Biological and immunological properties of the nerve growth factor from bovine seminal plasma: Comparison with the properties of mouse nerve growth factor

~~ruroscimce Vol. 8, No. 2. pp. 375 to 387, 1983 Printed m Great Bntain

0306-4522/X3/020375-13$03.00/0

Pergamon PressLtd 0 1983IBRO

BIOLOGICAL AND IMMUNOLOGICAL PROPERTIES OF THE NERVE GROWTH FACTOR FROM BOVINE SEMINAL PLASMA: COMPARISON WITH THE PROPERTIES OF MOUSE NERVE GROWTH FACTOR G.

P. HARPER,* Y.-A. BARDE,D. EDGAR, D. GANTEN,’ F. HEFTI,? R. HEUMANN,K. W. NAUJOKS,H. ROHRER, J. E. TURNER,: and H. THOENEN

Department of Neurochemistry, ‘Department of Pharmacology,

Max Planck Institute for Psychiatry, 8033 Martinsried/Munich; and University of Heidelberg, 6900 Heidelberg, Federal Republic of Germany

Abstract-The biological activities of Nerve Growth Factor (NGF) purified from bovine seminal plasma have been compared with those of NGF from mouse submandibular glands in a variety of systems: maintenance of survival in uitro and stimulation of nerve fibre outgrowth from sensory, sympathetic and parasympathetic neurons of the embryonic chick; maintenance of survival in vitro, stimulation of nerve fibre outgrowth and specific induction of tyrosine hydroxylase in neonatal rat sympathetic neurons; stimulation of nerve fibre outgrowth and cellular hypertrophy and specific induction of choline acetyltrapsferase in pheochromocytoma PC12 cells; induction of tyrosine hydroxylase in bovine adrenal medullary cells; and stimulation of nerve fibre outgrowth from explants of goldfish retinae. In all cases, the two NGFs had the same effects qualitatively and quantitatively, and with identical dose-dependencies. The results indicate that the wide range of biological effects and target cells delineated in detail for mouse NGF can justifiably be attributed to other NGF proteins, and that they are not exclusively restricted to the mouse NGF molecule. Furthermore, as bovine NGF is free of the renin contaminants so difficult to remove from mouse NGF, the above biological activities can truly be assigned to the NGF mol&ule. Immunologically, however, mouse and bovine NGFs differ substantially. This is demonstrated by the relatively poor ability of antisera against bovine NGF to inhibit the activity of mouse NGF in vitro, and by the incomplete nature of the immunosympathectomy caused in rats by treatment with antisera to bovine NGF, in contrast to the extensive immunosympathectomy caused in these animals by the administration of comparable quantities of antisera against mouse NGF. Clearly, the biochemical features of the NGF molecules responsible for their biological effectiveness and for their predominant antigenic properties are different.

Nerve Growth Factor (NGF) was originally discovered as a macromolecular agent produced by certain sarcoma cells and having dramatic effects on the sensory and sympathetic neurons of the tumour-carrying host animal (for review, see ref. 37). An essential step in the subsequent more detailed examination of this phenomenon was the establishment of a sensitive biological assay for NGF: thus, the tumour factor was able to stimulate the rapid outgrowth of a halo of nerve fibres from sensory (dorsal root) ganglia dis-

* To whom reprint requests should be addressed. t Present address: Department of Preclinical Research, Sandoz AG, CH-4002 Base], Switzerland. ‘j’Permanent address: Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103, U.S.A. Abbreviations: NGF, Nerve Growth Factor; BU, biological unit (of NGF); TH, tyrosine hydroxylase; ChAT, choline acetyltransferase; HPLC, high performance liquid chromatography; ACTH, adrenocorticotrophic hormone; EDTA, ethylenediaminetetraacetate; CSF, cerebrospinal fluid. 375 NST. 8/2-J

sected from &day chick embryos, and cultured in a plasma clot.38 This assay enabled the detection of much more potent sources of NGF in the submandibular glands of mice37*51 and in the venoms of all snakes so far examined.’ In recent years, many other neurotrophic agents have been reported (for review, see ref. 9) that have, however, been distinguished from NGF by various criteria. The convention has generally been accepted that, in order to be identified specifically as an NGF, an agent has to be able to stimulate fibre outgrowth in the standard biological assay, and this activity should be completely inhibited by specific antibodies raised against one of the ‘classical’ NGFs described above.” According to these criteria, the list of sources of NGF has recently been extended to include the prostate glands of the guinea pig,” rabbit and bu11,30other accessory sex organs of the bull (M. E. Schwab & G. P. Harper, unpublished data), and the seminal plasmas of the bull and some other species.28 The biological characterization of NGF purified from the mouse. submandibular gland has been

extended in many investigations so that now it is known that NGF acts on a variety of neural target cells to produce effects that are apparent morphologically and biochemically (for reviews, see refs 29. 31. 56; and more detailed discussion below). However, virtually no further work has been carried out on the biological properties of other NGFs, and it is not clear whether the wide range of biological effects produced by mouse NGF in many different target cells is a general property of the NGF group of proteins, or if they are restricted exclusively to the mouse protein. If the latter proved to be the case, it would argue for the existence of more than one functional active centre in the mouse NGF molecule. We therefore report here the results of a comparative study of the biological properties of NGFs purified from bovine seminal plasma2’ and mouse submandibular glands. 51 We have also studied these two purified NGFs using immunological methods, in view of previous indications using unfractionated tissue extracts,27.29-3 1 that mammalian NGFs have different immunological properties.

