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Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures
Journal of Neuroscience Methods 75 (1997) 199 – 205
Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures Sabine Fuhrmann *, Matthias Kirsch, Konstantin Wewetzer, Hans-Dieter Hofmann Institute of Anatomy, Uni6ersity of Freiburg, P.O. Box 111, D-79001 Freiburg, Germany Received 5 January 1997; accepted 21 March 1997
Abstract In this study we describe a large-scale screening cell ELISA protocol which is suitable for the characterization of exogenic factor effects in mixed central nervous system (CNS) culture. The main novelty of the assay is that it permits the measurement of cellular responses in populations comprising as little as 2–4% of the total cell number. For standardization of the assay, we employed antibodies against opsin and microtubule-associated protein (MAP2) which label distinct retinal cell classes. Embryonic chick retinal neurons were grown in microtiter plates and directly processed for detection of antibody binding on the same plate. Binding of the antibodies was saturable and the ELISA signal was proportional to the number of immunoreactive cells comprising 2–4% and 16% of the total cell number with opsin and MAP2 antibodies, respectively. A minimum of 2000 opsin-positive cells could be reliably determined. Using our cell ELISA protocol, we demonstrate a developmental increase of both cell markers which reflected an increase in the number of opsin-positive cells but an enhanced expression per cell in the case of MAP2. We also show that growth-promoting activity—the presumed chick ciliary neurotrophic factor (CNTF) — stimulated the expression of opsin in retinal cultures (EC50: 2.3 pM) and that a corresponding activity is specifically expressed in the developing retina. Our results show that the cell ELISA protocol allows the rapid screening for distinct, low-percentage cell populations responding to exogenous factors in mixed CNS cultures. © 1997 Elsevier Science B.V. Keywords: Cell ELISA; Opsin; Photoreceptor cell; Growth promoting activity; MAP2
1. Introduction Primary cultures of peripheral nervous tissue represent frequently used model systems for the study of extrinsic influences on neuronal development and function. Well-defined culture systems have been established from the peripheral nervous system (PNS) where relatively homogeneous neuronal populations are localized in different types of ganglia. These cultures have been used in bioassays for investigating the effects of neurotrophic proteins on specific classes of neurons (Varon and Adler, 1981). Dissociated culture systems have also been developed for various parts of the central nervous system (CNS), * Corresponding author. Tel.: +49 761 2035077; fax: + 49 761 2035054. 0165-0270/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 2 7 0 ( 9 7 ) 0 0 0 7 3 - 3
but, unlike PNS cultures, they display substantial heterogeneity with regard to the phenotypes of their neuronal and non-neuronal cellular components. Thus a given neuronal phenotype in culture is frequently associated with a low number of cells and specific effects on individual neuronal subpopulations can only be determined by the use of selective markers. Alterations of cell number in response to extrinsic signals (e.g. growth factors) are commonly determined by immunostaining and subsequent visual evaluation (counting, morphometrical analysis) of the labeled cells (Beck et al., 1993; Alexi and Hefti, 1993). This kind of evaluation is time-consuming and limits the number of factors and cell types which can be analyzed. Due to the increasing number of growth factors and other regulatory molecules with potential functions in the CNS and the availability of new cell type-specific markers these limi-
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tations have become more relevant. Therefore, we have adapted the cell ELISA technique (Sedgwick and Czerninsky, 1992) to establish a simple but sensitive screening method for analyzing changes in distinct neuronal subpopulations grown in mixed CNS culture. Whereas previously described cell ELISA protocols for CNS cultures were based on the detection of highabundant molecules and served to quantify overall cell survival or neurite outgrowth (Ip et al., 1991; Doherty et al., 1984), the present protocol was designed for the analysis of growth factor effects on specific cell types comprising only a minor part of all cells. As a model system, we used embryonic chick retinal cultures which were grown on a precoated microtiter plate for 1–3 days. Cells were fixed on the same plate and then processed according to Wewetzer et al. (1996) to quantify the expression of two marker proteins (microtubule-associated protein (MAP2), opsin) which identify different retinal cell populations (Tucker and Matus, 1987; Fuhrmann et al., 1995). Previously, it has been shown that the in vitro development of opsin-positive rod photoreceptors from postmitotic precursors is promoted by mammalian ciliary neurotrophic factor (CNTF; Fuhrmann et al., 1995). Using the cell ELISA protocol we now demonstrate that growth-promoting activity (GPA), the presumed chick CNTF homologue, which was purified and cloned from chick eye tissue (Nishi and Berg, 1981; Barbin et al., 1984; Leung et al., 1992) has identical effects and that a corresponding stimulating activity is present in the retina and pigment epithelium.
