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Nerve growth factor in medium conditioned by embryonic chicken heart cells
Int. J. Devl. Neuroscience. Vol. 4, No. I. pp. 41~19, 1986.
0736-5748186 $03.(10+0.00 Pergamon Press Ltd. © 1986 ISDN
Printed in Great Britain.
NERVE
G R O W T H F A C T O R IN M E D I U M C O N D I T I O N E D E M B R Y O N I C C H I C K E N H E A R T CELLS
BY
GUNII.I.A NORRGRI{N*'~" a n d TED EBENDAI.* :Uppsala University. Department of Zoology, Box 561. S-751 22 Uppsala, Sweden, and +BioCell Laboratories HB, Box 17, Uppsala I. Sweden ( A ccepled 31 A tlgust 1985 )
A b s t r a c t - - T h e present report demonstrates that embryonic chicken heart cells in culture release different nerve growth promoting factors to their culture medium, one which is biologically and immunologically similar to mouse gland [3NGF. Serum-free heart cell conditioned medium thus promoted neurite outgrowth from sympathetic and ciliary ganglia and supported survival of dissociated ciliary neurons. The addition of affinity purified antibodies against mouse [3NGF does substantially but not completely inhibit the fibre outgrowth from sympathetic ganglia, but does not to any extent diminish the effects on the parasympathetic neurons. The chicken N G F recovered from polyacrylamide gels after electrophoresis greatly enhanced sympathetic fibre outgrowth, an activity completely suppressive by anti-[3NGF antibodies. We conclude that a chicken N G F is being produced by the embryonic heart cells in culture, and that this factor may be produced also in the embryo to fulfill a role in heart innervation. K e y words: Cell culture, Conditioned medium, Chicken embryo, Ciliary neurons, Sympathetic ganglion, Nerve growth factor.
Nerve growth factor ([3NGF) is a polypeptide which plays a fundamental role in the development and maintenance of adrenergic and sensory neurons, s'2~4° Several non-neuronal cells (both transformed and non-transformed) have been found to synthesize and secrete a factor which is biologically and immunologically similar to the well characterized mouse submandibular gland N G F . 4"6"lS"lg"22-2~''2x'3q~-32"36"42 In addition, media conditioned by cells in culture have been shown to contain also other factors, 3'5"2° a m o n g them one maintaining parasympathetic ciliary neurons. 17,~ We demonstrated recently that embryonic chicken heart cells cultured under serum-free conditions released nerve growth promoting substances for both sympathetic and parasympathetic ciliary neurons. 2'~ An early report by Young et al. 43 indicated that primary chicken e m b r y o fibroblasts produced a NGF-like factor. On the other hand, Helfand and coworkers ~v have reported that medium conditioned over chicken e m b r y o heart cells ( H C M ) did not contain any NGF. In the study presented here, we have characterized the apparent NGF-like activity and separated it from the activity affecting ciliary neurons. It is demonstrated that the chicken heart cells in culture produce a factor immunologically and biologically similar to 13NGF from the mouse submandibular gland. MATERIALS AND METHODS Materials
Unless otherwise stated, all material for cell culture was obtained from Flow Laboratories and chemicals and reagents from Sigma. Equipment for c h r o m a t o g r a p h y and electrophoresis came from Bio-Rad and Pharmacia. Mouse [3NGF was prepared as described ~~2 and radiolabelling was performed with Na~eSl using chloramine-T to a specific activity of about 30,000 cpm/ng. Rabbit anti-(mouse) 13NGF antiserum was prepared in rabbits by multiple intradermal injections of purified I3NGF and the antibodies were affinity purified. 1~,12,3~ Cell culture and conditioned media
Primary chicken e m b r y o cells derived from hearts of 9-day-old embryos (White Leghorn strain) were prepared and cultured as described previously. > Cells were grown on Cytodex ~ 3 Address correspondence to: Dr. Ted Ebendal, D e p a r t m e n t of Zoology, Uppsala Unisersitv. Box 561. S-751 22 Uppsala, Sweden. ,4bbreviations: H ( ' M , heart conditioned medium: NGF, nerve growth factor.
41
42
(;. Norrgren rind T. Ebendal
microcarriers (Pharmacia) in Dulbecco's modification of Eagle's Medium (DME) and Ham's F 10 supplemented with non-essential amino acids, 2 mM glutamine, 1 mg/ml human ~erum albumin (a gift from Dr. J. Curling, Pharmacia), 10 i~g/ml fibronectin (a gift from E Ekrc, KABI), 25 ~g/ml transferrin (P. Ekre, KABI) and I i~g/ml insulin ( N O V O ) for 3 days before the conditioned medium was collected. Media were harvested from 600 ml microcarricr cultures, ~<' concentrated 80-fold and dialyzed against BME (Eagle's Basal Medium) by pressure dialysis over Pellicon P T G C (Millipore) membranes (cut-off tool. wt 10,000), and thereafter sterile filtered through Millipore G V filters.
Bioassay Nine-day-old chicken embryo ciliary and sympathetic ganglia or dissociated neurons were used to estimate the nerve growth activities present in the concentrated heart cell conditioned medium (HCM). Details of this technique have been reported previously. 9-13 For titration of nerve growth activity the test samples were diluted with B M E (series of two-fold dilutions) and added on top of the collagen gels (giving a further two-fold dilution). Ganglion cultures were examined after 2 days and the number of outgrowing neurites scored using a scale from zero (no fibres) to 5 (very dense fibre halo), or as biological units (1 BU being present per ml at optimum fibre density). Dissociated ciliary neurons were initially counted and the number of surviving cells determined after 2 days. To test for NGF-specific fibre response, test samples were incubated overnight (4°C) with 2 I~g/ml of affinity purified anti-13NGF antibodies (10-fold higher concentration than needed to block 1 BU of activity of the homologous antigen) and the samples were added to the nerve cell cultures.
