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Characterization of antisera raised against cultured rat sympathetic neurons
Neuroscience Vol.5, pp.2239to 2245 Pergamon Press Ltd 1980. Rintcdin GreatBritain 0 iBR0
0306_45Ui8O/lntt-2239M2.0010
CHARACTERIZATION OF ANTISERA RAISED AGAINST CULTURED RAT SYMPATHETIC NEURONS VIRGINIA M. LEE,’ M. L. SHELANSKI’and L. A. GREENE’ Departments of Neuropathology, Harvard Medical School, and Pharmacology, New York University School of Medicine, U.S.A. Abstract-Antisera were prepared against monolayer cultures of newborn rat sympathetic neurons and characterized by “Cr release microcomplement fixation, cytotoxicity and indirect immunofluorescence assays. After absorption with rat liver, kidney, spleen and thymus to remove activity against common rat antigens, the antisera retained activity against cultured sympathetic neurons, brain and adrenals. Indirect immuno~uorescence showed that the antigens recognized were widely distributed in the brain, while in the adrenaf gland they were limited to the meduliary cells. Absorption with the brain completely removed the activity of the antisera against sympathetic neurons; in contrast, partial activity was retained after repetitive absorption with adrenals. The antisera were also characterized with respect to recognition of the PC12 clonal line of rat pheochromocytoma cells. Under normal growth conditions, PC12 cells resemble their normal counterparts, adrenal chromaffin cells, while after treatment with nerve growth factor, they acquire the differentiated properties of sympathetic neurons. Both untreated and treated cells were recognized by the antisera and indirect histofluorescence on living cells indicated that at least some of the antigens on these cells (as well as on sympathetic neurons) are exposed on the cell surface. However, differences were noted in recognition of nerve growth factor treated and untreated PC12 ceils. For example, the former, but not the latter were recognized in cytotoxicity assays. Also, absorption of the antisera with treated cells removed all activity against rat sympathetic neurons, whereas repetitive absorption with untreated PC12 cells removed only part of the activity. Such findings indicate that PC12 cells treated with nerve growth factor and sympathetic neurons exhibit antigenic similarities and that our antisera can be used to identify, isolate, characterize and localize neural-specific surface antigens.
ONE approach
pathetic neurons including neurite out~owth, electrito identify and characterize cell surface cal excitability and presence of synaptic vesicles components of neurons is the use of specific antisera prepared against neural tissue (cf. reviews by FIELDS, (GREENE & TISCHLEIR,1976; DICHTER, TISCHLER & 1976 and BOCK, 1978). Among the challenges of such an approach is obtaining homogeneous and defined populations of neuronal cells with which to prepare and characterize antisera. To this end, several studies have employed cell lines that express neuronal phenotypes (AKE~ON & HERXHMAN, 1974; SCHACHNER, 1974; FIELDS, 1976; STALLCUP & COHN, 1976). For example, we have studied the antigenic properties of the PC12 clonal line of rat pheochromocytoma cells (LEE, SHELANSKI & GREENE, 1977; LEE, GREENE & SHELANSKI, 1980). Such cells display many differen-
tiated characteristics of normal adrenal medullary chromaffin cells (GREENE & TIXHLER, 1976; GREENE & REIN, 1977). When PC12 cells are treated with nerve growth factor (NGF), however, they cease mitosis and acquire a number of properties of sym-
Current addresses: ’ Department of Neuropathology, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. ’ Department of Pharmacology, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A. ~~~re~juf~o~s: AsRS, antisera against rat sympathetic neurons; NGF, nerve growth factor; PC12+, PCIZ-, PC12 pheochromocytoma cells grown in the presence and absence, respectively, of NGF.
GREENE, 1977; TISCHLER& GREENE, 1978). After absorption with non-neural tissue, antisera against PC1 2 cells either untreated with NGF (PC12 - cells) or treated with NGF (PC12+ cells) recognize antigens that are specific to neural and/or adrenal tissue (LEE er al., 1977). Moreover, indirect i~munofluorescent staining and complement-mediated cytotoxicity assays indicated that some of these antigens are present on the cell surface. Furthermore, such studies revealed that As against PC12 cells also recognize components that are present on the surface of sympathetic neurons. In addition to cell lines, defined populations of neurons can also be used to derive useful antisera. Cultured s~pathetic neurons appear to be a particularly attractive system. These can be obtained in reasonable numbers and can be maintained in monolayer culture essentially free of non-neuronal cells (MAINS & PATIXRSON,1973; ESTRIDGE& BUNGE, 1978). In addition, since sympathetic neurons appear to share a number of phenotypic properties with PC12 cells, comparison of antisera directed against these two cell types might establish (a) the degree of antigenic similarity between sympathetic neurons and PC12 cetls, (b) the degree to which such a similarity is altered by treating PC12 cells with NGF and (c) the
2239
2240
VIH~IC.IAH. 1.~. M I,.
SHELANKI
suitability of using PC1 2 cells as a practical source for purification of such neuronal surface molecules. The purpose of the present study is to describe the preparation of antisera against cultured rat sympathetic neurons (AsRS) and the characterization of such antisera by microcomplement fixation, complementmediated cytotoxicity and indirect immunofluorescence.
EXPERIMENTAL
PROCEDURES
Primary cultures of dissociated rat sympathetic neurons were prepared from superior cervical ganglia following procedures slightly modified from those described for cultures of chick sympathetic ganglia (GREENE, 1977). Briefly, ganglia were dissected from neonatal (I-4 days) rats. pooled and treated for 45 min at 37’ with 0.1% trypsin (DIFCO 1:250) in 2ml of Ca*+-Mg’+-free phosphate
buffered saline. Following this, the trypsin solution was removed, the ganglia were washed 3 x with culture medium (85% RPM1 1640, lo”/; horse serum, 5% fetal calf serum and 50ngm/ml NGF) and dissociated by vigorous trituration through a Pasteur pipette. Cells were plated in collagen-coated 35mm dishes at a concentration of eight ganglia/dish. Cytosine arabinoside (10 p(M)was added from
and L.. A i;Kt
I h,
“Cr formed
release microcomplemcnl tixatior: i~>\;~\; in <.. pi’ as described previously (Wrc;/~ 1 I 1%';. !!IVP!: REYS. MCCUNE, CHESS, HERMANN. MALESKA. Mnz... )‘.i.i;HAM, SC.HLO~~MA~& SrRo~it>c;t:a. 1Y7h) fiohq:;i \)I:( ~1 the bulk-absorbed whole AsRS antisera v er-e I;)un:l ISI tic anti-complementary. cxperimcntr uerc c,irricti IW! \irt/: !li!* y-globulin fraction of the annsent. This wds prcp,rtcd I/\ previously described (WII.I.IAMS & Ctwst.. 10671 ,411 411~. tions of this fraction were from a 2 mg ml stock ‘;l:iuti
Complement-mediated cytotoxic acttvities of dnttsera were assayed as described elsewhere (LI,I. et (ii.. lY80.1. Briefly. dissociated primary rat sympathetic neuron\. PC12+ cells or PC‘12 - cells were plated on collagencoated microtiter plates. Both sympathetic neuron\ md PC12+ cells were used when abundant neurites wcrc cvident (generally 2 4 days after plating) Cell dcathq ~.crc scored on a subjective scale of 1 4. wtth 4 representtng over 75:,, destruction and 1 + less that] 75”,, destruction (see LEL er al., 1980 for further details). Scoring M~C independently performed by two observers.
