Relationship between camp induced inhibition of human meningioma cell proliferation and autocrine secretion of interleukin-6

Life Sciencs, Vol. 58, No. 16, pp. 1323-132!9,19!X copyright 0 1996 Iskevier science Inc. Printed in the USA. All rights tescnd rm&32n5/96 slmo t .ca

PI1 SOO24-3205(96)00098-7

RELATIONSHIP

CELL

BETWEEN

PROLIFERATION

cANP

INDUCED INRIBITION OF RUNAN NBNINGIONA AND AUTOCRINE SECRETION OF INTERLEUKIN-6

Anita Hiittner, Ting Lei, Rudolf Fahlbusch, Uwe Schrell, and Eric F. Adams Department of Neurosurgery, Kopfklinikum, University of ErlangenNiirnberg, 91054 Erlangen, Germany (Received in final

form February 12, 1996)

Summary Previous studies have suggested that activation of the adenylyl cyclase - CAMP system in meningiomas results in decreased mitosis. We have used meningioma cell culture to further investigate this phenomenon and to examine the potential role played by interleukin-6 (IL6). Incubation of cultured meningioma cells for 4-6 days with cholera increase and both of which toxin theophylline, levels, markedly stimulated IL6 intracellular CAMP secretion and inhibited cell growth rate. Similar effects 8-bromo-CAMP. In were observed with contrast, a neutralisingpolyclonalantibody against IL6 significantly stimulated meningioma proliferation and reduced the inhibitory effects of 8-bromo-CAMP. These results support the concept that IL6 acts as an autocrine / paracrine inhibitory factor for meningioma proliferation, and that the inhibition exerted by elevated intracellular CAMP levels may be at least partially mediated via increased secretion of the cytokine. Key Words: meningiomas,

interleukind,

cyclic adenosine

3’

5 ’ -monophosphate,

autocrine inhibition of growth

With the ultimate aim of developing adjuvant medical therapies, much effort in recent years has been directed at fully understanding the biochemistry of meningiomas, particularly those factors involved in eliciting alterations in cell proliferative rate (l-7). Most studies have been directed at determining the receptor status of meningiomas and the effect of the respective ligands, such as steroids and somatostatin, on cell growth. More recently, attention has been drawn to examining the intracellular second messenger transduction systems involved in meningioma cell mitosis (8-11). These studies have suggested that, althoughmembrane phosphatidylinositol, protein kinase C and calcium are associated with mitogenesis, adenylyl cyclase activity and CAMP production causes growth retardation (8). The mechanism by which intracellular production of CAMP negatively regulates meningioma proliferation has not been deciphered. However, Corresponding author: Dr. E. F. Adams, Neuroendokrinologisches Labor, Kopfklinikum, Schwabachanalage 6, 91054 Erlangen, Germany. Tel: 9131 854430; Fax: 9131 854436