Characterization of nerve growth activity Molecular characteristics were evaluated by treatment with various enzymes and with heat. Concentrated HCM was heated in a waterbath for 30 rain to 60°C and then cooled on ice before analysis for biological activity. In another experiment HCM was incubated at 37°C for 2 hr with 100 ~g/ml of trypsin. Thereafter soybean trypsin inhibitor was added to a final concentration of 500 ~g/ml before being tested in bioassay. Hyaluronidase was dissolved in 10 mM sodium phosphate buffer, 0.15 M NaCI, pH 5.1, at 100 units/ml and added to HCM at a final concentration of 5 units/ml. This mixture was incubated for 6 hr at 37°C. Concentrated HCM was also brought to pH 2.5 with 1 M HCI. After 1 hr at room temperature samples were neutralized with 1 M NaOH.
Electrophoresis For preparative electrophoresis, polyacrylamide gradient gels (2-16%, Pharmacia) were used in a 0.09 M Tris, 0.08 M boric acid buffer (pH 8.4). Concentrated HCM (0.5 ml) was loaded onto the gel and the sample run for approximately 300 Vhr. Thereafter elution of proteins was performed by slicing the gel into 5 mm wide strips which were homogenized in 5(X) ILl of 0.02 M TrisHCI buffer (pH 8) and agitated overnight (4°C). Supernatants obtained after centrifugation were dialyzed for 3 days against BME before being tested in the bioassay or in a radioimmunoassay.
Radioimmunoassay A double antibody solid phase assay was used with the second antibody (anti-rabbit IgG, BioCell Lab. HB) covalently bound to CNBr-activated agarose particles. One hundred ill of affinity purified antibodies (0.1 I,tg/mi) against mouse [3NGF and I(XI ill of test samples or [3NGF standard (0 to 100 ng) in phosphate buffered saline (PBS, pH 7.6, 0.5% Tween) were incubated for 16 to 20 hr at room temperature. Then 1 ng of ~251-[3NGF in 100 p,I of the PBS buffer were added and incubation continued for another 16 to 20 hr. Finally 2 ml of the second antibody suspension were added. After 30 rain (20°C) the mixture was centrifuged and the radioactivity in the immunoprecipitate measured in a LKB gamma counter. Non-specific binding, determined by incubation in the presence of excess unlabelled [BNGF (16% of the total binding) was subtracted. RESULTS Medium conditioned over chicken embryo heart cells and then concentrated 80-fold was biologically active in stimulating the survival of dissociated ciliary neurons (Fig. 1). The response
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Fig. I. Survival of dissociated ciliary neurons as a function of concentration of the serum-free heart cell conditioned medium. The fraction of neurons remaining after 2 days in a collagen matrix (100 gl) is shown, Eight different samples (100 i,tl) were added to the matrices (final dilution in the culture of the 80 × HCM is given) and the results are stated as mean ± S.E.M. To test the influence of anti-lSNGF antibodies four samples were incubated with the affinity purified immunoglobulin (Ig), 2 ~g/ml, 4°C, before being added to cultures. A dose-dependent relationship between the concentration and survival is seen with no influence from the antibodies.
is clearly dose-dependent, and not inhibited by antibodies to nerve growth factor (Fig. 1). Neurite outgrowth from ciliary (Fig. 2) and sympathetic (Fig. 3) ganglia were also stimulated. The addition of affinity purified antibodies to 13NGF had different effects on the neurite outgrowths. No inhibition was seen in the ciliary outgrowth (Fig. 2). For the sympathetic ganglia a marked inhibition in outgrowth was found (Fig. 3). The anti-13NGF antibodies were present at a concentration 10 times higher than required for total inhibition of 1 BU of mouse ~NGF, and the addition of even higher amounts did not further suppress the residual sympathetic fibre outgrowth evoked by the concentrated HCM. Thus, 600 ml medium conditioned by the heart cells (concentrated to 8 ml) was estimated to carry an anti-NGF suppressable biological activity equivalent to about 40-60 BU of NGF. Under the assumption that 1 BU equals 10 ng of [3NGF protein '2a" this corresponds to 0.4-0.6 I,tg of the active substance (in 16(1 mg total protein). Addition of trypsin to HCM resulted in loss of these biological activities whereas no inactivation occurred after heating to 60°C, repeated freeze-thawing, exposure to acid (pH 2.5), 6 M urea or hyaluronidase. Medium concentrated without prior conditioning (to serve as control) totally htcked the growth-promoting effects reported here. To characterize the NGF-like activity further, concentrated heart cell conditioned medium was electrophorezed on 2-16% gradient polyacrylamide gels under native conditions. Active material migrated to the anode at pH 8.4 and eluates from the four top slices (high molecular weight region) of the gel gave high recoveries of NGF-like activity (Fig. 4), but only 5% of applied protein. No activity stimulating survival or neurite outgrowth in ciliary neurons were recovered from the polyacrylamide gels. Figure 5a shows the typical NGF-like response in a sympathetic ganglion to the eluated material, an activity completely blocked by antibodies to mouse [3NGF (Figs 4 and 5b). Staining of parallel gels with Coomassie Brilliant Blue revealed no distinct protein bands in the upper region of the gel but showed diffusely smeared materials, probably lipoproteins. Immunoblots incubated with anti-NGF antibodies visualized a broad zone of NGF-like reactivity corresponding to the slices from which N G F activity was recorded (not shown). The amount of NGF-like material eluted from the polyacrylamide gels was determined in a double antibody solid phase radio-immunoassay. The values obtained in the radioimmunoassay correlate well with the amount of NGF-like activity estimated from the bioassay experiments. Thus, 35 and 59 ng/ml were determined by RIA in two independent experiments. Corresponding
44
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Fig. 2. Fibre growth density scored in whole ciliary ganglia in culture. Conditions otherwise as in Fig, 1. In this case too the anti-~NGF antibodies fail to suppress the response in the ciliary neurons.