the next day onward to eliminate dividing non-neuronal cells. For both immunization and for microcomplement fixation assays, the ganglion cell cultures were used when all non-neuronal cells were eliminated (usually after 7- 10 days of culture in complete medium containing 10 pM cytosine arabinoside). At this time, the neurons had produced a dense network of neurites. For cytotoxicity assays, cells were plated in collagen-coated microtiter dishes (Falcon No. 3034) and assayed as previously described (LEE et al., 1980). For indirect immunofluorescence, cells were plated onto collagen-coated glass coverslips and were used after 2 days in vitro (LEE er al., 1977). PC12 cells were maintained in culture with (PC12+ cells) or without (PC12cells) 50 ngm/ml 2.5 S NGF (BOCCHINI & ANG~LETTI, 1969) as previously described (GREENE & TISCHLER, 1976). For cytotoxicity assays, indirect immunofluorescence and microcomplement fixation assays, PC12 cultures were prepared as previously described (Lrf: ct al.. 1977: 1980).
Antisew Antisera against the cultured rat sympathetic neurons (AsRS) were prepared in guinea-pigs by procedures previously described for pheochromocytoma cells (LEE et al.. 1977; 1980). Briefly, about IO6 neurons were washed free of culture medium, scraped from the dishes, emulsified with adjuvant and injected into guinea-pigs at weekly intervals. Complete Freund’s adjuvant was used for the first injection and incomplete adjuvant for all subsequent injections. The antisera were collected after the 7th immunization. Heat inactivation and bulk absorption of the antisera with rat kidney, liver, spleen and thymus were performed as described elsewhere (LEE et al.. 1977). The results reported in this paper were from unpooled antisera obtained from two guinea-pigs. Each yielded qualitatively identical results. The preparation and characterization of antisera against PC12 cells have been previously given (Lee et al., 1977: 1980).
Indirect immunofluorescence on frozen tissue sections and on living cells were performed as described in our previous study on PC12 cells (LEF et ul.. 1977)
Absorption Antisera were absorbed with particulate fracttons of various tissue preparations (see text) using techniques given in detail elsewhere (LEE et al., 1977; 1980). Generally, antisera (at a I :4 dilution) were absorbed with a tissue pellet at a ratio (serum :tissue, v;‘v) of 2 : 1.
RESULTS
Microcomplement
jixation
After bulk absorption with non-neuronal rat tissue. AsRS ?;-globulin (stock solution 2 mg/ml) had an antigenie titer (50% fixation point) in the microcomplement fixation assay of between 1 :20 and 1 :40 against homogendtes or particulate fractions of cultured rat sympathetic neurons (Table 1). Both particulate and supernatant fractions (prepared by a 100,000 g centrifugation for 1 h) of the homogenate were recognized to the same extent on a per mg protein basis. These titers were significantly higher than the ‘background’ (1 :8) obtained with AsRS y-globulin in the absence of target tissue. Also, sympathetic neurons and other tissues at up to 25 pg protein per sampie alone did not fix complement. To test the specificity of the AsRS y-globulin, particulate fractions of homogenates of various rat organs were also tested as targets. Of the tissues tested, only adrenal and brain were recognized to a significant extent (Table 1).
FIG. 1. Indirect immunofluorescent staining of rat adrenal and cerebellum with antisera to rat sympathetic neurons. (A) Sagittal section of cerebellum stained with AsRS (1%). The pattern of fluorescence shows weak staining of the molecular layer (m.), a more intense, diffuse staining of the granule cell layer (g.) and intense staining of the Purkinje cell layer (P.). (B) Cross section of adrenals stained with AsRS (1:20). Note that staining is limited to the medulla. Magnification x 192 in both A and B.
2241
FIG. 2. Indirect immunofluorescent staining of cultured rat sympathetic neurons, Pc‘l2- and PC‘12a cells with antisera to rat sympathetic neurons, Panels A, C, E and G show phase-contrast micrographs of cells while panels B, D, F, H and I show same fields photographed under fluorescence optics. (,4 and B) Rat sympathetic neurons (2 days in culture) stained with AsRS (I ~160).(C and D) PC12 - cells stained with AsRS (1:160). Note limitation of staining to caps. (E and F) PCIZ- cells stained with AsPC12+ (1 :?.oO),Note uniform distribution of staining. {G, H and I) PClZ+ cells (pre-treated with NGF for about 10 days and then passaged onto cover slips for 2 days in the presence of NGF) stained with AsRS (I :i60). Panel H shows detail of staining of neurites while panel I shoKs detail of staining af cell hodles. Magnification x 3 IV 2242
2243
Sympathetic neuron antiserum TABLE
I.
Y-GLOBULIN
MICRO-COMPLEMENT FROM
ANTISERA
AGAINST
FIXATION TO RAT
VARIOUS
ACTIVITIES
SYMPATHETIC
2,
TABLE
OF
OF
NEURONS
NEURONS
TARGETS
RESIDUAL
Y-GLOBULIN
AFTER
AGAINST REPETITIVE
Titer*
Target
TABLE
3.
RESIDUAL
TIC NEURONS
cYTOTOXIC
AGAINST
VARIOUS
LATE
CULTURED
FRACTIONS
RAT
None Cultured rat
None Cultured rat sympathetic neurons PClZ+ cells PC12- cells Brain Adrenal Adrenal and Brain
I :2cLl :40
Adrenal and brain
The ability of AsRS to recognize cell surface antigens was tested by means of complement-mediated cytotoxicity assays (Table 3). When tested against cultured rat sympathetic neurons, AsRS had a cytotoxic titer (i.e. the dilution at which 50% of target cells were killed) of between 19 and 1:16. Destruction of cell bodies as well as neurites was evident. In the absence of complement, AsRS had no cytotoxic activity, even at a dilution of 1: 1. AsRS was also tested against PC12+ and PC12- cells. For PC12+ cells, cytotoxicity was present against both cell bodies and neurites, and at a titer similar to that against sympathetic OF ANTISERA
TO RAT
AFTER ABSORPTION
WITH
SYMPATHEPARTICU-
TISSUES
Rat sympathetic neurons
PClZ+
cells PClZ-
cells
1 :I6 0
I :I6
0
0
NT
0 1:6t 0
0 0 NT NT
NT 0 NT NT
0
NT
NT
* Final dilution of AsRS yielding 2+ (50%) complement-mediated cytotoxicity. Assays were carried out after three repetitive absorptions with indicated tissue. Experiment was repeated three times with identical results. t Titer unchanged after two additional repetitive absorptions. NT = not tested.