EXPERIMENTAL

PROCEDURES

Preparation of nerve growth factors and antisera Mouse NGF was purified by the method of Bocchini & Angeletti,” as modified by Suda et aLt5 An antiserum against mouse NGF was raised in sheep as described by Suda et a1.55 For some experiments, antibodies were purified from this antiserum by affinity chromatography.54 Bovine NGF was purified from seminal plasma by the method of Harper et al.‘* Rabbit antisera against bovine and mouse NGFs were raised by injecting a total of SO-100 pg NGF per rabbit in multiple subcutaneous sites; complete Freund’s adjuvant (Gibco) was used for initial immunizations, and incomplete adjuvant for subsequent boosts. Animals were boosted (5&100 pg NGF per boost) every 4-6 weeks to achieve and maintain satisfactory titres (see Fig. 8). The NGF biological assay was performed according to minor modifications of the method described by Levi-Montalcini et al.,3s and antiserum titres were determined4 as the inverse of the maximum dilution completely inhibiting the response in the biological assay to 4-5 ng purified NGF per millilitre culture medium. This concentration of NGF produces optimal nerve fibre outgrowth, and is defined as 1 Biological Unit (BU) per ml. Antibody titres were defined as the amount of purified antibodies (per ml culture medium) inhibiting 1 BU NGF/ml. Tissue culture procedures

Details of most of the culture systems used have previously been published. Dissociated and purified neurons from chick embryo sensory ganglia were cultured according to the method of Barde et al.;’ and those from chick embryo sympathetic ganglia by the method of Edgar et a1.16 Dissociated and purified neurons were prepared from the ciliary (parasympathetic) ganglia of I-day-old chick embryos as follows: ganglia were incubated for 20min at 37°C with 0.1% trypsin in Ca2+- and Mg2+-free phosphate buffered saline. After the incubation, ganglia were washed twice with F14 medium (Gibco) containing 10% horse

serum. The ganglia were dissociated by gentle trlturatlcrn VI a siliconized Pasteur pipette. Non-neuronal cells present 111 the cell suspension were removed by preplating for 3.5 11aj described by Barde et (I/.~ Cells were then added to Falcc111 multiwell tissue culture plates (16 mm diameter wells) at ;I density of 6000-8000 cells per well. The culture wells had previously been coated with polyornithine (Sigma. type I--B) and pretreated“’ overnight with undiluted heart-conditioned medium. which was prepared as described previously.48 The cell suspension (60 ~1) was added to 600 /tl F14 medium containing IO”/,, horse serum and containing between 10 ng/ml and 1 pgjrnl of bovine NGF. 10 ng’ml of mouse NGF (final concentrations) or 600 /lI of heart-condltioned medium. After I h and after 2 days in culture, cells were counted by scanning strips (at least 8”,, of the surface) with a phase-contrast microscope at x 125 magnification. Dissociated and purified neurons from neonatal rat sympathetic ganglia were cultured as described by F. Hefti. H. Gnahn, M. E. Schwab & H. Thoenen (in preparation). Pheochromocytoma (PC1 2) cells were a subclone (FR-P9) that reacts rapidly to the presentation of NGF, and they were cultured as described by Heumann et ~1.~~Cellular

volume was measured electrically according to Heumann et a/.,33 using a flow cytometric pulse height analyser, as described by Kachel et ~1.~’ Adrenal medullary cells were prepared from the glands of calves as described by Naujoks et al.44 Explants of goldfish retinae were prepared and cultured as originally described by Landreth & Agranoff.30 and modified by Turner ef a1.57 The references quoted should also be consulted for the detailed methods of the morphological and biochemical analyses made (see also figure legends). Enzyme measurement.> Tyrosine hydroxylase (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3_hydroxylating), EC 1.14.3.a) was measured (in rat sympathetic neurons and following immunosympathectomy in uiuo) by the method of Levitt et a1..39 as modified by Edgar et a1.,‘6 and in bovine adrenal medullary cells according to the method of Mueller et a1.43 (see also ref. 44); choline acetyltransferase (acetyl-CoA: choline

0-acetyltransferase, EC 2.3.1.6) was measured by the method of Fonnum;” and lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27) was measured according to Johnson & Whittaker. For all studies, the enzymic reactions were proportional to time and to the amount of sample. Protein used the method of Lowry et al.@ (bovine as standard) or the dye-binding assay (bovine y-globulin as standard).

the incubation determinations serum albumin of Bradford”

Renin activities Renin activity was measured in vitro by incubating samples (0.1-l pg protein) with purified angiotensinogen,” in the presence of inhibitors of angiotensinases and other

degradative enzymes,53 and then determining the amount of angiotensin I liberated using a sensitive and specific radioimmunoassay. 53 The angiotensinogen used was purified” from rat plasma for tests of samples from mice and guinea pigs (see Table 2), and from sheep plasma for tests on bovine samples; possible species differences in the renin-angiotensinogen reaction should therefore be minimized. Similar assays were made folbwing 12 h diabysis uersus a 0.1 M citrate-O.2 M disodium hydrogen phosphate buffer, pH 3.3, to allow full activation of any prorenin