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Fig. 3. Density of sympathetic fibre outgrowth in response to × 8 0 concentrated heart cell conditioned medium. The assay is based on blind scoring of the fibre halo around whole sympathetic ganglia in a collagen matrix. Conditions otherwise as stated in Fig. t. In contrast to the ciliary fibre outgrowth (Fig. 2), the sympathetic fibre outgrowth is reduced but not completely stopped by the anti-,BNGF antibodies.
data from bioassay were 40 and 60 ng/ml (4 and 6 BU). After correction for the 4-fold dilution introduced during slicing of the gel, this implies that 1.3-1.9 p,g of chicken NGF protein are present in 600 mi of conditioned medium (thus 2-4 times the amount estimated from the crude HCM). DISCUSSION This report characterizes NGF-like material in serum-free medium conditioned for 3 days by embryonic chicken heart cells in culture, and distinguishes this material from an activity (or activities) stimulating fibre outgrowth and neuron survival in the parasympathetic ciliary
N G F in chicken h e a r t c o n d i t i o n e d m e d i u m
Fig. 5. (a) Sympathetic ganglion responding to material eluted from a polyacrylamide gel (cf. Fig. 4). Micrograph taken after 2 days in culture. Darkfield. (b) The same as (a) but with the sample incubated with anti-13NGF immunoglobulin (2 p,g/ml) before bioassay. Note total reduction in fibre outgrowth. Scale b a r - 0.5 ram.
45
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Fig. 4. Sympathetic fibre growth response to eluates from polyacrylamide ( P A A ) gels after native electrophoresis of x 80 HCM. The eluates tested (2 independent experiments) are diluted about fourfold compared with the loaded material. Bovine serum albumin (1 mg/ml) was added to the samples as a carrier. The gel eluate is completely blocked by antibodies to ~NGF. The eluatcs lacked growthstimulating activity for ciliary neurons.
ganglion. Cultured chicken cells were early reported to synthesize NGF-like material. 42"43 O t h e r investigators have, on the other hand, refuted the presence of N G F in medium conditioned by chicken cells, t7 So far, the role of N G F in the neuronal development of the chicken is implied only by the facts that sensory and sympathetic neurons in v i v o and in vitro express receptors for N G F , ~435"3~ that these neurons also have the ability to transport the factor retrogradely 7 and that chicken irides ~ seem to parallel the rat irides jl'L~ by an increase of NGF-like activity during culture. The stimulating activity for parasympathetic neurons is probably similar to the ciliary factor found in extracts made from chicken organs. ~'m This heterogeneity is in line with cells producing more than one type of nerve growth activity (or different cell populations, each contribution one activity). 2° Evidence of the heterogeneity of trophic activities has also been provided from studies of organ extracts from the chicken. 33 Furthermore, it has been demonstrated that neurons have multiple and changing requirements from growth factors, 2"~4 implying that production of different factors may well have relevance for regulation of neuronal development. The affinity purified antibodies to mouse 13NGF used here demonstrate that the chicken N G F carries at least some antigenic determinants in c o m m o n with the mouse protein. Especially after polyacrylamide gel electrophoresis, the N G F activity increased and the inhibitory effects of the anti-J3NGF antibodies became evident (Figs 4 and 5). Since the NGF-activity was repeatedly eluted from a wide zone of the gel corresponding to high molecular weight ( > 100,000) fractions wc believe that the 13NGF migrated towards the anode in association with negatively charged carrier molecules since IBNGF in itself is a basic protein. Possibly, hydrophobic interactions can account for the smeared-out distribution of the NGF-like activity. Earlier failures to detect NGF-like activity in chicken heart cell conditioned medium ~7 can possibly be explained by the fact that fractionation was not carried out, or by examination of lowproducing cultures (inadequate cell density or culture conditions). Nevertheless, it is worth noting that also in v i v o the recognition between anti-(mouse) 13NGF antibodies and chicken N G F is apparently low since i m m u n o s y m p a t h e c t o m y has not been successful in the chicken embryo. 4" The cell densities used here were about 5 x 1()~ cells per ml of medium. 2'~ Thus about 300 million cells were present in a 600 ml culture. Since a 3-day conditioning period resulted in about 1.5 t-tg NGF-protein, the average production per cell was about 2 fg of N G F per day. This figure is in excellent agreement with the 2.5 fg of N G F produced per day by m o u s e heart fibroblasts in culture. ~5
48
(J. Norrgren and T. Ebendal
The study described here establishes that cells derived from the chicken embryo can produce a factor with physiological and immunological properties similar to mouse NGF. The chicken N G F has several properties in c o m m o n with NGFs from other species. The partially purified factor was not sensitive to acid, heating to 60°C, urea and hyaluronidase but was sensitive to trypsin. With the identification of a chicken h o m o l o g u e to mouse [3NGF present in the heart cell conditioned medium, it is tempting to believe that the chicken N G F is not only secreted under culture conditions 27"4° but is also a product of chicken cells in t'ivo. Preliminary evidence using a e D N A probe for mouse I3NGF 37"4j has also identified a h o m o l o g o u s sequence in the chicken genome. In conclusion, evidence presented in this paper suggests that [3NGF may play a role in the neuronal development of the chicken. ~ Acknowledgements--We thank Dr F. Blomberg, BioCell Laboratories HB, Uppsala, for his many valuable suggestions. The advice given by Dr B. Obrink. Department of Medical and Physiological Chemistry, Uppsala University, is gratcful!y acknowledged. We thank Mr B. Molin, Mrs A. Kylberg and Mrs S. SOderstrOm for technical assistance and Mrs Vibeke Nilsson for preparing the figures and typing the manuscript. Financial support was received from the Swedish Natural Science Research Council (grants B-BU 4(124-102, S-FO 4024-1011.