Cytotoxicity
OF VARIOUS
I :1t
FRAC-
TISSUES
Residual titer*
syinpathetic neurons PCl2f cells PCIZ- cells Adrenal Brain
NEURONS
PARTICULATE
Tissue used for absorption
Residual titer* using as target: Tissue used for absorption
ACTIVITY
SYMPATHETIC
SYMPATHETIC
WITH
OF VARIOUS
FIXATION RAT
adrenals, AsRS retained a partial titer against rat sympathetic neurons. Such results indicate that (i) the antigens recognized by AsRS are also present in PC12+ cells and brain, and (ii) that some, but not all of these antigens are present in significant amounts in PC1 2 - cells and adrenals.
ACTIVITY TARGETS
TO
* Final dilution of AsRS y-globulin yielding 50% fixation. Assays were carried out after three repetitive absorptions with indicated tissue. Experiment was repeated three times with identical results. t Titer unchanged after two additional repetitive absorptions.
* Final dilution of AsRS y-globulin yielding 50% fixation. Ranges represent results from +8 or ++3 separate experiments. Since NGF-responsive PC12 pheochromocytoma cells share a number of properties with sympathetic neurons and are a useful model neuronal system, these cells were also tested as targets for AsRS y-globulin. Both NGF-treated (PC12+) and nontreated (PC12-) cells showed significant levels of fixation (Table 1). However, while the titer against PC12+ cells was similar to that against sympathetic neurons, the titer against PC12- cells was significantly lower. Cross-absorption experiments were carried out to test the extent to which antigens recognized by AsRS were shared with other targets. Accordingly, AsRS was absorbed 3-5 times with particulate fractions of rat sympathetic neurons, PC12+ cells, PC12- cells, rat adrenal or rat brain. In each case, after the antiserum was absorbed with a given tissue the antigenic titer against that tissue was lost. Table 2 shows the residual titers of AsRS y-globulin against rat sympathetic neurons after the various cross-absorptions. No antigenic activity was detectable after absorption with PC12+ cells or with brain. In contrast, even after multiple absorption with PC12- cells or with
ANTISERA
ABSORPTIONS TIONS
5 I:8 1:2&l :40+ 1:I&1 :20+ + 1:2@1:30++ < I:8 I1:8 < I:8 < 1:8 1:2(&l :40+ 1:10-l :20+
None (control) Cultured rat sympathetic neurons Adrenal Brain Liver Kidney Thymus Spleen PC12 + cells PC12- cells
MICROCOMPLEMENT
FROM
2244
VIRGINIA H. LE.
M. L. SHELANSKI and
neurons. In contrast, even at a dilution of 1: 1. AsRS had no detectable cytotoxic activity against PCIZcells. Cross-absorption experiments were carried out to test the extent to which antigens recognized by AsRS in the cytotoxicity assay are shared with other cell types (Table 3). After two absorptions with PCl2+ cells, AsRS lost all cytotoxic activity against rat sympathetic neurons as well as against PC12+ cells. However, while repetitive absorption with PC1 2 cells greatly reduced the cytotoxic titer against rat sympathetic neurons, a significant cytotoxicity was still retained. Also, the cytotoxic activity of AsRS against cultured sympathetic neurons was completely removed after absorption with particulate fractions of brain; after repetitive absorption to constant titer with particulate fractions of adrenals, in contrast, the cytotoxicity titer was only partially decreased. Immunojhorescent
localization
Indirect immunofluorescent localization of antigens recognized by AsRS were carried out on 10 /*m frozen tissue sections of brain and adrenal. In the adrenal gland, specific staining was present in the medulla, but totally absent in the cortex (Fig. 1). In the brain, staining was widespread and diffuse in the gray matter. There were. however, indications of differences in staining by different cell types. For example, as shown in Fig. 1. in the cerebellum, staining was particularly intense in the Purkinje cell layer. Controls in the same tissues with pre-immune serum or with second antibody alone gave no staining. Also, AsRS did not stain frozen sections of liver or spleen. Localization of antigens recognized by AsRS was also carried out on living cultured sympathetic neurons, PCl2+ cells and PCl2cells. Under the conditions of such experiments, the antisera presumably do not enter the cells and staining is limited to surface antigens. With cultured sympathetic neurons, specific staining was uniformly distributed on both cell bodies and neurites (Fig. 2B). A similar staining pattern was also observed with antiserum against PC12 + cells. AsRS also stained PC12 cells. However, in this case, staining was not uniform. As shown in Fig. 2D. staining of PC12 - cells appeared to be limited to a cap. With PC12 + cells (Fig. 2H and I) non-uniform localization of staining was less pronounced than on PCIZcells, but was also evident. In contrast. as previously reported (LEE et al., 1977) antiserum against PC12 cells produced an evenly distributed stain over the entire cell surface (Fig. 2F).