377

Properties of bovine nerve growth factor (reninogen) contained in the samples.20 Before assay, the samples were dialysed back to pH 7.5 (12 h; 0.1 M citrate phosphate buffers3). Whole bovine seminal plasma and a crude extract of the guinea pig prostate and coagulating gland complex were prepared as previously described.28+30 Purified mouse NGF5” was further purified by three passages over an HPLC-gel filtration column (I-125, Waters) in 50mM phosphate buffer, pH 6.0 at a flow-rate of 0.5 ml/min. The NGF-containing fractions (analysed by the standard biological assay) were pooled for re-chromatography under the same conditions. Renin activity was also measured in uiuo by injecting samples (200 pg purified bovine NGF in 200 ,& containing 0.1% bovine serum albumin), via a chronic cannula, into the third brain ventricle of adult beagles. Dogs were not nephrectomized and were on a normal diet (including a normal sodium content). Blood samples were removed with a syringe containing inhibitors of enzymes capable of degrading renin, angiotensin or ACTH,53 and 20mM EDTA to prevent coagulation, from a cannula inserted into the femoral vein; they were centrifuged at 4°C to remove blood cells, and stored at -20°C until used to measure ACTH5* renin activitys3 and angiotensin 114’ levels. Parallel experiments involved the administration (0.5 units in 5 ~1) of purified hog kidney renin, purchased from NBC, Cleveland, Ohio. Artificial cerebrospinal fluid or phys~ologica1 saline was used as the vehicle for all intraventricular injections. Similar experiments were performed in rats (see Table 3). Cerebrospinal fluid (CSF) was collected as described by Speck et aLS3 and the concentrations of angiotensinogen and angiotensin I determined by radioimmunoassay.2’~53

Fig. 1. Dose-dependency of the survival in vitro of sensory neurons from the IO-day-old chick embryo on the presence of NGF from the mouse (&--0) or from bovine seminal plasma (e--@). Also shown (A) is the survival in the presence of 1ng/ml of both of the NGFs together. Neurons, essentially free of non-neuronal cells, were plated at a density of 8800 cells per 16 mm tissue culture well, and cultured for 48 h in the presence of the given eoncentrations of NGF before counting.8 Essentially no neurons survive in the absence of NGF under these culture conditions. Results are the means of 3 determinations; the vertical bars indicate the S.E.M. (except where this is less than the width of the symbol).

Immunosympathectomy Rats were injected subcutaneously (at the nape of the neck) with a single dose of 50~1 antiserum during day 2 after birth, or with 50~1 per day on days 2-6 following birth. Control animals received pre- or non-immune sera from the appropriate species. Rats from a number of litters were mixed for each experiment, and cross-fostered to randomize maternal influences. Food and water was available ad libitum, and a 12 h light-dark cycle was maintained. Animals were killed 8 or 30 days following the final injection by exposure to ether or by cervical dislocation. Tissues were rapidly dissected and homogenized with ground-glass Kontes homogenizers in ice-cold 50mM Tris acetate buffer, pH 6.0, containing 0.1% (v/v) Triton X-100, Homogenates were centrifuged at 15,000 rpm (27,000 g ; 4°C) for 20 min and the supernatants were assayed for tyrosine hydroxylase activity.‘6*39 RESULTS: BIOLOGICAL

ACTIVITIES

Intact sensory ganglia from embryonic chicks The stimulation of nerve fibre outgrowth from dorsal root ganglia of 8-day-old chick embryos is the classical biological assay for NGF proteins. No differences were observed in the extent or in the dosedependence of the neurite outgrowth in response to bovine and mouse NGFs; maximal fibre outgrowths were therefore induced by 2-5 ng of either NGF per

miililitre culture medium. Dissociated chick sensory neurons Both NGFs maintained alive in vitro the same proportion of neurons isolated from lo-day-old chick sensory ganglia, with the same dose-dependency (Fig. 1). Though neither of the NGFs was able to maintain the survival of all the neurons dissociated from the ganglia (see ref. 8), the addition of maximally effective doses of both NGFs to the same cultures failed to maintain a higher proportion of neurons, though other neurotrophic factors (see ref. 8) can maintain the neurons that fail to respond to NGF. It can, therefore, be concluded that either mouse or bovine NGF can maintain in vitro the same sub-population of neurons at this developmental stage. As neurons are identified in this system by the property of producing axonal processes, it is also cIear that both mammalian NGFs were equally able to stimulate neurite outgrowth from these sensory neurons. Furthermore, the ability of either NGF to maintain sensory neurons alive in vitro declines with the age of the chick embryo from which they are dissected, as previously shown for mouse NGF.’ Thus, 5 ng mouse NGF/ml maintained for 48 h around 5yi, of the neurons from l&day-old chick sensory ganglia (control cultures contained, after 48 h in vitro, around 1% of the neurons plated), while the same concentration of bovine NGF maintained around 6”/;;of the neurons plated (5400 per tissue culture well). Dissociated chick sympathetic neurons

As for sensory neurons, bovine and mouse NGFs

G. P. Harper

17s

6’1cii

by this time.

In contrast. 111the presence of ~OLIIK bIg;‘ml) or mouse NGF (lOng,ml). or in control cultures containing no additibcs.
I$

10

-/,vy

0 g-t

01

1 NGFconcentration

10

100

( ng/ml 1

Fig. 2. Dose-dependency of the survival in cirro of sympathetic neurons from the 13-day-old chick embryo on the presence of NGF from the mouse (W-0) or from bovine seminal plasma (w- -0). Also shown (A) is the survival in the presence of 10 ng/ml of both of the two NGFs together. Neurons, essentially free of non-neuronal cells, were plated at a density of around 5000 cells per 35 mm tissue culture dish, and cultured for 48 h in the presence of the given concentrations of NGF before counting.” Essentially no neurons survive in the absence of NGF under these culture conditions. Results are the means of 3 determinations; the vertical bars indicate the S.E.M. The ED,, for either NGF preparation was 0.6 ng NGF!ml culture medium.

maintained alive in vitro the same sub-population of sympathetic neurons from 13-day-old chick embryos, with the same dose-dependency (Fig. 2). The pattern and extent of neurite outgrowth was indistinguishable in the presence of the two mammalian NGFs. Dissociated

chick parasympathetic

neurons

In the presence of heart-conditioned medium, 98 ) 2% of the neurons dissociated from the ciliary ganglia and counted 1 h after plating (when most had already produced processes) were still present after 2 days in culture. All the cells had produced nerve fibres

P

-1,

1

1

0.1

KU0 1 lm 10 NGFconcentmtion ( q/ml 1 Fig. 3. Dose-dependency of the survival in oitro of rat sympathetic neurons on the presence of NGF from the mouse (O-_-O) or from bovine seminal plasma (e--O). Cells were plated at a density of 10’ cells per 16 mm tissue culture dish, and cultured for 7 days in the presence of the given concentrations of NGF before counting, the medium being changed every 24 h (F. Hefti et al., in preparation). Cell counts are the mean values from 6-10 dishes per point; the vertical bars indicate the S.E.M.; where this is not shown, the S.E.M. is less than the width of the symbol.