2. Materials and methods
2.1. Cell culture Cultures were prepared from embryonic chick retinae (stage 33–34 according to Hamburger and Hamilton, 1951) as previously described (Fuhrmann et al., 1995). Briefly, retinae were carefully dissected free of pigment epithelium and then incubated for 10 min in Ca2 + / Mg2 + -free Hank’s balanced salt solution (HBSS). The tissue was treated with 0.25% trypsin (Sigma) in HBSS for 25 min at 37°C followed by inactivation of the enzyme with culture medium containing 5% heat-inactivated fetal calf serum (FCS). Retinae were washed once with culture medium containing 1% FCS and then dissociated by gentle trituration through a flame-narrowed glass pipette. Cells were seeded on poly-L-lysine (0.1 mg/ml) coated microtiter plates (96-well, Becton Dickinson) at a density of 10 ×104 cells per well if not indicated otherwise. For light microscopic studies, cells were grown on poly-L-lysine coated coverslips (14 mm diameter placed in a 24-multiwell plate) at a density of 10 or 20× 104 cells/cm2. Culture medium consisted of
Dulbecco’s modified Eagles medium (DMEM) supplemented with 2 mM glutamine, 100 U/ml penicilline, 100 U/ml streptomycin and 1% FCS. Cultures were grown for 1–3 days at 37°C in 5% CO2/95% air. Factors and cell extracts were added prior to seeding of the cells. Recombinant rat CNTF was kindly supplied by Regeneron Pharmaceuticals and recombinant GPA by Drs R. Nishi and H. Rohrer. For the preparation of cell extracts, retina, tectum, and remaining brain were dissected from 15 day-old embryonic chick (stage 41). The tissues were homogenized in ice-cold deionized water, centrifuged for 1 h at 100 000× g and stored frozen at − 80°C before use. Protein content was determined according to Lowry et al. (1951).
2.2. Cell ELISA Cells were washed once in DMEM, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.35, for 20 min and carefully washed with PB. Membrane permeabilization and blocking of nonspecific binding was done by treatment with 1% Triton X-100 and 10% normal goat serum (NGS) in PB for 30 min followed by rinsing in PBS (Serva) with 0.05% Tween20 (PBS/Tween). Cells were incubated for 1 h in 60 ml/well with the primary monoclonal antibodies in PB containing 1% bovine serum albumin (BSA) and 1% NGS: anti-MAP2, 1:100 000; rho-4D2, 1:400. The antiopsin antibody (rho-4D2) has been raised against bovine rhodopsin and was kindly provided by R.S. Molday (for characterization of the antibody see Molday, 1989). The antibody against MAP2 was purchased from Sigma. Cultures were then incubated with biotinylated goat anti-mouse antibody (Biotrend; 1:350) for 1 h followed by a 20 min incubation in streptavidin-conjugated peroxidase (Amersham; 1:1000), both diluted in PB/1% BSA. Each incubation step was followed by three washes in PBS/Tween. Finally, the plates were developed with 0.1% o-phenylendiamine (OPD), 0.03% H2O2 in 100 mM sodium citrate (pH 5.0; 100 ml per well). The conversion of OPD to its oxidized product was stopped after 20 min by addition of 50 ml 12.5% H2SO4. Product formation was determined by measuring the optical density at 492 nm using a microplate reader (Titertek). Data were corrected for blank values obtained in the absence of the first antibody.