REFERENCES 1. Barbin G., Manthorpe M. and Varon S. (1984) Purification of the chick eye ciliary neuronotrophic factor. J. Neurochem. 43, 1468--1478. 2. Barde Y.-A., Edgar D. and Thoenen H. (19811) Sensory neurons in culture: changing requirements for survival factors during embryonic development. Proc. hath. Acad. Sci.. U.S.A. 77, 1199-1203. 3. Barde Y.-A., Edgar D. and Thonen H; (1983) New neurotrophic factors. Amt. Rev. Physiol. 45, 6(11--612. 4. Barklis E. and Perez-Polo J. R. (1981) S-180 cells secrete nerve growth factor protein similar to 7S-nerve growth factor. J. Neurosci. Res. 6, 21-36. 5. Berg D. K. (1984) New neuronal growth factors. Ann. Rev. Neurosci. 7, 149-17t). 6. Brachet P. and Dicou E. (1984) L cells potentiate the effect of the extracellular NGF activity in co-cultures with PC 12 pheochromocytoma cells. Exp. Cell Re.~. 150, 234-241. 7. Brunso-Bechtold J. K. and Hamburger V. (19791 Retrograde transport of nerve growth factor in chicken embryo. Proc. hath. Acad. Sci.. U.S.A. 76, 1494-1496. 8. Cohen S., Levi-Montalcini R. and Hamburger V. (1954) A nerve growth-stimulating factor isolated from sarcomas 37 and 180. Proc. hath. Acad. Sci.. U.S.A. 40, 11114-1(]18. 9. Ebendal T., Hedlund K.-O. and Norrgren G. (1982) Nerve growth factors in chick tissues. J. Neurosci. Re.~. 8, 153164. 1(1. Ebendal T., Norrgren G. and Hedlund K.-O. (1983) Nerve growth-promoting activity in the chick embryo: quantitative aspects. Med. Biol. 61, 65-72. 11. Ebandal T., Olson L. and Seiger ,~. (1983) The level of nerve growth factor (NGF) as a function of innervation. A correlative radioimmunoassay and bioassay study of the rat iris. Exp. Cell Res. 148, 311-317. 12. Ebendal T., Olson L., Seiger A. and Belew M. (1984) Nerve growth factors in chick and rat tissues. In Celhdarand Molecular Biology o f Neuronal Development led. Black I. B.), pp. 231-242. Plenum, New York. 13. Ebendal T., Olson L., Seiger ,,~. and Hedlund K.-O. (19811) Nerve growth factors in the rat iris. Nature, Lond, 286, 25-28. 14. Edgar D., Barde Y.-A. and Thoenen H. ( 1981 ) Subpopulations of cultured chick sympathetic neurones differ in their requirements for survival factors. Nature, Lond. 289, 294-295. 15. Furukawa Y., Furukawa S., Satoyoshi E. and Hayashi K. (1984) Nerve growth factor secreted by mouse heart cells in culture. J. biol. Chem. 259, 1259-1269. 16. Hamburger V., Brunso-Bechtold J. K. and Yip J. W. ( 1981 ) Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor. J. Neurosci. I, 6(I-71. 17. Helfand S. L., Riopelle R. J. and Wessells N. K. (19781 Non-equivalence of conditioned medium and nerve growth factor for sympathetic, parasympathetic and sensory neurons. Exp. Cell Res. ! 13, 39-45. 18. Helfand S. L.. Smith G. A. and Wessells N. K. (1976) Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia. Devl Biol: 50, 541-547. 19. Kim !. S. and Pantazis N. J. (1985) Biological and biochemical properties of nerve growth factor synthesized by mouse S-180 cells in culture. E.ff~. Celt Res. 156, 391-404. 20. Lindsay R. M. (1979) Adult rat brain astrocytes support survival of both NGF-dependent and NGF-insensitive neurones. Nature, Lond. 282, 8(1-82. 21. Levi-Montalcini R. and Hamburger V. ( 1951 ) Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J. exp. Zool. 116, 321-361. 22. Longo A. M. (1978) Synthesis of nerve growth factor in rat glioma cells. Devl Biol. 65, 26(1-270. 23. Longo A. M. and Penhoet E. E. (1974) Nerve growth factor in rat glioma cells. Proc. hatH. Acad. Sci., U.S.A. 71, 2347-2349. 24. Murphy R. A., Oger J., Saide J. D.. Bhmchard M. H.. Arnason B. G. W., Hogan C., Pantazis N. J. and Young M. (1977) Secretion of nerve growth factor by central ncrw~us system glioma cells in culture. J. ('ell BioL 72, 769-773. 25. Murphy R. A., Pantazis N. J., Arnason B. G. W. and Young M. (1975) Secretion of a nerve growth factor by mouse neuroblastoma cells in culture. Proc. ttaln. Acad. Sci.. U.S.A. 72, 1895-1898. 26. Murphy R. A., Singer R. H., Saido J. D., Pantazis N. J., Blanchard M. H., Byron K. S., Arnason B. G. W. and Young M. (19771 Synthesis and secretion of a high molecular weight form of nerve growth factor by skeletal muscle cells in culture. Proc. n~lttt. Acad. Sci., U.S.A. 74, 449(vM-5(~).