(IOngm-1

Sympathetic neurons isolated from the superior cervical ganglia of neonatal rats (approximately 35-40”;1 of those irk tico) required 10 ng mouse NGF per ml (Fig. 3) to survive in ritro. These neurons also put out nerve fibres in response to the mouse NGF. By increasing the NGF concentration from IO to lOGQng/ml. a characteristic and specific induction of the activity of tyrosine hydroxylase by W,, was ohserved, whereas the activity of the non-specific marker lactate dehydrogenase was essentially unchanged (Table 1). In contrast, higher doses of bovine NGF were required to support the survival of rat sympathetic neurons, so that maximal survival was only observed in the presence of 30-IOOng bovine NGF per ml (Fig. 3). Neurite outgrowth was qualitatively indistinguishable from that induced by mouse NGF. However, studies of the induction of tyrosine hydroxylase from cultures maintained at long bovine NGF per ml would be meaningless, as any effect would include a component from differential effects on neuronal survival. The induction of tyrosine hydroxylase was therefore examined by increasing the bovine NGF concentration from 30ng;ml to 3 pg;ml, i.e. by the same factor of IO@fold as used for mouse NGF. The specific activity of the specific neuronal marker tyrosine hydroxylase increased by about X0”;, (Table i), while lactate dehydrogenase was again unaffected. Examination in the plasma clot (chick sensory ganglia) biological assay for NGF of culture media containing mouse and bovine NGFs. with or without exposure to the rat sympathetic neurons, readily revealed the cause of the apparent differences in potency of the two NGFs. Whereas all the mouse NGF added to rat sympathetic neuronal cultures (for 24 h) remained biologically active in the subsequent assay, only around 10’; of the bovine NGF added to the rat cultures could be detected in a subsequent biological assay. Moreover, the same results were obtained if the two NGFs were simply diluted into the media used to culture the rat neurons, i.e. exposure to the rat neurons themselves was not necessary to observe an inactivation of up to 9Ou/, of the bovine NGF added. Subsequent experiments revealed that part of the inactivation of bovine NGF was due to binding to the methyl cellulose component of the culture medium, but that the other components of the medium (L15 containing 5% rat serum) inactivated most of the bovine NGF added. Pheochromocytoma Mouse

essentially

and

PC1 2 cells

bovine

NGFs

the same increases

were able to induce in the specific activities

Properties of bovine nerve growth factor

379

Table 1. Effect of bovine and mouse NGFs on tyrosine hydroxylase specific activity in cultured rat sympathetic neurons

Treatment

Lactate dehydrogenase* pmol/pg protein/min

Tyrosine hydroxylase* pmol/pg protein/min

Cells maintained for 7 days with 10 ng mouse NGF/ml

62 f 9 (4)

415 + 61(4)

Cells maintained for 5 days in 10 ng mouse NGF/ml; then for 2 days in 1 pg mouse NGF/ml

96 + 4t (9)

433 * 17 (9)

Cells maintained for 7 days with 30 ng bovine NGF/ml

67 k 7(4)

323 + 38 (4)

Cells maintained for 5 days in 30 ng bovine NGF/ml; then for 2 days in 3 pg bovine NGF/ml

119 * 1st (4)

319 + 65 (4)

* Values are the means + S.E.M.; the number of determinations is given in brackets. t Significantly different from the appropriate control cultures, P < 0.05 by r-test.

of choline acetyltransferase (ChAT) in PC12 cells, with the dose-dependencies also being virtually indistinguishable (Fig. 4). Moreover, 48 h exposure of these cells to 1 pg mouse NGF per ml culture medium increased the mean cellular volume by 537& while 1 pg bovine NGF per ml increased the mean cellular volume by 46%. Subjective assessments of the nerve fibre outgrowth from these cells also indicated that the extent and dose-dependence of the effect was the same for the two NGFs.

0 L, 0

1

I

x1 loo NGF concentration f ng/ml 1

,

1MKl

Fig. 4. Dose-dependency of the induction of choline acetyltransferase (ChAT) in pheochromocytoma (PC12) cells, clone FR-P9, by NGF from the mouse (0-O) or from bovine seminal plasma (e--O). Cells were plated at a density of 10’ cells per 16 mm tissue culture dish, and cultured for 3 days in the presence of the given concentrations of NGF.32 Each point is the mean of 4 determinations; the vertical bars indicate the S.E.M.

Bovine adrenal medullary cells Bovine and mouse NGFs were able to stimulate increases in the specific activity of tyrosine hydroxylase to around the same extent in these cells, and with

‘1

2’\ 0

r 30

Ku

300 NGFconcentrotlon 1ng/ml I

, 1666

Fig. 5. Dose-dependency of the induction of tyrosine hydroxylase in bovine (calf) adrenal medullary cells by NGF from the mouse (0-O) or from bovine seminal plasma (O---O). Cells were plated at a density of around 2 x IO5 cells/cm’, cultured for I day in medium containing 10% calf serum, and then cultured for 2 days in fully defined medium in the presence of the given concentrations of NGF.““ Each point is the mean of 5 determinations, the vertical bars indicate the S.E.M.

