Grovith factors fail to protect rat oligodendrocytes against humoral injury in vitro

ELSEVIER

Neumscience Letters 183 (1995) 75-78

NEUgOSCIHCE L~HS

Growth factors fail to protect rat oligodendrocytes against humoral injury in vitro N e i l J. S c o l d i n g a,*, D. Alastair C o m p s t o n b aUniw'rsity of Cambridge Neurology unit, Addenbrooke's HospitaL Hills Road, Cambridge CB2 2QQ, UK bMRC Cambridge Centrefor Brain Repair, University Forvie Site, Robinson Way, Cambridge CB2 2SR, UK Received 28 September 1994; revised version received 28 Oetober 1994; aecepted 28 Oetober 1994

Abstract

CNS growth factors pmtect neurons and glia against a wide variety of insults in vitro and in vivo by mechanisms which include buffering toxic rises in inlracellular calcium. Cytosolic calcium elevation also plays a key mie in complement injury, but the possibility that growth factors protect against antibody-mediated complement attack has not hitherto been addressed. In multiple sclerosis, antibodies and complement appear to contribute to the selective targeting and damage of oligodendrocytes and myelin. Here we have investigated the possibility that growth factors active in oligodendrocyte development and differentiation might pmtect these cells against injury mediated by antibody and complement in vitro. None was found to be protective.

Keywords:Gmwth factor.,;;Complement; Oligodendrocytes; Multiple sclerosis; Antibody; Protection

Growth factors acting in the central nervous system were initially characterised in relation to their roles in neurite extension and c,eU maintenance in vitro; their importance in precursor proliferation and maturation and in tropic signalling subsequently emerged [2,11,28]. More recently, attention has focused on the involvement of growth factors in oeil death and injury. Natural cell death or apoptosis, an active and ubiquitous process vital for the development and maintenance of tissues, may be an inevitable and universal consequence of growth factor deprivation [3,20]. In addition to this physiological role, the protective effects of growth~factors in vivo and in vitro against pathological neural (and other) cell damage by soluble mediators of injury are increasingly recognised [15]. CNS growth factors protect neural cells against a wide range of injurious agents, including glutamate and other excitotoxins, ischaemia, glucose deprivation, 1-methyl-4pbenyl-l,2,3,6-tetrahydropyridine (MPTP), free oxygen radicals, cyanide, iron and fl-amyloid peptide [15]. Modulation and buffering of rises in intracellular calcium within target cells, whicb play a universal role in toxic * Corresponding author, Tel.: +44 223 217 091; Fax: +44 223 336 941.

cell death [23], appear to represent the common mechanism underlying these diverse protective effects [15]: calcium-buffering proteins are induced by growth factors [7,15], and elevated expression of calbindin or other calcium-buffering proteins correlates with resistance to excitotoxicity [14]. Calcium influx into target cells also plays a key role in complement-induced oeil damage [16]; this has been studied in particular in oligodendrocytes [9,25,29], and raises the possibility that CNS growth factors might protect oligodendrocytes against complement attack. Interest in oligodendrocyte cell biology and injury derives not least from its application to the clinical problem of multiple sclerosis, where immunological damage targets the oligodendrocyte-myelin unit [26]. Although the precise mechanisms of injury remain to be defined, and Tlymphocytes and their products undoubtedly play a pivotal role [22,26], there is substantial evidence for the additional involvement of humoral factors, including complement and antibodies directed against oligodendrocyte-myelin antigens [26,27]. We have previously studied antibody and complement mediated attack of cultured neonatal rat oligodendrocytes in order to explore the oligodendrocyte response to injury in vitro and, by extrapolation, in demyelinating disease [9,25]. Here, we extend