DISCUSSION The present work presents the characteristics of antisera (AsRS) prepared against neuronal cultures of newborn rat sympathetic ganglia. After bulk absorption with a variety of tissues, AsRS still recognized neuronal components. As shown by cytotoxicity
I.. 12 timr.ur
assays and indirect immunofluorescencc io~.&lzau~.~l on living sympathetic neurons in culture it h\i some of these components are exposed c)n th,, surfact~ of cell bodies and processes. AsRS also r~:~ogni~cti brain tissue, and absorption experiment\ i:>dicatcd that all of the antigens recognized by thi\ :r:ltism~l!: are also present in brain. Such findings suppc\r th;lt, while AsRS may recognize components \p>Glic 1~: neural tissue, it does not rccognizo ;rntigenL. tlh;tt ,!r~ specific to sympathetic neurons. Our histofluorescence studies indicated thirt 111~distribution of antigens recognized hy AsRS uithin the brain is not uniform. Not only was staining substantially higher in gray malter than in white matter. hr~t there was also evidence for enhanced staining witfun gray matter such as in the Purkinje cell layer of the cerebellum. Since AsRS recognizes at least ssvsral different components. the results of such locahzation ixperiments must be interpreted with care 4 final analysis will await the preparation of monospecific antibodies against single components ~nong me several recognized by AsRS. Complement fixation and immunofluorcscent staining revealed that AsRS recognizes adrenal medullary cells. Such commonality of antigenic composition between sympathetic neurons and adrenal chromaffin cells is not suprising in light of the common embryonic orgins and similarity in neurotransmitter propel-ties and chemosensitivity of the two cell types. In addition to such similarities, however. the absorption data indicate that AsRS also recognizes components present in sympathetic neurons and brain that are not shared with adrenals. Our studies show not only that AsRS recognizes PC12 pheochromocytoma cells, but also reveals antigenie differences between PC12 - and PC12 + cells For example, by microcomplement fixation the titer of AsRS against PC12+ cells was similar to that against sympathetic neurons but was significantly higher than that against PC1 2 - cells. Also. PlsRS had potent cytotoxic activity against PCl2+ cells. hut none against PClZcells. Furthermore. the :ictivity of AsRS against sympathetic neurons was total!! rcmoved by absorption with PC13+ cells. but only pal-tially decreased after repetitive ahsorptions :I ith PC1 2 - cells. Of related significance. micrncomplement fixation and immunohistolluoresccnce assay’s reveal that antisera against PC12cells (AsKI_’ ) and PC1 2 + cells (AsPCl2 -t ) both recognize r-at \ynpathetic neurons (LEE ct ~1.. 1977: 1980). In parallel with the present results, however, AsPCiZ 1 Iltd cytotoxic activity against these neurons. while AsPCl2did not (LEE et ill.. 1980). Our findings thus suggest that PC12 cells bear great antigenic resemblance to rat sympathetic neurons and that the antigens recognized by AsRS arc totally shared with PCl2+ cells, but only partially shared Rith P(‘12 -cells. Such findings are consistent with previous studies that suggest that PC12 - closely I-ecrmble their normal counterpart, adrenal chromatlin ~~~11s.
Sympathetic neuron antiserum while PC12 + cells more closely resemble symapthetic neurons. Indirect immunofluorescence staining indicated that at least some of the antigens recognized by AsRS on PC12 cells are present on the cell surface. However, in contrast to the uniform staining obtained with sympathetic neurons, staining of PC12 - cells (and to a lesser extent, of PC12+ cells) showed evidence of localization. This apparent non-uniformity of staining could reflect the true distribution of antigens or could result from the redistribution of antigens in response to treatment with antiserum. The ultimate goals of our neuroimmunological studies are to identify, localize, characterize and determine the functional role of specific markers on neural surface membranes. The present findings have provided useful reagents and suggested several experimental opportunities. First, AsRS has been shown to recognize (and can therefore be used to identify and
2245
isolate) neural-specific antigens as well as antigens that are shared with adrenal medullary cells. Second, the demonstration that the antigens recognized by AsRS are also present on the PC12 clonal cell line suggest that the latter will be a convenient and practical source from which to isolate neural and adrenal medullary antigens for characterization and preparation of monospecific antisera. In a subsequent report we shall describe the identification of some of the specific proteins that are recognized by antisera against cultured sympathetic neurons and PC 12 cells. Acknowledgements-We thank MS M.-N. CHOU and D. FALLOWSand Mr G. SALOMON for their expert assistance. Supported by grants from the U.S.P.H.S (NS 11557 and NS 15076), National Foundation-March of Dimes, McKnight Foundation and Sloan Foundation. Dr LEEwas the recipient of NRSA post-doctoral fellowship No. NS 05276-03.
REFERENCES AKESONR. & HER~CHMAN H. (1974) Modulation of cell-surface antigens of a murine neuroblastoma. Proc. natn. Acad. Sci. U.S.A. 71, 187-191. BOCCHINIV. & ANGELETTI P. U. (1969) The nerve growth factor: Purification as a 30,000-molecular-weight protein. Proc. natn. Acad. Sci. U.S.A. 64, 787-794.
BOCKE. (1978) Nervous system proteins. J. Neurochem. 30, 7-14. DICHTERM. A.. TISCHLERA. S. & GREENEL. A. (1977) Nerve growth factor-induced change in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature, Land. 268, 501-504. ESTRIDGEM. & BUNGER. (1978) Compositional analysis of growing axons from rat sympathetic neurons. J. Cell Biol. 79, 138-155. FIELDSK. L. (1976) Brain-specific cell-surface antigens. In Membranes and Disease (eds BOLISL., HOFFMANJ. F. & LEAF A.), pp. 369-377. Raven Press, New York. GREENE L. A. (1977) Quantitative in oitro studies on the nerve growth factor requirements of neurons-I. Sympathetic neurons. Deal Biol. 58, 96105. GREENEL. A. & REING. (1977) Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res. 129, 263-274. GREENEL. A. & TISCHLERA. S. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. natn. Acad. Sci. U.S.A. 73, 2424-2428. HUMPHREYS R. E., MCCUNEJ. M., CHESSL., HERRMANH. C., MALENKAD. J., MANND. L., PARHAMP., SCHLO~.SMAN S. F. & STROMINGER J. L. (1976) Isolation and immunologic characterization of a human, B-lymphocyte-specific, cell surface antigen. J. erp. Med. 144, 98-112. LEE V. H., GREENEL. A. & SHELANSKI M. L. (1980) Differential cytotoxic activities of antisera against NGF-treated and untreated clonal pheochromocytoma cells. Neuroscience 5, 1979-1987. LEE V., SHELANSKI M. L. & GREENEL. A. (1977) Specific neural and adrenal medullary antigens detected by antisera to clonal PC12 pheochromocytoma cells. Proc. natn. Acad. Sci. U.S.A. 74, 5021-5025. MAINS R. E. & PATTERSONP. H. (1973) Primary cultures of dissociated sympathetic neurons-I. Establishment of long-term growth in culture and studies of differentiated properties. J. Cell Biol. 59. 329-345. SCHACHNER M. (1974) NS-1 (nervous-system antigen-l), a glial-cell-specific antigenic component of the surface membrane. Proc. natn. Acad. Sci. U.S.A. 71, 1795-1799. STALLCUP,W. B. & COHN M. (1976) Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system. Exp. Cell Res. 98, 285-297. TISCHLERA. S. & GREENEL. A. (1978) Morphological and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Lab. Invest. 39, 77-89. WIGZELLH. (1965) Quantitative titrations of mouse H-2 antibodies using Cr “-labelled target cells. Transplantation 3, 42343 1. WILLIAMSC. A. & CHASEM. W. (1967) Methods in Immunology and Immunochemistry, Vol. I, pp. 315-332. Academic Press, New York. (Accepted 24 June 1980)