N G F in c h i c k e n h e a r t c o n d i t i o n e d m e d i u m
49
27. Murphy R. A., Watson A. Y. and Rhodes J. A. (1984) Biological sources of nerve growth factor. Appl. Neurophysiol. 47, 3 3 4 2 . 28. Norrgren G., Ebendal T., Belew M.. Jacobson C.-O. antt Porath J. (19811) Release of nerve growth factor by human glial cells in culture. Exp. Uell Res. 130, 31-39. 29. Norrgren G., Ebendal T. and Wikstr6m H. (1984) Production of nerve growth-stimulating factor(s) from chick embryo heart cells. Use of Cytodcx c" 3 microcarricrs and serum-free media. Exp. (.'ell Res. 152, 427-435. 30. Pantazis N. J. (1983) Nerve growth factor synthesized by mouse fibroblast cells in culture: absence of ¢~ and -,/ subunits. Biochemist O" 22, 4264-4271. 31. Pantazis N. J., Blanchard M. H., Arnason B. G. W. and Young M. (1977) Molecular properties of the nerve growth factor secreted by L cells. Proc. hath. Acad. Sci.. U.S.A. 74, 1492 1496. 32. Perez-Polo J. R., Hall K., Livingston K. and Westlund K. (1977) Steroid induction of nerve growth factor synthesis in cell culture. Li['e Sci. 21, 1535-1544. 33. Riopelle R. J. and Cameron D. A. ( 1981 ) Neurite growth promoting factors of embryonic chick----ontogeny, regional distribution, and characteristics. J. Neurobiol. 12, 175 lbI6. 34. Rohrer H. and Barde Y.-A. (1982) Presence and disappearance of nerve growth factor receptors on sensory neurons in culture. Devl Biol. 89, 309 315. 35. Rohrer H.. Thoenen H. and Edgar D. (1983) Presence of nerve growth factor receptors and catecholamine uptake in sub-populations of chick sympathetic neurons: correlation with surviwd faclor requirements in culture, l)evl Biol. 99, 34-4t~. 36. Schwartz J. P., Chuang D.-M. and Costa E. (1977) Increase in nerve growth factor content of C6 glioma cells by the activation of a [3-adrenergic receptor. Brain Res. 137, 369-375. 37. Scott J., Selby M., Urdea M., Quiroga M., Bell G. I. and Rutter W. J. (1983) Isolation and nucleotids sequence of a eDNA encoding the precursor of mouse nerve growth fuctor. Nature, Lond. 302, 538-541~. 38. Stoeckel K., Gagnon C., Guroff G. and Thoenen H. (1976) Puritication of nerve growth factor antibodies by affinity chromatography. J. Neurochem. 26, 12(17-121 I. 39. Sulter A., Riopel[e R. J., Harris-Warrick R. M. and Shooter E. M. (1979) Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J. hiol. (.Tzem. 254, 5972-5982. 411. Thoenen H. and Barde Y.-A. (198(I) Physiology of nerve growth factor. Physiol. Rev. 60, 1284-1335. 41. UIIrich A., Gray A., Berman C. and Dull T. J. (1983) Human [3-nerve growth factor gene sequence highly homologous to th~n of mouse. Nature, Lond. 303, 821-825. 42. Varon S., Raiborn C. and Burnham P. A. (1974) hnplication of a nerve growth factor-like antigen in the support derived by ganglionic neurons from their homologous gila in dissociated cultures. Neurohiology 4, 317 327. 43. Young M., Oger J., Blanchard M. H., Asdourian H., Amos H. and Arnason B. G. W. (1975) Secretion of a nerve growth facto~ by primary chick fibroblast cultures. Science 1517, 361-362.
0736-5748186 $03.(10+0.00 Pergamon Press Ltd. © 1986 ISDN
Printed in Great Britain.
NERVE
G R O W T H F A C T O R IN M E D I U M C O N D I T I O N E D E M B R Y O N I C C H I C K E N H E A R T CELLS
BY
GUNII.I.A NORRGRI{N*'~" a n d TED EBENDAI.* :Uppsala University. Department of Zoology, Box 561. S-751 22 Uppsala, Sweden, and +BioCell Laboratories HB, Box 17, Uppsala I. Sweden ( A ccepled 31 A tlgust 1985 )
A b s t r a c t - - T h e present report demonstrates that embryonic chicken heart cells in culture release different nerve growth promoting factors to their culture medium, one which is biologically and immunologically similar to mouse gland [3NGF. Serum-free heart cell conditioned medium thus promoted neurite outgrowth from sympathetic and ciliary ganglia and supported survival of dissociated ciliary neurons. The addition of affinity purified antibodies against mouse [3NGF does substantially but not completely inhibit the fibre outgrowth from sympathetic ganglia, but does not to any extent diminish the effects on the parasympathetic neurons. The chicken N G F recovered from polyacrylamide gels after electrophoresis greatly enhanced sympathetic fibre outgrowth, an activity completely suppressive by anti-[3NGF antibodies. We conclude that a chicken N G F is being produced by the embryonic heart cells in culture, and that this factor may be produced also in the embryo to fulfill a role in heart innervation. K e y words: Cell culture, Conditioned medium, Chicken embryo, Ciliary neurons, Sympathetic ganglion, Nerve growth factor.
Nerve growth factor ([3NGF) is a polypeptide which plays a fundamental role in the development and maintenance of adrenergic and sensory neurons, s'2~4° Several non-neuronal cells (both transformed and non-transformed) have been found to synthesize and secrete a factor which is biologically and immunologically similar to the well characterized mouse submandibular gland N G F . 4"6"lS"lg"22-2~''2x'3q~-32"36"42 In addition, media conditioned by cells in culture have been shown to contain also other factors, 3'5"2° a m o n g them one maintaining parasympathetic ciliary neurons. 17,~ We demonstrated recently that embryonic chicken heart cells cultured under serum-free conditions released nerve growth promoting substances for both sympathetic and parasympathetic ciliary neurons. 2'~ An early report by Young et al. 43 indicated that primary chicken e m b r y o fibroblasts produced a NGF-like factor. On the other hand, Helfand and coworkers ~v have reported that medium conditioned over chicken e m b r y o heart cells ( H C M ) did not contain any NGF. In the study presented here, we have characterized the apparent NGF-like activity and separated it from the activity affecting ciliary neurons. It is demonstrated that the chicken heart cells in culture produce a factor immunologically and biologically similar to 13NGF from the mouse submandibular gland. MATERIALS AND METHODS Materials
Unless otherwise stated, all material for cell culture was obtained from Flow Laboratories and chemicals and reagents from Sigma. Equipment for c h r o m a t o g r a p h y and electrophoresis came from Bio-Rad and Pharmacia. Mouse [3NGF was prepared as described ~~2 and radiolabelling was performed with Na~eSl using chloramine-T to a specific activity of about 30,000 cpm/ng. Rabbit anti-(mouse) 13NGF antiserum was prepared in rabbits by multiple intradermal injections of purified I3NGF and the antibodies were affinity purified. 1~,12,3~ Cell culture and conditioned media
Primary chicken e m b r y o cells derived from hearts of 9-day-old embryos (White Leghorn strain) were prepared and cultured as described previously. > Cells were grown on Cytodex ~ 3 Address correspondence to: Dr. Ted Ebendal, D e p a r t m e n t of Zoology, Uppsala Unisersitv. Box 561. S-751 22 Uppsala, Sweden. ,4bbreviations: H ( ' M , heart conditioned medium: NGF, nerve growth factor.