2.3. Immunocytochemistry Cultures grown on coverslips for three days were fixed for 30 min and washed in PB. After permeabilization and blocking of nonspecific binding (as described above), cells were incubated with anti-MAP2 (1:500) or rho-4D2 antibody (1:50) in PB containing 1% NGS overnight at 4°C. Visualization of the antibodies was performed by successive treatment of cells with biotiny-
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Fig. 1. Immunolabeling of dissociated cultures of embryonic chick retina (E8) by cell type-specific antibodies. Cultures were grown for 3 days in vitro at a cell density of 20×104 cells/well. (A) Fluorescence micrograph demonstrating MAP2-positive cells. (B) Same visual field as in A shown with phase contrast optics. (C) Immunfluorescence staining with anti-opsin antibody (rho-4D2). (D) Phase contrast micrograph showing the same visual field as in C. Scale bar, 40 mm.
lated goat anti-mouse antibody and fluoresceine isothiocyanate (FITC)-conjugated avidin. Cultures were mounted in potassium iodide/glycerol and viewed with a Zeiss Axiovert equipped with epifluorescence optics. To determine the proportion of cells expressing MAP2 and opsin, the total number of cells and the number of immunopositive cells were counted along one diameter of each coverslip. A minimum of 2300 cells per coverslip was evaluated in this way. Determinations were performed in triplicate and each experiment was repeated at least twice.
3. Results For the standardization of the cell ELISA protocol, we used two different antibodies: anti-MAP2 antibody which recognizes cone photoreceptors and a small subpopulation of amacrine cells in the intact retina (Tucker and Matus, 1987; not shown) and anti-opsin antibody which specifically labels rod photoreceptors (Fuhrmann et al., 1995). Fig. 1 shows chick retinal cultures immunofluorescence-stained for MAP2 and opsin after 3 days of culture demonstrating the marked difference in the number of cells labeled with the two antibodies.
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The anti-MAP2 antibody recognized 15 – 16% of all cells in different experiments and 1.6 – 4.4% of all cells were opsin-positive. To determine the appropriate conditions for the cell ELISA, cultures of different seeding densities were incubated after 3 days in vitro with serial dilutions of anti-MAP2, anti-opsin and control antibodies. With anti-MAP2 antibody, saturating sigmoid dose response curves with half maximal signals at dilutions of 5 –10 × 105 were observed at all cell densities tested (3, 5 and 10× 104 cells/well corresponding to 4.8, 8 and 16× 103 MAP2-positive cells/well, respectively) and the magnitude of the signals was proportional to the cell density (Fig. 2A). With anti-opsin antibody a saturable dose response curve was obtained only at the highest seeding density used (10 × 104 cells/well) which corresponded to approximately 3×103 opsin-positive cells/well. The
Fig. 2. Titration of antibody binding in chick retinal cultures determined by cell ELISA. Cultures were grown for 3 days in vitro at different cell densities. Antibody binding of MAP2 (A) and of rho-4D2 (B) are demonstrated at cell densities of 3× 104 (), 5 ×104 () and 10× 104 ( ) cells/well. Values represent means9 S.D. (N = 3).
Fig. 3. Quantification of opsin and MAP2 expression in retinal cultures by cell ELISA grown under different conditions and for different time periods. Cultures were grown either for 1 day (1 DIV) or 3 days (3 DIV) without treatment (open bars) or with 5 ng/ml CNTF (hatched bars). Values represent means9 S.D. (n = 6− 12).