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it is known that meningiomas secrete the cytokine, interleukin-6 (IL6), which may act in an autocrine / paracrine fashion to inhibit proliferative rate (12, 13). We present here further evidence in support of this hypothesis and that the effects of CAMP on meningioma growth may be mediated by increased IL6 secretion. Specifically, we have investigated the effect of substances able to increase intracellular CAMP levels (cholera toxin (CT), theophylline and 8-bromo-CAMP) on IL6 secretion and, in parallel, meningioma cell growth in-vitro. Additionally, growth rate was determined in the presence and absence of a neutralising antibody against IL6 in order to further elucidate the potential function of the cytokine in an autocrine or paracrine system. Materials and Methods Experiments were performed on 8 meningiomas (removed from 5 females and 3 males, age range 44-77 years; histological subtypes: 4 meningotheliomatous, 3 transitional and 1 fibroblastic). Surgically removed tissue was dispersed with collagenase, placed into culture, the cells grown to confluence and then passaged (passage 1 cells). Details of the culture procedure and medium used, including confirmation that the cultured cells retain their meningioma-like characteristics, have been described in full (1, 2). Equal aliquots of passage 1 cells (0.5 X 105) were distributed into 25 cm2 tissue culture flasks (Greiner, Frickenhausen, FRG) and allowed to attach and equilibrate for 24 hours. Culture medium was then removed and replaced with fresh medium containing varying dilutions of either CT, theophylline (Sigma Chemie, Deisenhofen, FRG) or 8-bromo-CAMP (Biologin, Bremen, Germany). Three cultures were used to test each variable, including controls, and medium was replenished each 2-3 days. Growth rates varied, depending on the tumour, with estimated doubling times of l-5 days. Cells were incubated for 4-6 days followed by collection of culture medium for assessment of IL6 content by ELISA adapted from IRMA technology (14). The number of cells in each flask was determined as described (1, 2) and IL6 secretion is expressed as ng or pg produced per lo6 cells during the time course of the experiment. The effect of a neutralising polyclonal antibody against IL6 (goat anti-IL6, National Institute for Biological Standards and Control, Potters Bar, U.K.) on meningioma proliferation was determined by assement of H3-thymidine uptake as previously described in detail (1, 2). Statistical significances were determined by unpaired t-tests using pooled estimates of error. Log transformation was used for the data depicted in Figure 1A and 2A, since some raw data variances differed significantly from the variance of the control group. Results Incubation of cultured human meningioma cells for 4 days with varying amounts of CT, a substance which directly activates adenylyl cyclase, resulted in significantly (P < 0.05) reduced cell growth (Figure 1, panel A) but markedly increased IL6 secretion by up to about 300 % (Figure 1, panel B). A similar observation was made by increasing intracellular CAMP levels with theophylline (lo mm01 / L) I a phosphodiesterase inhibitor. Theophylline inhibited meningioma cell growth by 26 % and stimulated IL6 secretion 4-fold (data not shown). Direct addition of 8-bromo-CAMP (25-100 Fmol / L) also significantly (P < 0.05) caused reduced cell growth with parallel stimulation of IL6 secretion (Figure 2). A polyclonal neutralising antibody against IL6 significantly (P < 0.05) stimulated H3-

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Interleukind,cAMP andHumanMeningiomas

Cholera toxin (ng

l325

I mL)

Fig. 1 Effect of varying doses of cholera toxin on (A) cell growth and (B) IL-6 secretion by cell cultures of a human menlngloma. Cultures were grown for 4 days after lnltially seedlng 0.5 X lo5 cells on day 0. Slmllar results were observed using cell cultures of 3 other menlnglomas. ** P c 0.01, l ** P < 0.001 v. control.

thymidine uptake by 4 meningiomas (Tumors A-D, Figure 3) in cell culture. The effect was variable ranging from 30 % (Tumor D) to 170 % (Tumours A and C) over control. The same polyclonal antibody abolished the inhibitory effects of 8-bromo-CAMP on H3-thymidine uptake (Figure 4). Discussion There is compelling evidence that in many tissues intracellular CAMP plays a role in controlling cellular proliferation although the precise effect is tissue-dependent. For example, in pituitary and thyroid tissues, increased levels of intracellular CAMP are associated with increased mitosis (15, 16), whereas CAMP induces inhibition of proliferation in pancreatic tumour cells (17). In meningiomas, increased activity of adenylyl cyclase induced by forskolin correlates with reduced rate of proliferation in-vitro, whereas inhibition of adenylyl cyclase by somatostatin resulted in increased mitosis (8). The present results confirm and extend these earlier findings. Thus, treatment of meningioma cells with CT, which directly activates adenylyl cyclase by ribosylating the G,o!subunit of the regulatory G-protein (15), resulted in markedly decreased proliferation in-vitro. Moreover, the proliferative rate was also reduced by indirectly and directly increasing intracellular CAMP Taken levels with theophylline and 8-bromo-CAMP, respectively. together, these findings strongly suggest that intracellular CAMP Additionally, is coupled to inhibition of mitosis in meningiomas.

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+J1.5 z B 1.0 6 .j

0.5

E 91 3

0

0

25 Gbromo-CAMP

50

100

(pm01

I

L)

Fig. 2 Effect of varying doses of b-bromo-CAMP on (A) cell growth and (B) IL-8 secretion by a menlngloma In culture. Cultures w$re Incubated for 8 days Cells per flask. Similar and were Initially seeded at a density of 0.5 X 10 results were found with 2 other menlngiomas. * P < 0.05, l ** P c 0.001 v. control.

NISAILB

NIS

AIL6

Flg. 3 Stimulatory effect of an antibody against IL-8 (AIL6, 1500 dllutlon) on H3 -thymidine uptake by cell cultures of 4 dlfferent menlnglomas (tumours A-D). NIS = non-immune serum (controls). * P < 0.05, ** P < 0.01, *** P 4 0.001 v. control.