0306_45Ui8O/lntt-2239M2.0010
CHARACTERIZATION OF ANTISERA RAISED AGAINST CULTURED RAT SYMPATHETIC NEURONS VIRGINIA M. LEE,’ M. L. SHELANSKI’and L. A. GREENE’ Departments of Neuropathology, Harvard Medical School, and Pharmacology, New York University School of Medicine, U.S.A. Abstract-Antisera were prepared against monolayer cultures of newborn rat sympathetic neurons and characterized by “Cr release microcomplement fixation, cytotoxicity and indirect immunofluorescence assays. After absorption with rat liver, kidney, spleen and thymus to remove activity against common rat antigens, the antisera retained activity against cultured sympathetic neurons, brain and adrenals. Indirect immuno~uorescence showed that the antigens recognized were widely distributed in the brain, while in the adrenaf gland they were limited to the meduliary cells. Absorption with the brain completely removed the activity of the antisera against sympathetic neurons; in contrast, partial activity was retained after repetitive absorption with adrenals. The antisera were also characterized with respect to recognition of the PC12 clonal line of rat pheochromocytoma cells. Under normal growth conditions, PC12 cells resemble their normal counterparts, adrenal chromaffin cells, while after treatment with nerve growth factor, they acquire the differentiated properties of sympathetic neurons. Both untreated and treated cells were recognized by the antisera and indirect histofluorescence on living cells indicated that at least some of the antigens on these cells (as well as on sympathetic neurons) are exposed on the cell surface. However, differences were noted in recognition of nerve growth factor treated and untreated PC12 ceils. For example, the former, but not the latter were recognized in cytotoxicity assays. Also, absorption of the antisera with treated cells removed all activity against rat sympathetic neurons, whereas repetitive absorption with untreated PC12 cells removed only part of the activity. Such findings indicate that PC12 cells treated with nerve growth factor and sympathetic neurons exhibit antigenic similarities and that our antisera can be used to identify, isolate, characterize and localize neural-specific surface antigens.
ONE approach
pathetic neurons including neurite out~owth, electrito identify and characterize cell surface cal excitability and presence of synaptic vesicles components of neurons is the use of specific antisera prepared against neural tissue (cf. reviews by FIELDS, (GREENE & TISCHLEIR,1976; DICHTER, TISCHLER & 1976 and BOCK, 1978). Among the challenges of such an approach is obtaining homogeneous and defined populations of neuronal cells with which to prepare and characterize antisera. To this end, several studies have employed cell lines that express neuronal phenotypes (AKE~ON & HERXHMAN, 1974; SCHACHNER, 1974; FIELDS, 1976; STALLCUP & COHN, 1976). For example, we have studied the antigenic properties of the PC12 clonal line of rat pheochromocytoma cells (LEE, SHELANSKI & GREENE, 1977; LEE, GREENE & SHELANSKI, 1980). Such cells display many differen-
tiated characteristics of normal adrenal medullary chromaffin cells (GREENE & TIXHLER, 1976; GREENE & REIN, 1977). When PC12 cells are treated with nerve growth factor (NGF), however, they cease mitosis and acquire a number of properties of sym-
Current addresses: ’ Department of Neuropathology, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. ’ Department of Pharmacology, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A. ~~~re~juf~o~s: AsRS, antisera against rat sympathetic neurons; NGF, nerve growth factor; PC12+, PCIZ-, PC12 pheochromocytoma cells grown in the presence and absence, respectively, of NGF.
GREENE, 1977; TISCHLER& GREENE, 1978). After absorption with non-neural tissue, antisera against PC1 2 cells either untreated with NGF (PC12 - cells) or treated with NGF (PC12+ cells) recognize antigens that are specific to neural and/or adrenal tissue (LEE er al., 1977). Moreover, indirect i~munofluorescent staining and complement-mediated cytotoxicity assays indicated that some of these antigens are present on the cell surface. Furthermore, such studies revealed that As against PC12 cells also recognize components that are present on the surface of sympathetic neurons. In addition to cell lines, defined populations of neurons can also be used to derive useful antisera. Cultured s~pathetic neurons appear to be a particularly attractive system. These can be obtained in reasonable numbers and can be maintained in monolayer culture essentially free of non-neuronal cells (MAINS & PATIXRSON,1973; ESTRIDGE& BUNGE, 1978). In addition, since sympathetic neurons appear to share a number of phenotypic properties with PC12 cells, comparison of antisera directed against these two cell types might establish (a) the degree of antigenic similarity between sympathetic neurons and PC12 cetls, (b) the degree to which such a similarity is altered by treating PC12 cells with NGF and (c) the
2239
2240
VIH~IC.IAH. 1.~. M I,.
SHELANKI
suitability of using PC1 2 cells as a practical source for purification of such neuronal surface molecules. The purpose of the present study is to describe the preparation of antisera against cultured rat sympathetic neurons (AsRS) and the characterization of such antisera by microcomplement fixation, complementmediated cytotoxicity and indirect immunofluorescence.
EXPERIMENTAL
PROCEDURES
Primary cultures of dissociated rat sympathetic neurons were prepared from superior cervical ganglia following procedures slightly modified from those described for cultures of chick sympathetic ganglia (GREENE, 1977). Briefly, ganglia were dissected from neonatal (I-4 days) rats. pooled and treated for 45 min at 37’ with 0.1% trypsin (DIFCO 1:250) in 2ml of Ca*+-Mg’+-free phosphate
buffered saline. Following this, the trypsin solution was removed, the ganglia were washed 3 x with culture medium (85% RPM1 1640, lo”/; horse serum, 5% fetal calf serum and 50ngm/ml NGF) and dissociated by vigorous trituration through a Pasteur pipette. Cells were plated in collagen-coated 35mm dishes at a concentration of eight ganglia/dish. Cytosine arabinoside (10 p(M)was added from
and L.. A i;Kt
I h,
“Cr formed
release microcomplemcnl tixatior: i~>\;~\; in <.. pi’ as described previously (Wrc;/~ 1 I 1%';. !!IVP!: REYS. MCCUNE, CHESS, HERMANN. MALESKA. Mnz... )‘.i.i;HAM, SC.HLO~~MA~& SrRo~it>c;t:a. 1Y7h) fiohq:;i \)I:( ~1 the bulk-absorbed whole AsRS antisera v er-e I;)un:l ISI tic anti-complementary. cxperimcntr uerc c,irricti IW! \irt/: !li!* y-globulin fraction of the annsent. This wds prcp,rtcd I/\ previously described (WII.I.IAMS & Ctwst.. 10671 ,411 411~. tions of this fraction were from a 2 mg ml stock ‘;l:iuti
Complement-mediated cytotoxic acttvities of dnttsera were assayed as described elsewhere (LI,I. et (ii.. lY80.1. Briefly. dissociated primary rat sympathetic neuron\. PC12+ cells or PC‘12 - cells were plated on collagencoated microtiter plates. Both sympathetic neuron\ md PC12+ cells were used when abundant neurites wcrc cvident (generally 2 4 days after plating) Cell dcathq ~.crc scored on a subjective scale of 1 4. wtth 4 representtng over 75:,, destruction and 1 + less that] 75”,, destruction (see LEL er al., 1980 for further details). Scoring M~C independently performed by two observers.