number of opsin-positive cells present at lower cell densities seemed to be too low to be accurately determined by the cell ELISA. Antigens which are not expressed by cultured retinal cells as determined by immunofluorescence (e.g. parvalbumin, 200 kD neurofilament protein) elicited no specific titrable signal in the cell ELISA (not shown) demonstrating the specificity of the signals observed with the anti-MAP2 and anti-opsin antibodies. To further assess the reliability of the cell ELISA, we used the same protocol in comparison with cell counts after immunofluorescence staining to study the in vitro development of rod photoreceptors and MAP2-positive cells in the presence and absence of recombinant rat CNTF. In agreement with results from cell counting (Fuhrmann et al., 1995), the cell ELISA indicated that no opsin-positive rods were present after 1 DIV, but started to differentiate between 1 and 3 DIV (Fig. 3). As also shown previously by immunocytochemistry, the expression of opsin immunoreactivity was markedly enhanced in the presence of 5 ng/ml CNTF (by 150– 300% in different experiments) reflecting an increase in the number of differentiating rod photoreceptors. Expression of MAP2 was clearly detectable at earlier stages (1 DIV) and was not influenced by CNTF (Fig. 3). Again, this corresponded with data from immunocytochemical experiments. However, the significant increase in MAP2 expression between 1 and 3 DIV measured by cell ELISA did not correlate with changes in the number of MAP2-positive cells determined by immunocytochemistry (1 DIV: 13.9%9 2.0; 3 DIV: 16.2%91.8). Rather, immunofluorescence experiments revealed an increase in the staining intensity of the labeled cells suggesting a higher MAP2 expression per cell (not shown).
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Non-retinal chick eye tissue has been shown to contain relatively high levels of a CNTF-like activity (Adler et al., 1979). The protein GPA which presumedly represents the chick CNTF and was more recently cloned from the same tissue shares only about 50% amino acid identity with mammalian CNTF (Leung et al., 1992). We used the newly established cell ELISA protocol to investigate, whether GPA can mimick the effects on cultured photoreceptors observed with mammalian CNTF (Fuhrmann et al., 1995; Kirsch et al., 1996) and whether a corresponding endogenous activity is present in the chick retina. As shown in Fig. 4, GPA promoted the expression of opsin in a dose-dependent manner. Half-maximal activity was observed at a concentration of 50 pg/ml corresponding to 2.3 pM. Very similar results were obtained by counting immunolabeled cells in parallel control experiments. The EC50 value was virtually identical to that determined for GPA (data not shown) in the ciliary ganglion cell survival assay (Barbin et al., 1984) and closely correlated with the EC50 (2.6 pM) measured for the CNTF effect on opsin expression in retinal cultures (Fuhrmann et al., 1995). The production of endogenous GPA-like, rod-promoting activity in chick tissue was studied by growing the retinal cultures in the presence of soluble extracts (17 – 100 mg protein/ml) prepared from E15 retina, pigment epithelium, tectum and brain. As shown in Fig. 5, extracts from embryonic retina and pigment epithelium stimulated the expression of opsin by 200–300%, whereas other extracts did not contain detectable levels of activity. 4. Discussion The results presented in this study show that the cell ELISA technique is suited to study cell type-specific
Fig. 4. Dose response curve for the stimulating effect of GPA on opsin expression in chick retinal cultures measured by cell ELISA (EC50: 2.3 pM). Chick retinal cultures were grown for 3 days under the concentrations indicated. Values represent means9 S.D. of 4 determinations.
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Fig. 5. Influence of nervous tissue extracts of embryonic chick (E15) on opsin expression determined by cell ELISA. Retinal cultures were grown for 3 days without additives (CON), in the presence of 5 ng/ml CNTF (CNTF), 18 mg/ml retina extract (R), 17 mg/ml pigment epithelium extract (PE), 100 mg/ml tectum extract (T) or 100 mg/ml remaining brain extract (B). Values represent means 9 S.D. of 3–4 determinations.