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l

2.5 P% g a+( 07 c 0 dF E x PE 7 2

+

2.0. 1.5

l

.

1.0, 0.5

1 O-

Control

AIL6

6-bromoCAMP

AIL6 6-br*omoCAMP

Fig. 4 Effect of an antibody against IL-6(AIL6,1:500 dilution) and 6-brOmOCAMP (100 gmol I L), alone and in combination, on ii3 -thymfdine uptake by Cell cultures of a human menlngloma. Similar resUltS were obtained In one other experiment. * P < 0.05, ** P < 0.01, l ** P c 0.001 v. control; + P c 0.01 v. 6-bromo-CAMP.

the present results extend this concept and indicate that the CAMPinduced inhibition of meningioma cell proliferation may be, at least in part, because of increased secretion of IL6. This is an important issue to address since there is growing evidence that human meningiomas produce a number of peptide cytokines and growth-factors which act in an autocrine / paracrine manner to modulate further cell proliferation (2, 13, 18), and it is conceivable that their secretion is controlled by the CAMP second messenger system. It is now well established that one of these autocrine factors is IL6 (13, / 181 amino-acid polypeptide originally shown to be 19) I a 180 secreted by cells of the immune system and to mediate immune and acute phase responses (20). IL6 has been shown to be secreted by many other cell types and to have a wide variety of effects and several studies provide strong evidence for an autocrine or paracrine role with both of IL6 in neoplastic cell growth, inhibitory and stimulatory effects on proliferation described, depending on the type of tumour (21-23). For meningiomas, there is some controversey concerning the role of IL6 in cell proliferation. Addition of exogenous IL6 to cultured human meningioma cells has been conflictingly reported to stimulate (19) or inhibit mitosis however, that examining the effects of (12). It is possible, exogenously added IL6 may not be a precise enough method for determining its role in cell proliferation since endogenous IL6 is also being produced, including in control cultures. Indeed, it has previously been suggested that the observed effects of exogenous IL6 on meningioma growth may be a reflection of a pharmacological rather than physiological response (19). For these reasons, a better approach is to decipher the effects of endogenously produced IL6 using neutralising antibodies and correlations of rate of growth with amount of IL6 produced. Using the latter method, our own previous findings have suggested that the endogenously produced IL6 exerts an autocrine inhibitory influence (13). This conclusion was based on negatively

the

observation

that

proliferative

rate

in-vitro

is

correlated with amount of IL6 secreted. The present results showing that neutralisation of the endogenously secreted IL6 with a polyclonal antibody leads to increased proliferative rate are supportive of the concept of an autocrine inhibitory loop. Furthermore, all the substances which increased intracellular CAMP