the next day onward to eliminate dividing non-neuronal cells. For both immunization and for microcomplement fixation assays, the ganglion cell cultures were used when all non-neuronal cells were eliminated (usually after 7- 10 days of culture in complete medium containing 10 pM cytosine arabinoside). At this time, the neurons had produced a dense network of neurites. For cytotoxicity assays, cells were plated in collagen-coated microtiter dishes (Falcon No. 3034) and assayed as previously described (LEE et al., 1980). For indirect immunofluorescence, cells were plated onto collagen-coated glass coverslips and were used after 2 days in vitro (LEE er al., 1977). PC12 cells were maintained in culture with (PC12+ cells) or without (PC12cells) 50 ngm/ml 2.5 S NGF (BOCCHINI & ANG~LETTI, 1969) as previously described (GREENE & TISCHLER, 1976). For cytotoxicity assays, indirect immunofluorescence and microcomplement fixation assays, PC12 cultures were prepared as previously described (Lrf: ct al.. 1977: 1980).
Antisew Antisera against the cultured rat sympathetic neurons (AsRS) were prepared in guinea-pigs by procedures previously described for pheochromocytoma cells (LEE et al.. 1977; 1980). Briefly, about IO6 neurons were washed free of culture medium, scraped from the dishes, emulsified with adjuvant and injected into guinea-pigs at weekly intervals. Complete Freund’s adjuvant was used for the first injection and incomplete adjuvant for all subsequent injections. The antisera were collected after the 7th immunization. Heat inactivation and bulk absorption of the antisera with rat kidney, liver, spleen and thymus were performed as described elsewhere (LEE et al.. 1977). The results reported in this paper were from unpooled antisera obtained from two guinea-pigs. Each yielded qualitatively identical results. The preparation and characterization of antisera against PC12 cells have been previously given (Lee et al., 1977: 1980).
Indirect immunofluorescence on frozen tissue sections and on living cells were performed as described in our previous study on PC12 cells (LEF et ul.. 1977)
Absorption Antisera were absorbed with particulate fracttons of various tissue preparations (see text) using techniques given in detail elsewhere (LEE et al., 1977; 1980). Generally, antisera (at a I :4 dilution) were absorbed with a tissue pellet at a ratio (serum :tissue, v;‘v) of 2 : 1.
RESULTS
Microcomplement
jixation
After bulk absorption with non-neuronal rat tissue. AsRS ?;-globulin (stock solution 2 mg/ml) had an antigenie titer (50% fixation point) in the microcomplement fixation assay of between 1 :20 and 1 :40 against homogendtes or particulate fractions of cultured rat sympathetic neurons (Table 1). Both particulate and supernatant fractions (prepared by a 100,000 g centrifugation for 1 h) of the homogenate were recognized to the same extent on a per mg protein basis. These titers were significantly higher than the ‘background’ (1 :8) obtained with AsRS y-globulin in the absence of target tissue. Also, sympathetic neurons and other tissues at up to 25 pg protein per sampie alone did not fix complement. To test the specificity of the AsRS y-globulin, particulate fractions of homogenates of various rat organs were also tested as targets. Of the tissues tested, only adrenal and brain were recognized to a significant extent (Table 1).
FIG. 1. Indirect immunofluorescent staining of rat adrenal and cerebellum with antisera to rat sympathetic neurons. (A) Sagittal section of cerebellum stained with AsRS (1%). The pattern of fluorescence shows weak staining of the molecular layer (m.), a more intense, diffuse staining of the granule cell layer (g.) and intense staining of the Purkinje cell layer (P.). (B) Cross section of adrenals stained with AsRS (1:20). Note that staining is limited to the medulla. Magnification x 192 in both A and B.
2241
FIG. 2. Indirect immunofluorescent staining of cultured rat sympathetic neurons, Pc‘l2- and PC‘12a cells with antisera to rat sympathetic neurons, Panels A, C, E and G show phase-contrast micrographs of cells while panels B, D, F, H and I show same fields photographed under fluorescence optics. (,4 and B) Rat sympathetic neurons (2 days in culture) stained with AsRS (I ~160).(C and D) PC12 - cells stained with AsRS (1:160). Note limitation of staining to caps. (E and F) PCIZ- cells stained with AsPC12+ (1 :?.oO),Note uniform distribution of staining. {G, H and I) PClZ+ cells (pre-treated with NGF for about 10 days and then passaged onto cover slips for 2 days in the presence of NGF) stained with AsRS (I :i60). Panel H shows detail of staining of neurites while panel I shoKs detail of staining af cell hodles. Magnification x 3 IV 2242
2243
Sympathetic neuron antiserum TABLE
I.
Y-GLOBULIN
MICRO-COMPLEMENT FROM
ANTISERA
AGAINST
FIXATION TO RAT
VARIOUS
ACTIVITIES
SYMPATHETIC
2,
TABLE
OF
OF
NEURONS
NEURONS
TARGETS
RESIDUAL
Y-GLOBULIN
AFTER
AGAINST REPETITIVE
Titer*
Target
TABLE
3.
RESIDUAL
TIC NEURONS
cYTOTOXIC
AGAINST
VARIOUS
LATE
CULTURED
FRACTIONS
RAT
None Cultured rat
None Cultured rat sympathetic neurons PClZ+ cells PC12- cells Brain Adrenal Adrenal and Brain
I :2cLl :40
Adrenal and brain
The ability of AsRS to recognize cell surface antigens was tested by means of complement-mediated cytotoxicity assays (Table 3). When tested against cultured rat sympathetic neurons, AsRS had a cytotoxic titer (i.e. the dilution at which 50% of target cells were killed) of between 19 and 1:16. Destruction of cell bodies as well as neurites was evident. In the absence of complement, AsRS had no cytotoxic activity, even at a dilution of 1: 1. AsRS was also tested against PC12+ and PC12- cells. For PC12+ cells, cytotoxicity was present against both cell bodies and neurites, and at a titer similar to that against sympathetic OF ANTISERA
TO RAT
AFTER ABSORPTION
WITH
SYMPATHEPARTICU-
TISSUES
Rat sympathetic neurons
PClZ+
cells PClZ-
cells
1 :I6 0
I :I6
0
0
NT
0 1:6t 0
0 0 NT NT
NT 0 NT NT
0
NT
NT
* Final dilution of AsRS yielding 2+ (50%) complement-mediated cytotoxicity. Assays were carried out after three repetitive absorptions with indicated tissue. Experiment was repeated three times with identical results. t Titer unchanged after two additional repetitive absorptions. NT = not tested.