Ci. I’. Harper VI (I/

tained very high levels of renin activity. Even purii!ing the mouse NGF by three passages over an HPLC-gel filtration column failed to eliminate c!rmpletely the renin contaminant (Table 2). Interestingly. a crude extract of the guinea pig prostate and coagulating gland complex also contained no significant renin activity. No increase. in plasma ACTH could be detected following the injection of purified bovine NGF Into the brain ventricle of dogs (i.e. the variation of ACTH

u 025

05 10 50 NGFconcentrotlon( ng/ml 1

M.0

Fig. 6. Dose-dependency of the nerve fibre outgrowth from explants of goldfish retina on the presence of NGF from the mouse (O---O) or from bovine seminal plasma (e--e). Optic nerves were crushed in ~ioo 7 days prior to explant culture. Retinae were cut into 600 pm square explants, plated in a minimum amount of medium for 24 h to obtain a good attachment, and then cultured for 7 days in the presence of the given concentrations of NGF.” The neurite growth index is the product of semi-quantitative assessments of nerve fibre length (1 unit per 200 PM outgrowth) and nerve fibre density (0_4).3h,5’ Results are the means for IS-18 explants; vertical bars indicate the S.E.M.

t

1

0 lul lKl l120l160lzcl l2ui +zcl l320 lime(Inin) lnt&bf injection

-40

0

\

broadly the same dose-dependencies (Fig. 5). Minor differences in Fig. 5 arise from the inherent variability ?f these primary cultures. NGF was not necessary for the survival of these cells in oitro. Goldjiish retinal explant cultures Explants of goldfish retinae were cultured 7 days after crushing the optic nerve in ~ivo; this treatment was necessary to stimulate subsequent neurite outgrowth and to optimize the subsequent responsiveness to NGF administration in vitro.36,s7 Both bovine and mouse NGFs were of equal potency and efficacy in stimulating the outgrowth of neurites from the explants (Fig. 6). An inherent aspect of these cultures is that attachment of the explants to the culture dishes is only maintained in the presence of agents able to keep the cells alive in vitro; no differences were observed between the two NGFs in this respect. Renin activities No could plasma plasma NGF

significant angiotensin I-generating activity be detected in unfractionated bovine seminal when incubated with an excess of sheep angiotensinogen (Table 2); in contrast, mouse purified by conventional procedures”” con-

Introve;ltrlculor InJectlon Fig. 7. Plasma adrenocorticotrophic hormone levels following the intraventricular injection of (a) purified bovine NGF, or (b) purified hog kidney renin. (a) Plasma ACTH levels following intraventricular injection of 200 pg purified bovine NGF, in: (0-O) dog 241; tW-+) dog 9032; (O-.-.-Cl) dog XX; (W.‘.’ ‘.R) dog 9423. (b) Plasma ACTH levels following intraventricular injection of 5 4 containing 0.5 units of purified hog kidney renin (NBC) in: (c----o) dog 1; (e--O) dog S-3-81; CU.-. -0) dog 4-3-81: and (H. W)dog 3-3-81. The vertical axis is at the same scale as Fig. 7(a) to facilitate comparison.

381

Properties of bovine nerve growth factor Table 2. Renin activity in vitro: release of angiotensin I from purified angiotensinogen Renin specific activity (pmol angiotensin I formed/mg protein/h)*

Sample

(4 81,000 f 4900(10) 14.9 + 0.2 (10)

Purified mouse NGFt Whole bovine seminal plasma Crude extract, guinea pig prostate and coagulating glands

5.9 f 0.1 (10)

(b) Purified mouse NGFt NGF after 1st HPLC] purification NGF after 2nd HPLC purification NGF after 3rd HPLCp purification

69,000 f 2000 (6) 16,300 k 140 (6) 1180 k 90(6) 3800 k 180(6) 2800 + 240(6)

* Renin activity was determined as described under ‘Experimental Procedures’. Results are means + S.E.M.; the number of determinations is given in brackets. None of the samples contained increased renin activity after acidification to activate prorenin (data not shown; 4 determinations per sample). t Prepared by the method of Suda et a1.55 $ See ‘Experimental Procedures’. 8 The single NGF-containing peak was arbitrarily divided for renin assays into 2 equal parts following the third HPLC purification step. The increase in renin activity compared to the material following 2 HPLC cycles probably reflects an elution of some renin previously binding to the column.

a decrease in the angiotensinogen content and an increase in the angiotensin I levels in the cerebrospinal fluid (CSF), and a small increase in the plasma ACTH content (Table 3). The intraventricular administration of hog kidney renin also increased the CSF levels of angiotensin I.

content with time was always within the normal range of values in control animals; Fig. 7a), whereas the injection of purified hog kidney renin in parallel experiments produced a marked temporary increase in ACTH levels (Fig. 7b). Moreover, in rats, intraventricular administration of purified5’ mouse NGF caused

Table 3. Effects in uiuo of intraventricular

Measuremen r

saline

injection in rats of mouse NGF

Intraventricular injection of: hog kidney reninf mouse NGFt

Cerebrospinal fluid angiotensinogen content (pm01 angiotensin I generated/ml CSF)

17.5 f 1.2(10)

1.9 & 0.2 (10)

n.d.

Cerebrospinal fluid angiotensin I content (fmol/ml CSF)

17.6 + 1.5 (10)

3400 * lOOO(10)

4500 f 500 (10)

Plasma ACTH content (pg/ml plasma)

185 k 70(8)

420 ? 140(8)

n.d.