41
42
(;. Norrgren rind T. Ebendal
microcarriers (Pharmacia) in Dulbecco's modification of Eagle's Medium (DME) and Ham's F 10 supplemented with non-essential amino acids, 2 mM glutamine, 1 mg/ml human ~erum albumin (a gift from Dr. J. Curling, Pharmacia), 10 i~g/ml fibronectin (a gift from E Ekrc, KABI), 25 ~g/ml transferrin (P. Ekre, KABI) and I i~g/ml insulin ( N O V O ) for 3 days before the conditioned medium was collected. Media were harvested from 600 ml microcarricr cultures, ~<' concentrated 80-fold and dialyzed against BME (Eagle's Basal Medium) by pressure dialysis over Pellicon P T G C (Millipore) membranes (cut-off tool. wt 10,000), and thereafter sterile filtered through Millipore G V filters.
Bioassay Nine-day-old chicken embryo ciliary and sympathetic ganglia or dissociated neurons were used to estimate the nerve growth activities present in the concentrated heart cell conditioned medium (HCM). Details of this technique have been reported previously. 9-13 For titration of nerve growth activity the test samples were diluted with B M E (series of two-fold dilutions) and added on top of the collagen gels (giving a further two-fold dilution). Ganglion cultures were examined after 2 days and the number of outgrowing neurites scored using a scale from zero (no fibres) to 5 (very dense fibre halo), or as biological units (1 BU being present per ml at optimum fibre density). Dissociated ciliary neurons were initially counted and the number of surviving cells determined after 2 days. To test for NGF-specific fibre response, test samples were incubated overnight (4°C) with 2 I~g/ml of affinity purified anti-13NGF antibodies (10-fold higher concentration than needed to block 1 BU of activity of the homologous antigen) and the samples were added to the nerve cell cultures.
Characterization of nerve growth activity Molecular characteristics were evaluated by treatment with various enzymes and with heat. Concentrated HCM was heated in a waterbath for 30 rain to 60°C and then cooled on ice before analysis for biological activity. In another experiment HCM was incubated at 37°C for 2 hr with 100 ~g/ml of trypsin. Thereafter soybean trypsin inhibitor was added to a final concentration of 500 ~g/ml before being tested in bioassay. Hyaluronidase was dissolved in 10 mM sodium phosphate buffer, 0.15 M NaCI, pH 5.1, at 100 units/ml and added to HCM at a final concentration of 5 units/ml. This mixture was incubated for 6 hr at 37°C. Concentrated HCM was also brought to pH 2.5 with 1 M HCI. After 1 hr at room temperature samples were neutralized with 1 M NaOH.
Electrophoresis For preparative electrophoresis, polyacrylamide gradient gels (2-16%, Pharmacia) were used in a 0.09 M Tris, 0.08 M boric acid buffer (pH 8.4). Concentrated HCM (0.5 ml) was loaded onto the gel and the sample run for approximately 300 Vhr. Thereafter elution of proteins was performed by slicing the gel into 5 mm wide strips which were homogenized in 5(X) ILl of 0.02 M TrisHCI buffer (pH 8) and agitated overnight (4°C). Supernatants obtained after centrifugation were dialyzed for 3 days against BME before being tested in the bioassay or in a radioimmunoassay.
Radioimmunoassay A double antibody solid phase assay was used with the second antibody (anti-rabbit IgG, BioCell Lab. HB) covalently bound to CNBr-activated agarose particles. One hundred ill of affinity purified antibodies (0.1 I,tg/mi) against mouse [3NGF and I(XI ill of test samples or [3NGF standard (0 to 100 ng) in phosphate buffered saline (PBS, pH 7.6, 0.5% Tween) were incubated for 16 to 20 hr at room temperature. Then 1 ng of ~251-[3NGF in 100 p,I of the PBS buffer were added and incubation continued for another 16 to 20 hr. Finally 2 ml of the second antibody suspension were added. After 30 rain (20°C) the mixture was centrifuged and the radioactivity in the immunoprecipitate measured in a LKB gamma counter. Non-specific binding, determined by incubation in the presence of excess unlabelled [BNGF (16% of the total binding) was subtracted. RESULTS Medium conditioned over chicken embryo heart cells and then concentrated 80-fold was biologically active in stimulating the survival of dissociated ciliary neurons (Fig. 1). The response
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Fig. I. Survival of dissociated ciliary neurons as a function of concentration of the serum-free heart cell conditioned medium. The fraction of neurons remaining after 2 days in a collagen matrix (100 gl) is shown, Eight different samples (100 i,tl) were added to the matrices (final dilution in the culture of the 80 × HCM is given) and the results are stated as mean ± S.E.M. To test the influence of anti-lSNGF antibodies four samples were incubated with the affinity purified immunoglobulin (Ig), 2 ~g/ml, 4°C, before being added to cultures. A dose-dependent relationship between the concentration and survival is seen with no influence from the antibodies.