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N.J Scolding, D.A. Compston / Neuroscience Letters 183 (1995) 75-78

the use of this model system to investigate strategies for protecting oligodendrocytes; specifically, we have systematically studied the potential of a wide variety of growth factors to protect oligodendrocytes against complement and antibody attack. Rat oligodendrocytes are uniquely susceptible to the lytic effects of syngeneic complement attack in the absence of antibody, a phenomenon which results from a combination of their ability directly to activate CI, the first component of the classical complement pathway, and a relative deficiency of one or more surface complement regulatory proteins [24]. Complement injury was therefore investigated by exposing oligodendrocytes (prepared and cultured from 7-day-old rat optic nerve as previously described [21]) on coverslips to serum for 40 min at 37°C; cell damage was assessed morphologically and after staining with propidium iodide (PI), which is normaUy excluded by healthy cells but penetrates damaged cell membranes, intercalates with nuclear DNA and becornes fluorescent. After injury, 100-200 oligodendrocytes per coverslip were counted and the proportion exhibiting nuclear PI fluorescence was used to indicate cell damage. Normal rat serum added at 1:8 dilution to cultures after 5-6 days in vitro rendered 98-100% oligodendrocytes PI permeable after 40 min at 37°C (Fig. 1) as previously

described [24]. Pre-incubation of oligodendrocytes for 24 or 48 h with ciliary neurotrophic factor (CNTF; 10 ng/ml) failed to protect against antibody-independent complement attack, as previously reported [12]. Pre-incubation with platelet-derived growth factor (PDGF; 10 ng/ml), insulin-like growth factor-1 (IGF-I; 100ng/ml), transforming growth factor-fl (TGF-fl; 5 ng/ml), epidermal growth factor (EGF; 10ng/ml), nerve growth factor (NGF; 50 ng/ml), or neurotrophin-3 (NT-3; 5 ng/ml), ail similarly failed to protect cells against complement attack; 98-100%} oligodendrocytes continued to be lysed on exposure to 1:8 normal rat serum. (PDGF, IGF-1, EGF, CNTF and basic fibroblast growth factor (bFGF) were supplied by agreement with Amgen Inc. and, with TGF-fl, also obtained commercially from R & D Systems Ltd, Minneapolis. NGF was purchased from Boehringer Mannheim, and NT-3 obtained by arrangement with Genentech.) When mature oligodendrocytes were pre-incubated with 10 ng/ml basic fibroblast growth factor (bFGF), a significant proportion of cells were observed to die over the next 24 h. bFGF did not induce death of astrocytes or immature oligodendrocytes, and no discernible effect on mature oligodendrocytes was observed during preincubation with any other growth factor (proliferation was not specifically investigated). This unexpected phenome-

Fig. 1. Oligodendrocytes exposed to complement attack. Oligodendrocytes were prepared and cultured from 7-day-old rat optic nerve dissected, digeste& triturated and plated onto poly-n-lysine coated glass coverslips as previously described [21]. After culture at 37°C in CO 2 using modified Dulbeeeo's medium with Bottenstein-Sato addifives [6] and containing only 0.5% foetal bovine serum, bipotential glial progenitors isolated in this way differentiate into oligodendrocytes, and after 5-6 days, at which rime cells were used for the current studies, these cultures routinely contain 85% maturc oligodendrocytes. Blood obtained by cardiac puncture from ether anaesthetised adult Wistar rats was left to clot at 0°C for 30 min and centrifuged (1000 x g for 30 min) to obtain serum. Cells on coverslips were exposed to serum for 40 min at 37°C and cell damage was then assessed morphologically and after staining with propidium iodide (PI), which is normally excluded by healthy cells but penetrates damaged oeil membranes, intercalates with nuclear DNA and becomes fluorescent. Healthy mature oligodendrocytes (a), after exposure to activated complement, lose their cell proeesses and develop swollen, granular cytoplasm and dense nuclei Ço). The damaged oeil membrane allows propidium iodide to leak into the oeil; intercalation with nuclear DNA then leads to fluorescence ((c), same field as (a)) which may be visualised using rhodarnine optics. (x400).