* Measurements were made as described under ‘Experimental Procedures’, samples being collected 5 min after intraventricular injection. Results are the means + S.E.M., the number of determinations is given in brackets: n.d. not determined. t 0.5 pg mouse NGF, prepared by the method of Suda er ~1.,‘~ in 0.5 ~1. $0.01 units of hog kidney renin, in 0.5 ~1.

G. P. Harper Table 4. Antiserum

or purified antibody titres for bovine conventional biological assay Immunizing antigen

Antiserum or antibody preparation

RESULTS:

and antibody

IMMUNOLOGICAL

and mouse

NGFs

111the

Tltre in biologlcal assay* vs mouse NGF bs bovine NGt_____~~~

NGF

I 17 ngjml

I17 ng, ml

mouse NGF

x 12,500

x 12,500

mouse

Antibodies from sheep Whole antiserum from sheep Whole antiserum from rabbit Rl Whole antiserum from rabbit R2 * Antiserum

el al

mouse

NGF

X 3000

X 30(K)

mouse

NGF

X 1000

X 1000

titres are defined

PROPERTIES

Antiserum titres in the conventional biological assay

As required by the operational definition of the NGF (see Introduction), the biological activity of either mouse or bovine NGF in the classical biological assay was completely inhibited by antisera to purified mouse NGF. The titres for a number of different antisera against mouse NGF were essentially identical when tested against mouse and bovine NGFs (Table 4). However, antisera raised against bovine NGF were much less potent in inhibiting the in vitro response to mouse NGF than they were effective in blocking the response to the homologous bovine NGF (Fig. 8). term

under

‘Experimental

Procedures’.

antisera to bovine NGF, or of bovine NGF against antisera to mouse NGF, were uniformly unsuccessful, though each antisera produced the expected immunoprecipitate bands when tested against their homologous antigens (data not shown).

A single injection into neonatal rats of antiserum against mouse NGF (sheep, see Table 4) caused the tyrosine hydroxylase levels in the superior cervical

Comparative immunodifisions

Attempts to produce precipitin bands by double immunodiffusion (Ouchterlony technique; see refs 27, 30 for experimental details) of mouse NGF against

:

i ‘1,

/ \ ‘1I : \\ : \ \\ : \ : ‘? /

’ : I : i I :

AOR VAS

/ i

/’ ,,A0

I / 6

~mmummhon

.

.

:-------

i\,

I41

T

6

10

12

14

16

18

20

22

Weekspost- immutnzotton

Fig. 8. Antiserum titres, as determined in the conventional biological assay for NGF, for antisera from rabbits R7 (full lines) and R8 (dotted lines), immunized with purified bovine NGF. Titres were determined for each bleed, Versus 1BUjml (see ‘Experimental Procedures’) of either bovine (0) or mouse (0) NGFs. The arrows indicate the times of boosting of the animals.

SCG AIR

VAS

Fig. 9. Total tyrosine hydroxylase content (per pair of organs) of the superior cervical ganglia (SCG). adrenal glands (ADR) and vasa deferentia (VAS) of rats following treatment with non-immune sera (controls; open columns) or with antisera (shaded columns) raised against mouse NGF, or against bovine NGF. The titres for these two antisera were x 12,500 for the antiserum against mouse NGF (determined in the classical biological assay tjersus 1 BU/ml mouse NGF) and x 18,750 for the antiserum against bovine NGF (determined versus I BUjml bovine NGF). All injections (50 ~1) were made subcutaneously on day 2 of life, and animals were killed 8 days later (antiserum to mouse NGF) or 30 days later (antiserum to bovine NGF). The number of determinations for each treatment are given in brackets; values are means, kS.E.M. (or the individual values where only 2 daterminations were made).

Properties of bovine nerve growth factor ganglion to decrease to undetectable values, while the levels in the adrenal glands increased substantially and those in the vas deferens were unaffected (Fig. 9). In contrast, a single injection of the same volume of an antiserum against bovine NGF (rabbit 8; week 13; Fig. 8, i.e. an antiserum having approximately the same titre in the biological assay when tested against its homologous NGF as the sheep anti-mouse NGF antiserum) caused only a relatively small (35%) permanent decrease in the levels of tyrosine hydroxylase in the superior cervical ganglion, while those in the adrenal gland and vas deferens were unaffected. Attempts were made to increase the effects of the antiserum to bovine NGF by making 5 injections of antiserum on succeeding days. While the results indicated a greater decrease in the tyrosine hydroxylase content of the superior cervical ganglia (30 days after the final injection) than was apparent after only a single injection of antiserum, and an increase in the adrenal tyrosine hydroxylase levels compared to control animals (given 5 injections of pre-immune serum), it is impossible to rely too heavily on these measurements (data not shown), as, in contrast to the effects of single injections, animals receiving 5 doses of antiserum to bovine NGF were substantially smaller than the corresponding control animals. Furthermore, their development of fur was appreciably retarded, especially at the site of the injection.