changes in mixed neuronal cultures. In view of the considerable heterogeneity of CNS cultures it was particularly important to demonstrate that this simple and rapid technique can quantitatively assess alterations in cell number or in the expression of differentiation markers in distinct cell types comprising only a minor subpopulation of the cultured cells. Previous cell ELISA protocols frequently included enzymatic detachment of cells and plating into microtiter plates prior to incubation with antibodies (Gaffar et al., 1989; Zhou et al., 1996; Sedgwick and Czerninsky, 1992). This method is more laborious and requires the availability of large numbers of cells. In addition, cell loss during replating and interference of the enzymatic treatment with antibody binding may reduce the sensitivity and reliability of the assay. For applying the cell ELISA technique to neuronal cultures it was important to fix and process the cells directly on the culture plates (Wewetzer et al., 1996) in order to preserve the neuronal processes, because changes in neurite formation or expression of neuritic proteins may contribute to the cellular responses to exogenous factors. Using high-abundant proteins (e.g. neurofilament proteins) as neuronal markers, cell ELISAs have been applied to quantify overall cell survival and/or neurite formation in neuronal monolayer cultures (Doherty et al., 1984; Ip et al., 1991). We now show that, by optimizing the protocol, the technique can be used for more sensitive and specific characterizations of developmental processes in neuronal cultures. By directly comparing results obtained by the cell ELISA technique with cell countings after immunofluorescence staining we could demonstrate that subpopulations comprising only 2–4% of the total cell number can be reliably detected as shown with the rod-specific anti-opsin antibody. For this marker anti-
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gen, the limit of sensitivity was reached at about 2000 immunoreactive cells per well. With other immunocytochemical markers which are expressed at higher levels per cell the sensitivity should be even higher. As shown by varying cell densities and by measuring opsin expression after different culture periods, cell ELISA signals were directly proportional to the number of immunoreactive cells. This was confirmed by studying the effect of CNTF which is known to increase the number of opsin-immunoreactive cells without detectably affecting the total number of cells in the cultures (Fuhrmann et al., 1995). The stimulatory effect of CNTF on opsin expression and the lack of an effect on MAP2 expression closely correlated with the results obtained by counting immunofluorescence-labeled cells demonstrating again that minor changes in cell numbers (2–4%) can be specifically monitored with the cell ELISA. It is trivial to say that changes in the cell ELISA signal do not necessarily indicate changes in the number of cells expressing a particular marker antigen, but could equally reflect an altered production of the antigen by the individual cell. This was the case with MAP2-positive cells which did not change in cell number during a 3 day culture period, whereas their specific cell ELISA signal increased substantially during this period. Although the direct interpretation of the cell ELISA data is hampered by this ambiguity, it does not diminish the usefulness of the technique. Rather, it is an advantage of the cell ELISA technique as compared to immunolabeling combined with cell counting that it allows to quantify changes in marker expression caused by either alterations in cell number or by changes in neuronal differentiation. Thus this protocol is particularly suited for the rapid screening of cellular responses in mixed CNS cultures. We have used the optimized cell ELISA protocol to investigate if chick retinal tissue contains a CNTF-like activity which could be related to the effects on retinal development observed in retinal cultures after addition of mammalian CNTF (Hofmann, 1988; Fuhrmann et al., 1995). Previous studies suggest that the recently cloned protein GPA represents the chick CNTF (Heller et al., 1993; 1995). Furthermore, in accordance with the observed in vitro effects of CNTF on retinal neurons, the expression of the CNTF receptor a-subunit in the developing chick retina (Heller et al., 1995) indicates a role of chick CNTF in retinal development. However, no GPA expression in the chick retina was found by Northern blot analysis (Leung et al., 1992). We now demonstrate that GPA can mimick the CNTF effect on chick photoreceptors very potently (EC50, 2.3 pM) stimulating opsin expression in chick retinal cultures. In addition, we show that a GPA-like activity is specifically expressed in the developing retina at the time of photoreceptor differentiation. Although the active molecule in the retinal extracts remains to be identified,
our results support the conclusion that GPA or a closely related factor is involved in the regulation of retinal development. In sum, our cell ELISA protocol proved to be useful for the characterization of exogenous effects on lowpercentage subpopulations in mixed CNS cultures and should thus be of general interest as a large-scale screening assay.
Acknowledgements The authors wish to thank M. Winter for the photographic work. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 505/A4.