N,J Scolding, D.A. Compston / Neuroscience Letters 183 (1995) 75-78

non will be described elsewhere (manuscript in preparation). Oligodendrocytes surviving bFGF exposure were no less sensitive to complement attack, and cells incubated for 24 h with lower concentrations of bFGF (2.5 ng/ml and 5.0 ng/ml) were not protected against complement attack. In order to exclude the possibility that partial oligodendrocyte protection had been overwhelmed by the supra-lethal doses of complement used above, complement attack at carefully controlled lower doses was investigated. The dose-response relationship for antibodyindependent complement attack varied slightly between different batches of rat serum; the effect was also labile at room temperature and very susceptible to repeated freezethaw cycles [24], and we therefore studied antibodymediated complement attack for this purpose. Dose titration experiments (not shown) established that exposure of 5-6-day-old oligodendrocytes to 1:4 anti-galactocerebroside antibody (Ranscht mAb supernatant, 1:4 in culture medium; 40 min at 37c'C) with 1:80 rabbit serum reliably and consistently caused sub-total (90-98%) lysis of oligodendrocyte populations; at this serum concentration, no antibody-independent lytic effect was apparent. Pre-incubation of oeils for 24 or 48 h with either PDGF, bFGF, IGF-1, CNTF, EGF, NGF, NT-3, or TGF-fl (concentrations as above) did not lower the proportion of oligodendrocytes lysed following antibody-mediated complement attack. These results clearly demonstrate that growth factors do not protect oligodendrocytes against supra-lethal or Iower dose antibody-independent or antibody-mediated complement attack. A comprehensive range of growth factors was tested, for each of which oligodendrocytes have been shown to possess functional receptors, either directly (using radio-labelling, immunostaining or molecular probing [ 10,18], although rat oligodendrocytes do hOt express high affinity NGF receptors), or indirectly by demonstrating biological effects; oligodendrocytes or their progenitors divide in response to bFGF, PDGF, IGF1, NGF, and NT-3 [1,4,19], and migrate towards PDGF [2], while IGF-I, EGF and TGF-fl induce differentiation [13,19]. CNTF, IGF-1, NT-3 and PDGF prevent apoptosis [4,5,12]; CNTF also protects oligodendrocytes against injury by tumour necrosis factor [12]. In patients with multiple sclerosis, antibodies directed against a variety of oligodendrocyte and myelin antigens are present in serum anti spinal fluid [17,26]. Blood brain barrier breakdown is an early event in the development of inflammatory demyelinating lesions (sec Ref. [26]), exposing oligodendrocytes to complement in addition to these antibodies, and a variety of complement activation products bas been demanstrated immunohistochemically within and around active lesions [8,25,26]. It is therefore likely that antibody and complement contribute to oligodendrocyte-myelin in.jury in multiple sclerosis. Agents which protect oligodendrocytes against humoral attack

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might usefully contribute to strategies designed to limit damage and encourage repair in multiple sclerosis, but the results here described exclude such a role for CNS growth factors. More generally, these findings may have a broader relevance for cell injury and protection. Growth factors protect neural cells against a wide range of toxic and injurious agents, predominantly by promoting calcium homeostasis. The importance of intracellular calcium transients in mediating complement injury is well established. Nevertheless, our results indicate that complement attack cannot be added to the list of cell injury mechanisms against which growth factors provide protection. We thank Genentech for the provision of neurotrophin-3 and Amgen Inc. for the provision of IGF- 1, bFGF, PDGF and CNTF. NJS is an MRC Clinician Scientist Fellow. [1] Althaus, H.H., Kloppner, S., Schmidt-Schultz, T. and Schwartz, P., Nerve growth factor induces proliferation and enhances fiber regeneration in oligodendrocytes isolated from adult pig brain, Neurosci. Lett., 135 (1992) 219-223. [2] Armstrong, R.C., Harvath, L. and Dubois-Dalcq, M.E., Type 1 astrocytes and oligodendrocyte-type 2 astrocyte progenitors migrate towards distinct molecules, J. Neurosci. Res., 27 (1990) 400-407. [3] Barde, Y.-A., Trophic factors and neuronal survival, Neuron, 2 (1989) 1525-1534. [4] Barres, B.A., Raff, M.C., Gaese, F., Bartke, I., Deschant, G. and Barde, Y.-A., A crucial role for neurotrophin-3 in oligodendrocyte development, Nature, 367 (1994) 371-375. [5] Barres, B.A., Schmid, R., Sendnter, M. and Raff, M.C., Multiple extracellular signais are required for long-term oligodendrocyte survival, Development, 118 (1993) 283-295. [6] Bottenstein, J.E. and Sato, G.H., Growth of a rat neuroblastoma line in serum free supplemented mcdium, Proc. Natl. Acad. Sci. USA, 76 (1979) 514-517. [7] Collazo, D., Takahashi, H. and McKay, R.D.G., Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus, Neuron, 9 (1992) 643-656. [8] Compston, D.A.S., Morgan, B.P. and Campbell, A.K. et al., lmmunocytochemical Iocalisation of the terminal complement complex in multiple sclerosis, Neuropathol. Appl. Neurobiol., 79 (1989) 78-85. [9] Compston, D.A.S., Scolding, N.J., Wren, D. and Noble, M., The pathogenesis of demyelinating disease: insights from cell biology, Trends Neurosci., 14 (1991) 175-182. [10] Hart, I.K., Richardson, W.D., Heldin, C-H., Westermark, B. and Raff, M.C., PDGF receptors on cells of the oligodendrocyte-type 2 astrocyte lineage, Development, 105 (1989) 595-603. [11] Lampson, L.A., Mechanisms and control of migration in the adult CNS, Brain Pathol., 4 (1994) 123-148. [12] Louis, J.-C., Magal, E., Takayama, S. and Varon, S., CNTF protection of oligodendrocytes against natural and tumour necrosis factor-induced death, Science, 259 (1993) 689-692. [13] Mackinnon, R.D., Piras, G., ida, J.A. and Dubois-Dalcq, M., A role for transforming growth factor beta in oligodendrocyte differentiation, J. Cell Biol., 121 (1993) 11397-11407. [14] Mattson, M.P., Rychlik, B., Chu, C. and Christakos, S., Evidence for calcium-redueing and excito-protective roles for the calciumbinding protein (calbindin D-28k) in cultured hippocampal neurons, Neuron, 6 (1991) 41-51.