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levels (CT, theophylline and 8-bromo-CAMP) not only reduced mitosis further emphasising an stimulated IL6 secretion, but also association between the two. In this respect, it is worth noting that somatostatin is able to inhibit the secretion of IL6 by meningioma cells (12), which may explain its mitogenic effect. Conversely, bioactive peptides such as vasoactive intestinal peptide and ILlB markedly increase IL6 secretion by meningiomas and tend to have inhibitory effects on cell growth (12). All of these studies therefore show an inverse relationship between amount of IL6 secretion and meningioma cell growth, giving credence to the concept that the cytokine acts as an autocrine or paracrine inhibitory factor for mitosis in this type of tumour. The degree to which this secreted IL6 influences growth may differ between meningiomas, as evidenced by the varying effect of the neutralising antibody (see Figure 3) and the wide range of secretory activity exhibited by different tumours (13, 19). Additionally, the maximal observed stimulatory effect of CT on the 4 meningiomas studied ranged from 2.7 to 5.4-fold (data not shown). To date, we have not observed any obvious correlation of these variable responses with histological subtype, although a much larger series of tumours will be required to fully investigate this. It remains unknown whether these effects of CAMP and IL6 are tumour specific or if similar phenomena also occur in normal meningial cells. Nevertheless, the identification of CAMP and IL6 as potential inhibitory factors for meningioma proliferation may prove to be the basis for developing novel modes of therapy, perhaps via drugs able to increase intracellular adenylyl cyclase activity, such as dopamine Dl-receptor agonists (24), or factors able to mimic the growth inhibitory effects of IL6. References 1. E.F. ADAMS, U.M.H. SCHRELL, R. FAHLBUSCH and P. THIERAUF, J. Neurosurg. 73 750-755 (1990). 2. E.F. ADAMS, T. TODO, U.M.H. SCHRELL, P. THIERAUF, M.C. WHITE M. and R. FAHLBUSCH, Int. J. Cancer 49 398-402 (1991). 3. U.M.H. SCHRELL, E.F. ADAMS, R. FAHLBUSCH, R GREB, G.JIRIKOWSKI, R. PRIOR and F.J. RAMALHO-ORTIGAO, J. Neurosurg. 73 743-749 (1990). 4. M. MAXWELL, T. GALANOPOULOS, T. HEDLEY-WHYTE, P.M. BLACK, and H.N. ANTONIADES, Int. J. Cancer 46 16-21 (1990). 5. A.S. WEISMAN, J.G. VILLEMURE and P-A. KELLEY, Cancer Res. 46 2545-2550 (1987). 6. J.C. REUBI, U. HORISBERGER, W. LANG, J.W. KOPER, R. BRAAKMAN and S.W.J. LAMBERTS, Am. J. Pathol. 134 337-344 (1989). 7. S.M. GRUNBERG, A.M. DANIELS, H. MUENSCH, J.R. DANIELS, L. BERNSTEIN, V. KORTES and M.H. J. WEISS, Neurosurg. 66 405-408 (1987). 8. J.W. KOPER, R. MARKSTEIN, C. KOHLER, D.J. KWEKKEBOOM, C.J.J. AVEZAAT, S.W.J. LAMBERTS and J.C. REUBI, J. Clin. Endocrinol. Metab. 74 543-547 (1992). 9. R.L. JENSEN, T.C. ORIGITANO, Y.S. LEE, M. WEBER and R. WURSTER, Neurosurgery 36 365-373 (1995). 10. T. TODO and R. FAHLBUSCH, J. Neurosurg. 80 890-896 (1994). 11. T. TODO and R. FAHLBUSCH, Acta Neurochir. 131 282-288 (1994). 12. E.F. ADAMS, T. TODO, B. RAFFERTY, J. MOWER and R. FAHLBUSCH, J. Endocrinol. 135 SUDDl. P86 (1992).

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13. T. TODO, E.F. ADAMS, B. RAFFERTY, R. FAHLBUSCH, T. DINGERMANN and H. WERNER, J. Neurosurg. 81 394-401 (1994). 14. B. RAFFERTY, J.A. MOWER, Y.S. TAKTAK and S. POOLE, Immunol. Methods 144 69-76 (1991). 15. F.H. BURTON, K.W. HASEL, F.E. BLOOM and J.G. SUTCLIFFE, Nature 350 74-77 (1991). 16. P. ROGER, M. TATON, J. VAN SANDE and J.E. DUMONT, J. Clin. Endocrinol. Metab. 66 1158-1165 (1988). 17. J. ISHIZUKA, R.D. BEAUCHAMP, B.M. EVERS, C.M. TOWNSEND and J.C. THOMPSON, Biochem. Biophys. Res. Commun. 185 577-581 (1992). 18. J.A. TAKAHASHI, H. MORI, M. FUKUMOTO, K. IGARASHI, M. JAYE, Y. ODA, H. KIKUCHI and M. HATANAKA, Proc. Natl. Acad. Sci. USA 87 5710-5714 (1990). 19. E. BOYLE-WALSH, I.A. HASHIM, V. SPEIRS, W.D. FRASER and M.C WHITE, Neuroscience Lett. 170 129-132 (1994). 20. J. LE and J.VILCEK, Lab. Invest. 61 588-602 (1989). 21. E.F. ADAMS, B. RAFFERTY and M.C. WHITE, Int. J. Cancer 49 118121 (1991). 22. J.K. HITZLER, H. MARTINEZ-VALDEZ, D.B. BERGSAGEL, M.D. MINDEN and H.A. MESSNER, Blood 78 1996-2004 (1991). 23. J. NEMUNAITIS, D.F. ANDREWS, D.Y. MOCHIZUKI, M.B. LILLY and J.W. SINGER Blood 74 1929-1935 (1989). 24. U.M.H. SCHRELL, R. FAHLBUSCH, E.F. ADAMS, P. NOMIKOS and M. REIF, J. Clin. Endocrinol. Metab. 71 1669-1671 (1990).