is clearly dose-dependent, and not inhibited by antibodies to nerve growth factor (Fig. 1). Neurite outgrowth from ciliary (Fig. 2) and sympathetic (Fig. 3) ganglia were also stimulated. The addition of affinity purified antibodies to 13NGF had different effects on the neurite outgrowths. No inhibition was seen in the ciliary outgrowth (Fig. 2). For the sympathetic ganglia a marked inhibition in outgrowth was found (Fig. 3). The anti-13NGF antibodies were present at a concentration 10 times higher than required for total inhibition of 1 BU of mouse ~NGF, and the addition of even higher amounts did not further suppress the residual sympathetic fibre outgrowth evoked by the concentrated HCM. Thus, 600 ml medium conditioned by the heart cells (concentrated to 8 ml) was estimated to carry an anti-NGF suppressable biological activity equivalent to about 40-60 BU of NGF. Under the assumption that 1 BU equals 10 ng of [3NGF protein '2a" this corresponds to 0.4-0.6 I,tg of the active substance (in 16(1 mg total protein). Addition of trypsin to HCM resulted in loss of these biological activities whereas no inactivation occurred after heating to 60°C, repeated freeze-thawing, exposure to acid (pH 2.5), 6 M urea or hyaluronidase. Medium concentrated without prior conditioning (to serve as control) totally htcked the growth-promoting effects reported here. To characterize the NGF-like activity further, concentrated heart cell conditioned medium was electrophorezed on 2-16% gradient polyacrylamide gels under native conditions. Active material migrated to the anode at pH 8.4 and eluates from the four top slices (high molecular weight region) of the gel gave high recoveries of NGF-like activity (Fig. 4), but only 5% of applied protein. No activity stimulating survival or neurite outgrowth in ciliary neurons were recovered from the polyacrylamide gels. Figure 5a shows the typical NGF-like response in a sympathetic ganglion to the eluated material, an activity completely blocked by antibodies to mouse [3NGF (Figs 4 and 5b). Staining of parallel gels with Coomassie Brilliant Blue revealed no distinct protein bands in the upper region of the gel but showed diffusely smeared materials, probably lipoproteins. Immunoblots incubated with anti-NGF antibodies visualized a broad zone of NGF-like reactivity corresponding to the slices from which N G F activity was recorded (not shown). The amount of NGF-like material eluted from the polyacrylamide gels was determined in a double antibody solid phase radio-immunoassay. The values obtained in the radioimmunoassay correlate well with the amount of NGF-like activity estimated from the bioassay experiments. Thus, 35 and 59 ng/ml were determined by RIA in two independent experiments. Corresponding
44
Norrgren and T. Ebendal
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Fig. 2. Fibre growth density scored in whole ciliary ganglia in culture. Conditions otherwise as in Fig, 1. In this case too the anti-~NGF antibodies fail to suppress the response in the ciliary neurons.
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Fig. 3. Density of sympathetic fibre outgrowth in response to × 8 0 concentrated heart cell conditioned medium. The assay is based on blind scoring of the fibre halo around whole sympathetic ganglia in a collagen matrix. Conditions otherwise as stated in Fig. t. In contrast to the ciliary fibre outgrowth (Fig. 2), the sympathetic fibre outgrowth is reduced but not completely stopped by the anti-,BNGF antibodies.
data from bioassay were 40 and 60 ng/ml (4 and 6 BU). After correction for the 4-fold dilution introduced during slicing of the gel, this implies that 1.3-1.9 p,g of chicken NGF protein are present in 600 mi of conditioned medium (thus 2-4 times the amount estimated from the crude HCM). DISCUSSION This report characterizes NGF-like material in serum-free medium conditioned for 3 days by embryonic chicken heart cells in culture, and distinguishes this material from an activity (or activities) stimulating fibre outgrowth and neuron survival in the parasympathetic ciliary
N G F in chicken h e a r t c o n d i t i o n e d m e d i u m
Fig. 5. (a) Sympathetic ganglion responding to material eluted from a polyacrylamide gel (cf. Fig. 4). Micrograph taken after 2 days in culture. Darkfield. (b) The same as (a) but with the sample incubated with anti-13NGF immunoglobulin (2 p,g/ml) before bioassay. Note total reduction in fibre outgrowth. Scale b a r - 0.5 ram.
45
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Fig. 4. Sympathetic fibre growth response to eluates from polyacrylamide ( P A A ) gels after native electrophoresis of x 80 HCM. The eluates tested (2 independent experiments) are diluted about fourfold compared with the loaded material. Bovine serum albumin (1 mg/ml) was added to the samples as a carrier. The gel eluate is completely blocked by antibodies to ~NGF. The eluatcs lacked growthstimulating activity for ciliary neurons.