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[15] Mattson, M., Cheng, B. and Smith-Swintosky, L., Neurotrophic factor mediated protection from excitotoxicity and disturbances in calcium and free radical metabolism, Semin. Neurosci., 5 (1993) 295-307. [16] Morgan, B.P., Effeets of the membrane attack complex of complement on nucleated oeils, Curr. Top. Microbiol. Immunol., 178 (1992) 115-140. [17] Olsson, T., Immunology of multiple sclerosis, Curr. Opin. Neurol. Neurosurg., 5 (1992) 195-202. [18] Otero, G.C. and Merriil, J.E., Cytokine reeeptors on glial cells, Glia, 11 (1994) 117-128. [19] Pfeiffer, S.E., Warrington, A.E. and Bansal, R., The oligodendrocyte and its many cellular proeesses, Trends Cell Biol., 3 (1993) 191-197. [20] Raff, M.C., Social controls on cell survival and death: an extreme view, Nature, 356 (1992) 397--400. [21] Raff, M.C., Miller, R.H. and Noble, M., A glial progenitor that develops in vitro into an astroeyte or an oligodendrocyte depending on culture medium, Nature, 303 (1986) 390-396. [22] Raine, C.S., Mulaple selerosis: a pivotal role for the T oeil in lesion deveiopment, Neuropathol. Appl. Neurobiol., 17 (1991) 265-274. [23] Schanne, F.A.X., Cane, A.B., Young, E.E. and Farber, J.L., Cal-

[24]

[25]

[26]

[27]

[28] [29]

cium dependence of toxic oeil death: a final common pathway, Science, 206 (1979) 700-702. Scoiding, N.J., Morgan, B.P., Houston, A., CampbeU, A.K., Linington, C. and Compston, D.A.S., Normal rat serum eytotoxicity against syngeneic oligodendrocytes: complement activation and attack in the absence of anti-myelin antibodies, J. Neurol. Sci., 89 (1989) 289-300. Scolding, N.J., Morgan, B.P., Houston, A., Linington, C., Campbell, A.K. and Compston, D.A.S., Vesicular removal by oligodendrocytes of membrane attack complexes formed by complement, Nature, 339 (1989) 620--622. Scolding, N.J., Zajicek, J.P., Wood, N. and Compston, D.A.S., The pathogenesis of demyelinating disease, Prog. Neurobiol., 43 (1994) 143-173. Shin, M.L. and Koski, C.L., The complement system in demyelination. In R.E. Martenson (FA.), Myelin: Biology and Chemistry, CRC Press, Boca Raton, FL, 1992, pp. 801-832. Thoenen, H., The chahging scene of neurotrophic factors, Trends Neurosci., 14 (1991) 165-170. Wood, A., Wing, M.G., Benham C.D. and Compston, D.A.S., Specific induction of intracellular calcium oscillations by complement membrane attack on oligodendroglia, J. Neurosci., 13 (1993) 3319-3332.