Cytotoxicity
OF VARIOUS
I :1t
FRAC-
TISSUES
Residual titer*
syinpathetic neurons PCl2f cells PCIZ- cells Adrenal Brain
NEURONS
PARTICULATE
Tissue used for absorption
Residual titer* using as target: Tissue used for absorption
ACTIVITY
SYMPATHETIC
SYMPATHETIC
WITH
OF VARIOUS
FIXATION RAT
adrenals, AsRS retained a partial titer against rat sympathetic neurons. Such results indicate that (i) the antigens recognized by AsRS are also present in PC12+ cells and brain, and (ii) that some, but not all of these antigens are present in significant amounts in PC1 2 - cells and adrenals.
ACTIVITY TARGETS
TO
* Final dilution of AsRS y-globulin yielding 50% fixation. Assays were carried out after three repetitive absorptions with indicated tissue. Experiment was repeated three times with identical results. t Titer unchanged after two additional repetitive absorptions.
* Final dilution of AsRS y-globulin yielding 50% fixation. Ranges represent results from +8 or ++3 separate experiments. Since NGF-responsive PC12 pheochromocytoma cells share a number of properties with sympathetic neurons and are a useful model neuronal system, these cells were also tested as targets for AsRS y-globulin. Both NGF-treated (PC12+) and nontreated (PC12-) cells showed significant levels of fixation (Table 1). However, while the titer against PC12+ cells was similar to that against sympathetic neurons, the titer against PC12- cells was significantly lower. Cross-absorption experiments were carried out to test the extent to which antigens recognized by AsRS were shared with other targets. Accordingly, AsRS was absorbed 3-5 times with particulate fractions of rat sympathetic neurons, PC12+ cells, PC12- cells, rat adrenal or rat brain. In each case, after the antiserum was absorbed with a given tissue the antigenic titer against that tissue was lost. Table 2 shows the residual titers of AsRS y-globulin against rat sympathetic neurons after the various cross-absorptions. No antigenic activity was detectable after absorption with PC12+ cells or with brain. In contrast, even after multiple absorption with PC12- cells or with
ANTISERA
ABSORPTIONS TIONS
5 I:8 1:2&l :40+ 1:I&1 :20+ + 1:2@1:30++ < I:8 I1:8 < I:8 < 1:8 1:2(&l :40+ 1:10-l :20+
None (control) Cultured rat sympathetic neurons Adrenal Brain Liver Kidney Thymus Spleen PC12 + cells PC12- cells
MICROCOMPLEMENT
FROM
2244
VIRGINIA H. LE.
M. L. SHELANSKI and
neurons. In contrast, even at a dilution of 1: 1. AsRS had no detectable cytotoxic activity against PCIZcells. Cross-absorption experiments were carried out to test the extent to which antigens recognized by AsRS in the cytotoxicity assay are shared with other cell types (Table 3). After two absorptions with PCl2+ cells, AsRS lost all cytotoxic activity against rat sympathetic neurons as well as against PC12+ cells. However, while repetitive absorption with PC1 2 cells greatly reduced the cytotoxic titer against rat sympathetic neurons, a significant cytotoxicity was still retained. Also, the cytotoxic activity of AsRS against cultured sympathetic neurons was completely removed after absorption with particulate fractions of brain; after repetitive absorption to constant titer with particulate fractions of adrenals, in contrast, the cytotoxicity titer was only partially decreased. Immunojhorescent
localization
Indirect immunofluorescent localization of antigens recognized by AsRS were carried out on 10 /*m frozen tissue sections of brain and adrenal. In the adrenal gland, specific staining was present in the medulla, but totally absent in the cortex (Fig. 1). In the brain, staining was widespread and diffuse in the gray matter. There were. however, indications of differences in staining by different cell types. For example, as shown in Fig. 1. in the cerebellum, staining was particularly intense in the Purkinje cell layer. Controls in the same tissues with pre-immune serum or with second antibody alone gave no staining. Also, AsRS did not stain frozen sections of liver or spleen. Localization of antigens recognized by AsRS was also carried out on living cultured sympathetic neurons, PCl2+ cells and PCl2cells. Under the conditions of such experiments, the antisera presumably do not enter the cells and staining is limited to surface antigens. With cultured sympathetic neurons, specific staining was uniformly distributed on both cell bodies and neurites (Fig. 2B). A similar staining pattern was also observed with antiserum against PC12 + cells. AsRS also stained PC12 cells. However, in this case, staining was not uniform. As shown in Fig. 2D. staining of PC12 - cells appeared to be limited to a cap. With PC12 + cells (Fig. 2H and I) non-uniform localization of staining was less pronounced than on PCIZcells, but was also evident. In contrast. as previously reported (LEE et al., 1977) antiserum against PC12 cells produced an evenly distributed stain over the entire cell surface (Fig. 2F).
