Boyce BF, Aufdemorte TB, Garrett IR, Yates AP, Mundy GR 1989 Effects of interleukin-I on bone turnover in normal mice. Endocrinology 125:1142-1150. 11. Van Damme J, Opdenakker G , Simpson RJ, Rubira MR, Cayphas S, Vink A, Billiau A, Van Snick J 1987 Identif‘ication of the human 26-kD protein, interferon beta,, as a B cell hybridoma/platmacytoma growth factor induced by interleukin-I and tumor necrosis factor. J Exp Med 165:914-919. 12. Kawano M. Hirano T, Matsuda T, Taga T , Horii Y. et al. 1988 Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332:83-85. 13. Houssiau F, Devogelaer J-P, Van Damme J, Nagant de Deuxchaisnes C , Van Snick J 1988 Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 31:784-788. 14. Brakenhoff J P J , De Groot ER, Evers RF. Pannerkoerk H , Aarden LA 1987 Molecular cloning and expression of hybridoma growth factor in Escherichro coli. J lmmunol 139: 41 16-4121. 15. O’Hara .I,Paul WE 1985 Production of a monoclonal antibody to and characerisation of a B cell stimulatory factor 1 . Nature 315:333-336. 16. Lerner UH 1987 Modifications of the mouse calvarial technique improve the responsiveness to stimulators of bonc resorption. J Bone Min Res 2:375-383. 17. Mosmann T 1983 Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J lmmunol Methods 6955-63. 18. Gowen M, Wood DD, Mundy GR, Russell RCG 1985 Studies on the control o f IL-1-stimulated bone resorption. In: Kluger MJ, Oppenheim J J , Powanda MC (eds) The Phytiologic, Metabolic and Immunologic Actions of Interleukin-I. Alan Liss, New York, pp. 85-93. 19. Lowick C , Van der Pluijm G, Hoekman K , Aarden L. Bijvoet 0, Papapoulos S 1989 Parathyroid hormone (PTH) and PTH-like protein stimulate interleukin 6 production by osteogenic cells: A possible role of interleukin 6 in osteoclastogenesis. Biochem Biophys Res Commun 162:1546- 1550. 20. Miyaura C , Ishimi Y , Jin C H , Hirano T, Kishimoto T, Suda T 1989 Interleukin-6: Its production by osteoblasts and activity to induce bone resorption. J Bone Min Res 4:S151. 21. Ogawa M, Clark SC 1988 Synergistic interaction between interleukin-6 and interleukin-3 in support of stem cell proliferation in culture. Blood Cells 14:329-337. 22. Feyen J H M , Elford P , Di Padova FE, Trechsel U 1989 Interleukin 6 is produced by bone and modulated by parathyroid hormone. J Bone Min Res 4:633-638.

AL-HUMIDAN ET AL.

8 23. Gowen M, Nedwin G Mundy G R 1986 Preferential inhibition of cytokine-stimulated bone resorption by recombinant interferon gamma. J Bone Min Res 1:469-474.

Address reprint requests to: Dr. Maxine Cowen Bath Instirule for Rheumatic Diseases Trim Bridge Barh BAI IHD, UK Received for publication May 31, 1989; in revised form June 4, 1990; accepted August 20, 1990.