DISCUSSION Actions C$nerve growth factor in neural systems Among the biological activities of mouse NGF investigated in detail in many laboratories are: (i) the ability to maintain alive in vitro, and to stimulate nerve fibre outgrowth from defined sub-populations of sensory’ and sympatheticr6 neurons at defined developmental stages of the chick embryo, and similarly for sympathetic neurons from neonatal rats; (F. Hefti, H. Gnahn, M. E. Schwab & H. Thoenen, in preparation); (ii) the inability to maintain alive in vitro, or to induce neurite outgrowth from embryonic chick parasympathetic neurons (for review, see ref. 31), a consequence of the absence of NGF-receptors on these cells;48 (iii) the ability to induce specifically increases in the levels of the neuronal marker tyrosine hydroxylase in rat sympathetic neurons (F. Hefti et al., in preparation) and in bovine adrenal medullary cells,44 and to induce increases in choline acetyltransferase in PC12 cells;” (iv) the ability to increase the cellular volume,25 indicative of a general metabolic stimulation, and to induce nerve fibre outgrowth,25,32 from PC12 cells; (v) the ability to stimulate nerve fibre outgrowth from explanted goldfish retinae.57 In all these systems (but see below), bovine NGF was indistinguishable from mouse NGF, both with respect to the extent of the effects, and to their dosedependencies. The only exception to this statement occurred in

383

the cultures of rat sympathetic neurons. However, it is clear that the rat neurons responded to the biologically effective concentrations of mouse and bovine NGFs in exactly the same manner. In considering the results of biological assays of media from cultures of rat sympathetic neurons, it should be recognized that this assay is only semi-quantitative; however, there was clearly an inactivation of a substantial portion of the bovine NGF following exposure to the medium used to culture the rat neurons. Thus, bovine NGF, but not mouse NGF, was subject to inactivation by binding to methyl cellulose and by binding to, or enzymic inactivation by, other components of the medium, presumably present in the rat serum. The data presently available do not allow a resolution between a binding and an enzymic mechanism for the latter inactivation. Harper et aLz8 have previously reported that bovine NGF is substantially ‘stickier’ even than the notoriously difficult mouse NGF. Mouse NGF is well-known for its ability to bind nonspecifically to surfaces4’ and to other proteins, including to serum components,4g,55 although the present data would suggest that the binding of mouse NGF to components in rat serum does not lead to any detectable inactivation. Preliminary data (H. Gnahn, unpublished results) suggest a similar inactivation of bovine NGF, but not of mouse NGF, occurs in rats in viuo, as judged by the induction of tyrosine hydroxylase activity in the superior cervical ganglia following NGF administration. Renin activities Recent studies have indicated that some of the biological activities originally attributed to mouse NGF, e.g. induction of ornithine decarboxylase in the central nervous system, stimulation of increased thirst and sodium appetite following intracerebral injection, etc., are in fact due to a contamination of standard mouse NGF preparations with renin,3*15*26.45*46 also present in large amounts in the mouse submandibular gland. The renin contaminant appears to be rather difficult to eliminate,’ 5,46 which is in agreement with the present results, in which even three HPLC-gel filtration cycles failed to remove all the renin activity. In contrast, even whole bovine seminal plasma was essentially renin-free (i.e. there was no generation in vitro of angiotensin I from purified angiotensinogen). The absence of renin activity in such unfractionated materials could, however, be explained as being due to the concomitant presence of renin inhibitors. Therefore, purified bovine NGF was also tested: when injected intraventricularly into dogs, purified bovine NGF failed to stimulate the release of ACTH from the pituitary gland. Renin in the central nervous system, by its formation in situ of angiotensin II, is able to stimulate the pituitary-adrenocortical axis by the enhanced release of ACTH.42 Such effects are shown in Table 3, following the intraventricular injection in rats of mouse NGF. The renin contamination in the mouse NGF preparation generates angiotensin

G. P. Harper

3x4

I from angiotensinogen, so that the cerebrospinal fluid concentrations of the substrate angiotensinogen fall, and those of angiotensin I rise. Angiotensin I is converted intracerebrally to angiotensin II. which then acts to cause plasma ACTH levels to rise slightly (Table 3). Thus, whereas rigorous precautions are necessary when using mouse NGF to ensure that the observed effects cannot be attributed to renin contaminants, the use of the renin-free bovine NGF allows the definite exclusion of such artefacts. It is likely, as the guinea pig prostate and coagulating gland complex also contains no detectable renin activity (Table 2) that the use of purified guinea pig NGF would also have this advantage. These results allow us to conclude that the ‘neural’ biological effects demonstrated in the present paper for mouse and bovine NGFs must be due to the NGF molecule and not to renin contaminants (see also ref. 46). Biological

activities of‘ nerve growth factors

The conclusion can therefore be drawn, from the marked parallelism of the biological activities of bovine and mouse NGFs, that probably all the members of the NGF group of proteins exhibit the wide range of effects and target cells delineated in detail for mouse NGF. As described in the Introduction, other evidence for this is virtually completely absent (other than effects on intact sensory ganglia from chick embryos). Just as mouse NGF is essential for the survival in vitro of sympathetic neurons from neonatal rats (see above), so the administration of exogenous mouse NGF to neonatal mice or rats can maintain in vivo excess neurons that normally die during development.31,56 The NGF from Vipera russelli was also reported to have this latter activity.6 Vipera russelli NGF could also maintain alive in vitro sensory neurons from g-day-old chick embryos,’ though non-neuronal cells were also present in these studies and possibly contributed to the maintenance effect (see ref. 8). Immunological

properties and immunosympathectomy

Though the biological activities of bovine and mouse NGFs are thus virtually indistinguishable, their immunological properties differ substantially. This is not apparent from the biological assay titres of antisera raised against mouse NGF (Table 4), but it is very clear when the titres for antisera raised against bovine NGF (Fig. 8) are considered. Clearly antisera against bovine NGF are only poorly effective at inhibiting the effects of mouse NGF. This ‘non-symmetry’ in the antiserum titres might indicate that major antigenie determinants present on the bovine NGF molecule are absent from the mouse NGF protein, while most of the main antigenic determinants of mouse NGF are also present on the bovine NGF molecule. The mechanism of action of NGF is thought to involve the production of ‘endogenous’ NGF by per-