DISCUSSION The present work presents the characteristics of antisera (AsRS) prepared against neuronal cultures of newborn rat sympathetic ganglia. After bulk absorption with a variety of tissues, AsRS still recognized neuronal components. As shown by cytotoxicity
I.. 12 timr.ur
assays and indirect immunofluorescencc io~.&lzau~.~l on living sympathetic neurons in culture it h\i some of these components are exposed c)n th,, surfact~ of cell bodies and processes. AsRS also r~:~ogni~cti brain tissue, and absorption experiment\ i:>dicatcd that all of the antigens recognized by thi\ :r:ltism~l!: are also present in brain. Such findings suppc\r th;lt, while AsRS may recognize components \p>Glic 1~: neural tissue, it does not rccognizo ;rntigenL. tlh;tt ,!r~ specific to sympathetic neurons. Our histofluorescence studies indicated thirt 111~distribution of antigens recognized hy AsRS uithin the brain is not uniform. Not only was staining substantially higher in gray malter than in white matter. hr~t there was also evidence for enhanced staining witfun gray matter such as in the Purkinje cell layer of the cerebellum. Since AsRS recognizes at least ssvsral different components. the results of such locahzation ixperiments must be interpreted with care 4 final analysis will await the preparation of monospecific antibodies against single components ~nong me several recognized by AsRS. Complement fixation and immunofluorcscent staining revealed that AsRS recognizes adrenal medullary cells. Such commonality of antigenic composition between sympathetic neurons and adrenal chromaffin cells is not suprising in light of the common embryonic orgins and similarity in neurotransmitter propel-ties and chemosensitivity of the two cell types. In addition to such similarities, however. the absorption data indicate that AsRS also recognizes components present in sympathetic neurons and brain that are not shared with adrenals. Our studies show not only that AsRS recognizes PC12 pheochromocytoma cells, but also reveals antigenie differences between PC12 - and PC12 + cells For example, by microcomplement fixation the titer of AsRS against PC12+ cells was similar to that against sympathetic neurons but was significantly higher than that against PC1 2 - cells. Also. PlsRS had potent cytotoxic activity against PCl2+ cells. hut none against PClZcells. Furthermore. the :ictivity of AsRS against sympathetic neurons was total!! rcmoved by absorption with PC13+ cells. but only pal-tially decreased after repetitive ahsorptions :I ith PC1 2 - cells. Of related significance. micrncomplement fixation and immunohistolluoresccnce assay’s reveal that antisera against PC12cells (AsKI_’ ) and PC1 2 + cells (AsPCl2 -t ) both recognize r-at \ynpathetic neurons (LEE ct ~1.. 1977: 1980). In parallel with the present results, however, AsPCiZ 1 Iltd cytotoxic activity against these neurons. while AsPCl2did not (LEE et ill.. 1980). Our findings thus suggest that PC12 cells bear great antigenic resemblance to rat sympathetic neurons and that the antigens recognized by AsRS arc totally shared with PCl2+ cells, but only partially shared Rith P(‘12 -cells. Such findings are consistent with previous studies that suggest that PC12 - closely I-ecrmble their normal counterpart, adrenal chromatlin ~~~11s.
Sympathetic neuron antiserum while PC12 + cells more closely resemble symapthetic neurons. Indirect immunofluorescence staining indicated that at least some of the antigens recognized by AsRS on PC12 cells are present on the cell surface. However, in contrast to the uniform staining obtained with sympathetic neurons, staining of PC12 - cells (and to a lesser extent, of PC12+ cells) showed evidence of localization. This apparent non-uniformity of staining could reflect the true distribution of antigens or could result from the redistribution of antigens in response to treatment with antiserum. The ultimate goals of our neuroimmunological studies are to identify, localize, characterize and determine the functional role of specific markers on neural surface membranes. The present findings have provided useful reagents and suggested several experimental opportunities. First, AsRS has been shown to recognize (and can therefore be used to identify and
2245
isolate) neural-specific antigens as well as antigens that are shared with adrenal medullary cells. Second, the demonstration that the antigens recognized by AsRS are also present on the PC12 clonal cell line suggest that the latter will be a convenient and practical source from which to isolate neural and adrenal medullary antigens for characterization and preparation of monospecific antisera. In a subsequent report we shall describe the identification of some of the specific proteins that are recognized by antisera against cultured sympathetic neurons and PC 12 cells. Acknowledgements-We thank MS M.-N. CHOU and D. FALLOWSand Mr G. SALOMON for their expert assistance. Supported by grants from the U.S.P.H.S (NS 11557 and NS 15076), National Foundation-March of Dimes, McKnight Foundation and Sloan Foundation. Dr LEEwas the recipient of NRSA post-doctoral fellowship No. NS 05276-03.
REFERENCES AKESONR. & HER~CHMAN H. (1974) Modulation of cell-surface antigens of a murine neuroblastoma. Proc. natn. Acad. Sci. U.S.A. 71, 187-191. BOCCHINIV. & ANGELETTI P. U. (1969) The nerve growth factor: Purification as a 30,000-molecular-weight protein. Proc. natn. Acad. Sci. U.S.A. 64, 787-794.
BOCKE. (1978) Nervous system proteins. J. Neurochem. 30, 7-14. DICHTERM. A.. TISCHLERA. S. & GREENEL. A. (1977) Nerve growth factor-induced change in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature, Land. 268, 501-504. ESTRIDGEM. & BUNGER. (1978) Compositional analysis of growing axons from rat sympathetic neurons. J. Cell Biol. 79, 138-155. FIELDSK. L. (1976) Brain-specific cell-surface antigens. In Membranes and Disease (eds BOLISL., HOFFMANJ. F. & LEAF A.), pp. 369-377. Raven Press, New York. GREENE L. A. (1977) Quantitative in oitro studies on the nerve growth factor requirements of neurons-I. Sympathetic neurons. Deal Biol. 58, 96105. GREENEL. A. & REING. (1977) Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res. 129, 263-274. GREENEL. A. & TISCHLERA. S. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. natn. Acad. Sci. U.S.A. 73, 2424-2428. HUMPHREYS R. E., MCCUNEJ. M., CHESSL., HERRMANH. C., MALENKAD. J., MANND. L., PARHAMP., SCHLO~.SMAN S. F. & STROMINGER J. L. (1976) Isolation and immunologic characterization of a human, B-lymphocyte-specific, cell surface antigen. J. erp. Med. 144, 98-112. LEE V. H., GREENEL. A. & SHELANSKI M. L. (1980) Differential cytotoxic activities of antisera against NGF-treated and untreated clonal pheochromocytoma cells. Neuroscience 5, 1979-1987. LEE V., SHELANSKI M. L. & GREENEL. A. (1977) Specific neural and adrenal medullary antigens detected by antisera to clonal PC12 pheochromocytoma cells. Proc. natn. Acad. Sci. U.S.A. 74, 5021-5025. MAINS R. E. & PATTERSONP. H. (1973) Primary cultures of dissociated sympathetic neurons-I. Establishment of long-term growth in culture and studies of differentiated properties. J. Cell Biol. 59. 329-345. SCHACHNER M. (1974) NS-1 (nervous-system antigen-l), a glial-cell-specific antigenic component of the surface membrane. Proc. natn. Acad. Sci. U.S.A. 71, 1795-1799. STALLCUP,W. B. & COHN M. (1976) Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system. Exp. Cell Res. 98, 285-297. TISCHLERA. S. & GREENEL. A. (1978) Morphological and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Lab. Invest. 39, 77-89. WIGZELLH. (1965) Quantitative titrations of mouse H-2 antibodies using Cr “-labelled target cells. Transplantation 3, 42343 1. WILLIAMSC. A. & CHASEM. W. (1967) Methods in Immunology and Immunochemistry, Vol. I, pp. 315-332. Academic Press, New York. (Accepted 24 June 1980)