ganglion. Cultured chicken cells were early reported to synthesize NGF-like material. 42"43 O t h e r investigators have, on the other hand, refuted the presence of N G F in medium conditioned by chicken cells, t7 So far, the role of N G F in the neuronal development of the chicken is implied only by the facts that sensory and sympathetic neurons in v i v o and in vitro express receptors for N G F , ~435"3~ that these neurons also have the ability to transport the factor retrogradely 7 and that chicken irides ~ seem to parallel the rat irides jl'L~ by an increase of NGF-like activity during culture. The stimulating activity for parasympathetic neurons is probably similar to the ciliary factor found in extracts made from chicken organs. ~'m This heterogeneity is in line with cells producing more than one type of nerve growth activity (or different cell populations, each contribution one activity). 2° Evidence of the heterogeneity of trophic activities has also been provided from studies of organ extracts from the chicken. 33 Furthermore, it has been demonstrated that neurons have multiple and changing requirements from growth factors, 2"~4 implying that production of different factors may well have relevance for regulation of neuronal development. The affinity purified antibodies to mouse 13NGF used here demonstrate that the chicken N G F carries at least some antigenic determinants in c o m m o n with the mouse protein. Especially after polyacrylamide gel electrophoresis, the N G F activity increased and the inhibitory effects of the anti-J3NGF antibodies became evident (Figs 4 and 5). Since the NGF-activity was repeatedly eluted from a wide zone of the gel corresponding to high molecular weight ( > 100,000) fractions wc believe that the 13NGF migrated towards the anode in association with negatively charged carrier molecules since IBNGF in itself is a basic protein. Possibly, hydrophobic interactions can account for the smeared-out distribution of the NGF-like activity. Earlier failures to detect NGF-like activity in chicken heart cell conditioned medium ~7 can possibly be explained by the fact that fractionation was not carried out, or by examination of lowproducing cultures (inadequate cell density or culture conditions). Nevertheless, it is worth noting that also in v i v o the recognition between anti-(mouse) 13NGF antibodies and chicken N G F is apparently low since i m m u n o s y m p a t h e c t o m y has not been successful in the chicken embryo. 4" The cell densities used here were about 5 x 1()~ cells per ml of medium. 2'~ Thus about 300 million cells were present in a 600 ml culture. Since a 3-day conditioning period resulted in about 1.5 t-tg NGF-protein, the average production per cell was about 2 fg of N G F per day. This figure is in excellent agreement with the 2.5 fg of N G F produced per day by m o u s e heart fibroblasts in culture. ~5
48
(J. Norrgren and T. Ebendal
The study described here establishes that cells derived from the chicken embryo can produce a factor with physiological and immunological properties similar to mouse NGF. The chicken N G F has several properties in c o m m o n with NGFs from other species. The partially purified factor was not sensitive to acid, heating to 60°C, urea and hyaluronidase but was sensitive to trypsin. With the identification of a chicken h o m o l o g u e to mouse [3NGF present in the heart cell conditioned medium, it is tempting to believe that the chicken N G F is not only secreted under culture conditions 27"4° but is also a product of chicken cells in t'ivo. Preliminary evidence using a e D N A probe for mouse I3NGF 37"4j has also identified a h o m o l o g o u s sequence in the chicken genome. In conclusion, evidence presented in this paper suggests that [3NGF may play a role in the neuronal development of the chicken. ~ Acknowledgements--We thank Dr F. Blomberg, BioCell Laboratories HB, Uppsala, for his many valuable suggestions. The advice given by Dr B. Obrink. Department of Medical and Physiological Chemistry, Uppsala University, is gratcful!y acknowledged. We thank Mr B. Molin, Mrs A. Kylberg and Mrs S. SOderstrOm for technical assistance and Mrs Vibeke Nilsson for preparing the figures and typing the manuscript. Financial support was received from the Swedish Natural Science Research Council (grants B-BU 4(124-102, S-FO 4024-1011.
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N G F in c h i c k e n h e a r t c o n d i t i o n e d m e d i u m
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27. Murphy R. A., Watson A. Y. and Rhodes J. A. (1984) Biological sources of nerve growth factor. Appl. Neurophysiol. 47, 3 3 4 2 . 28. Norrgren G., Ebendal T., Belew M.. Jacobson C.-O. antt Porath J. (19811) Release of nerve growth factor by human glial cells in culture. Exp. Uell Res. 130, 31-39. 29. Norrgren G., Ebendal T. and Wikstr6m H. (1984) Production of nerve growth-stimulating factor(s) from chick embryo heart cells. Use of Cytodcx c" 3 microcarricrs and serum-free media. Exp. (.'ell Res. 152, 427-435. 30. Pantazis N. J. (1983) Nerve growth factor synthesized by mouse fibroblast cells in culture: absence of ¢~ and -,/ subunits. Biochemist O" 22, 4264-4271. 31. Pantazis N. J., Blanchard M. H., Arnason B. G. W. and Young M. (1977) Molecular properties of the nerve growth factor secreted by L cells. Proc. hath. Acad. Sci.. U.S.A. 74, 1492 1496. 32. Perez-Polo J. R., Hall K., Livingston K. and Westlund K. (1977) Steroid induction of nerve growth factor synthesis in cell culture. Li['e Sci. 21, 1535-1544. 33. Riopelle R. J. and Cameron D. A. ( 1981 ) Neurite growth promoting factors of embryonic chick----ontogeny, regional distribution, and characteristics. J. Neurobiol. 12, 175 lbI6. 34. Rohrer H. and Barde Y.-A. (1982) Presence and disappearance of nerve growth factor receptors on sensory neurons in culture. Devl Biol. 89, 309 315. 35. Rohrer H.. Thoenen H. and Edgar D. (1983) Presence of nerve growth factor receptors and catecholamine uptake in sub-populations of chick sympathetic neurons: correlation with surviwd faclor requirements in culture, l)evl Biol. 99, 34-4t~. 36. Schwartz J. P., Chuang D.-M. and Costa E. (1977) Increase in nerve growth factor content of C6 glioma cells by the activation of a [3-adrenergic receptor. Brain Res. 137, 369-375. 37. Scott J., Selby M., Urdea M., Quiroga M., Bell G. I. and Rutter W. J. (1983) Isolation and nucleotids sequence of a eDNA encoding the precursor of mouse nerve growth fuctor. Nature, Lond. 302, 538-541~. 38. Stoeckel K., Gagnon C., Guroff G. and Thoenen H. (1976) Puritication of nerve growth factor antibodies by affinity chromatography. J. Neurochem. 26, 12(17-121 I. 39. Sulter A., Riopel[e R. J., Harris-Warrick R. M. and Shooter E. M. (1979) Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J. hiol. (.Tzem. 254, 5972-5982. 411. Thoenen H. and Barde Y.-A. (198(I) Physiology of nerve growth factor. Physiol. Rev. 60, 1284-1335. 41. UIIrich A., Gray A., Berman C. and Dull T. J. (1983) Human [3-nerve growth factor gene sequence highly homologous to th~n of mouse. Nature, Lond. 303, 821-825. 42. Varon S., Raiborn C. and Burnham P. A. (1974) hnplication of a nerve growth factor-like antigen in the support derived by ganglionic neurons from their homologous gila in dissociated cultures. Neurohiology 4, 317 327. 43. Young M., Oger J., Blanchard M. H., Asdourian H., Amos H. and Arnason B. G. W. (1975) Secretion of a nerve growth facto~ by primary chick fibroblast cultures. Science 1517, 361-362.