PI ril

ipheral effector-organs, and a retrograde axonal transport of the NGF back to the innervating neuronal cell bodies (for reviews set refs 31,56). The evidence for the production of NGF by the effector-organs, however, is at present only indirect. as the levels are too low to be detected by current NGF assays. Thus. this model relies on indirect evidence. the most compelling of which is the fact that the administration to neonatal mammals of antibodies to (mouse) NGF causes the destruction of the sympathetic neurons (immunosympathectomy). by inactivating the ‘endogenous’ NGF that is essential at this developmental stage for the survival in rice of the sympathetic neurons. The time-course of immunosympathectomy following a single injection of antibodies was previously determined by Goedert et u/.‘*.*’ Thus. after the administration of antibodies to mouse NGF. the levels of tyrosine hydroxylase (TH), an enzyme localized exclusively to the neurons in sympathetic ganglia. were permanently and almost fully reduced within 2 days of injection.22.24 Neuronal cell death, as judged morphologically, lagged slightly behind the decrease in TH levels,24 so the present studies used the irreversible decreases in TH content at least 8 days after injection as a measure of the extent of neuronal cell death (see ref. 22). As a consequence of the destruction of the sympathetic nervous system by antiserum against (mouse) NGF, the adrenal medulla is activated,’ so the levels of TH in this organ increase.13 The adrenergic innervation of the vas deferens is, for unknown reasons, insensitive to immunosympathectomy, so the TH levels in this organ are unaffected.23 All these effects were confirmed in the present studies, following the administration of antiserum against mouse NGF (Fig. 9). However, the antiserum to bovine NGF produced a relatively low degree of immunosympathectomy. though 30 days postinjection were allowed in case the destruction of sympathetic neurons by this antiserum should be slower. The survival of the greater part of the sympathetic nervous system prevented the compensatory activation of the adrenal medulla, so adrenal TH did not increase. and the adrenergic innervation of the vas deferens was also unaffected. The latter data are interesting because bovine NGF is produced by accessory sex organs innervated by the ‘short’ type of adrenergic neuron supplying the vas deferens. This suggests that these ‘short’ adrenergic neurons are not resistant to the effects of antiserum against mouse NGF (see refs 31,37) because of the production of a different type of NGF by their effector-organs. The ability of an antiserum against NGF to cause immunosympathectomy in rats depends, according to the model delineated above, directly on its ability to inhibit the NGF endogenous to rats. As antisera to bovine NGF only poorly inhibit in vitro the biological activity of mouse NGF (Fig. 8). it is not surprising that they are also relatively poor in inhibiting irt r:ico

Properties of bovine nerve growth factor the biological activity of rat NGF, which is likely to resemble closely the mouse NGF molecule. Therefore antisera against bovine NGF produce a poor immunosympathectomy in rats. Repeated doses of antiserum against bovine NGF, while leading to marked non-specific effects on body growth, etc. (see Results), clearly were able to reduce TH levels in the sympathetic ganglia further (indicative of the death of more neurons), with adrenal TH then becoming stimulated in compensation; vas deferens TH remained unaffected. It should be emphasised that it was not the purpose of the present investigation to determine the ideal injection protocol for obtaining as complete an immunosympathectomy as possible. It is likely that this would require isolating the antibodies against bovine NGF by affinity chromatography.54 It is, however, justified to compare the effects of two whole antisera having approximately the same titres when tested in vitro against their respective NGFs (Table 4; Fig. 8). Previous studies2*4.5,‘3.50 have demonstrated limited immulological cross-reactivities in vitro (i.e. in the classical biological assay) or in uivo (i.e. in causing immunosympathectomy) between NGFs from the mouse and from snake venoms. Furthermore, the inability of antisera against mouse NGF (Table 4) to distinguish in the biological assay between mouse and bovine NGF has already been observed using other mammalian NGFs.~‘~~*~~~~~~However more highly resolving immunochemical techniques (comparative immunodiffusions, 2-site radioimmunoassays) have demonstrated that substantial immunological differences can be observed between the mammalian NGFs even using these antisera to mouse NGF.27,2g*30 As cross-reactivity shown here, the immunological between mouse and bovine NGFs is low enough to

385

prevent successful cross-immunoprecipitation; the use of the 2-site radioimmunoassay to quantify the degree of cross-reactivity2g~30 indicated that only around 10% of the antibodies purified from the sheep antiserum (Table 4) could effectively recognize bovine NGF in this assay system. Interestingly, the present and previous studies 27~2g,30indicate that guinea pig NGF is immunologically more similar to mouse NGF than is bovine NGF, which is consistent with the ability of antisera against guinea pig NGF to produce as complete an immunosympathectomy (in mice) as do antisera against mouse NGF.” CONCLUSIONS Mouse and bovine NGFs are extremely similar in their biological activities, but immunologically they differ substantially. These data would seem to support the earlier hypothesis 2g.30,3’ that the NGF molecules contain a highly conserved active site, which binds to the NGF cell-membrane receptor and prompts the subsequent biological effects, while the remaining parts of the NGF molecule are not under such conservative evolutionary restraint, and have therefore diverged more fully. Acknowledgements-GPH was supported by the Deutsche Forschungsgemeinschaft (grant Th 270/l); FH was a Fellow of the Alexander von Humboldt-Stiftung; JET was the recipient of an N.I.H. Research Career Development Award (NS 00338). We thank E. Lamperti for his help with the immunosympathectomy experiments. We are very grateful to the Priif- und Besamungsstation MiinchenGrub e.V.; Besamungsvereinigung Nordwiirttemberg e.V.; Besamungsstation Herbertingen e.V.; Rinderproduktion Niedersachsen GmbH; and Besamungsverein Neustadt an der Aisch e.V., for supplies of bovine seminal plasma.

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(Accepted

24 August 1982)