References Adler R, Landa KB, Manthorpe M, Varon S. Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons. Science 1979;204:1434 – 6. Alexi T, Hefti F. Trophic actions of transforming growth factor a on mesencephalic dopaminergic neurons developing in culture. Neuroscience 1993;55:903 – 18. Barbin G, Manthorpe M, Varon S. Purification of the chick eye ciliary neuronotrophic factor (CNTF). J Neurochem 1984;43:1468 – 78. Beck KD, Knu¨sel B, Hefti F. The nature of the trophic action of brain-derived neurotrophic factor, des(1 – 3)-insulin-like growth factor-1, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture. Neuroscience 1993;52:855 – 66. Doherty P, Dickson JG, Flanigan TP, Walsh FS. Quantitative evaluation of neurite outgrowth in cultures of human foetal brain and dorsal root ganglion cells using an enzyme-linked immunoadsorbent assay for human neurofilament protein. J Neurochem 1984;42:1116 – 22. Fuhrmann S, Kirsch M, Hofmann H-D. Ciliary neurotrophic factor promotes chick photoreceptor development in vitro. Development 1995;121:2695 – 706. Gaffar SA, Li Z, Epstein AL. A live cell enzyme-linked immunosorbent assay for detecting human hepatoma membrane antigens. Hybridoma 1989;8:331 – 6. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol 1951;88:49–92. Heller S, Huber J, Finn TP, Nishi R, Rohrer H. GPA and CNTF produces similar effects in sympathetic neurons but differ in receptor binding. Neuroreport 1993;5:357 – 60. Heller S, Finn TP, Huber J, Nishi R, Geißen M, Pu¨schel A, Rohrer H. Analysis of function and expression of the chick GPA receptor (GPARa) suggests multiple roles in neuronal development. Development 1995;121:2681 – 93. Hofmann H-D. Development of cholinergic retinal neurons from embryonic chicken in monolayer cultures: Stimulation by glial cell-derived factors. J Neurosci 1988;8:1361 – 9. Ip NY, Li Y, van de Stadt I, Panayotatos N, Alderson RF, Lindsay RM. Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures. J Neurosci 1991;11:3124–34. Kirsch M, Fuhrmann S, Wiese A, Hofmann H-D. CNTF exerts opposite effects on in vitro development of rat and chick photoreceptors. Neuroreport 1996;7:697 – 700.
S. Fuhrmann et al. / Journal of Neuroscience Methods 75 (1997) 199–205 Leung DW, Parent AS, Cachianes G, Esch F, Coulombe JN, Nikolics K, Eckenstein FP, Nishi R. Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons. Neuron 1992;8:1045–53. Molday RS. Monoclonal antibodies to rhodopsin and other proteins of rod outer segments. Prog Retinal Res 1989;8:173 – 209. Nishi R, Berg DK. Two components from eye tissue that differentially stimulate the growth and development of ciliary ganglion neurons in cell culture. J Neurosci 1981;1:505–13. Sedgwick JD, Czerninsky C. Detection of cell-surface molecules, secreted products of single cells and cellular proliferation by enzyme immunoasssay. J Immunol Methods 1992;150:159 – 75. Tucker RP, Matus AI. Developmental regulation of two micro-
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tubule-associated proteins (MAP2 and MAP5) in the embryonic avian retina. Development 1987;101:535 – 46. Varon S, Adler R. Trophic and specifying factors directed to neuronal cells. Adv Cell Neurobiol 1981;2:115 – 63. Wewetzer K, Heiniger C, Seilheimer B. An improved cell-ELISA for the differential screening of antibodies against cell surface molecules of viable adherent Schwann cells. J Immunol Methods 1996;191:171 – 8. Zhou L, Zhang S-R, Yue BYJT. Adhesion of human trabecular meshwork cells to extracellular matrix proteins. Invest Ophtalmol Vis Sci 1996;37:104 – 13. Lowry OH, Rosenbrough NY, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265 – 75.
Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures Sabine Fuhrmann *, Matthias Kirsch, Konstantin Wewetzer, Hans-Dieter Hofmann Institute of Anatomy, Uni6ersity of Freiburg, P.O. Box 111, D-79001 Freiburg, Germany Received 5 January 1997; accepted 21 March 1997
Abstract In this study we describe a large-scale screening cell ELISA protocol which is suitable for the characterization of exogenic factor effects in mixed central nervous system (CNS) culture. The main novelty of the assay is that it permits the measurement of cellular responses in populations comprising as little as 2–4% of the total cell number. For standardization of the assay, we employed antibodies against opsin and microtubule-associated protein (MAP2) which label distinct retinal cell classes. Embryonic chick retinal neurons were grown in microtiter plates and directly processed for detection of antibody binding on the same plate. Binding of the antibodies was saturable and the ELISA signal was proportional to the number of immunoreactive cells comprising 2–4% and 16% of the total cell number with opsin and MAP2 antibodies, respectively. A minimum of 2000 opsin-positive cells could be reliably determined. Using our cell ELISA protocol, we demonstrate a developmental increase of both cell markers which reflected an increase in the number of opsin-positive cells but an enhanced expression per cell in the case of MAP2. We also show that growth-promoting activity—the presumed chick ciliary neurotrophic factor (CNTF) — stimulated the expression of opsin in retinal cultures (EC50: 2.3 pM) and that a corresponding activity is specifically expressed in the developing retina. Our results show that the cell ELISA protocol allows the rapid screening for distinct, low-percentage cell populations responding to exogenous factors in mixed CNS cultures. © 1997 Elsevier Science B.V. Keywords: Cell ELISA; Opsin; Photoreceptor cell; Growth promoting activity; MAP2
1. Introduction Primary cultures of peripheral nervous tissue represent frequently used model systems for the study of extrinsic influences on neuronal development and function. Well-defined culture systems have been established from the peripheral nervous system (PNS) where relatively homogeneous neuronal populations are localized in different types of ganglia. These cultures have been used in bioassays for investigating the effects of neurotrophic proteins on specific classes of neurons (Varon and Adler, 1981). Dissociated culture systems have also been developed for various parts of the central nervous system (CNS), * Corresponding author. Tel.: +49 761 2035077; fax: + 49 761 2035054. 0165-0270/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 0 2 7 0 ( 9 7 ) 0 0 0 7 3 - 3
but, unlike PNS cultures, they display substantial heterogeneity with regard to the phenotypes of their neuronal and non-neuronal cellular components. Thus a given neuronal phenotype in culture is frequently associated with a low number of cells and specific effects on individual neuronal subpopulations can only be determined by the use of selective markers. Alterations of cell number in response to extrinsic signals (e.g. growth factors) are commonly determined by immunostaining and subsequent visual evaluation (counting, morphometrical analysis) of the labeled cells (Beck et al., 1993; Alexi and Hefti, 1993). This kind of evaluation is time-consuming and limits the number of factors and cell types which can be analyzed. Due to the increasing number of growth factors and other regulatory molecules with potential functions in the CNS and the availability of new cell type-specific markers these limi-
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tations have become more relevant. Therefore, we have adapted the cell ELISA technique (Sedgwick and Czerninsky, 1992) to establish a simple but sensitive screening method for analyzing changes in distinct neuronal subpopulations grown in mixed CNS culture. Whereas previously described cell ELISA protocols for CNS cultures were based on the detection of highabundant molecules and served to quantify overall cell survival or neurite outgrowth (Ip et al., 1991; Doherty et al., 1984), the present protocol was designed for the analysis of growth factor effects on specific cell types comprising only a minor part of all cells. As a model system, we used embryonic chick retinal cultures which were grown on a precoated microtiter plate for 1–3 days. Cells were fixed on the same plate and then processed according to Wewetzer et al. (1996) to quantify the expression of two marker proteins (microtubule-associated protein (MAP2), opsin) which identify different retinal cell populations (Tucker and Matus, 1987; Fuhrmann et al., 1995). Previously, it has been shown that the in vitro development of opsin-positive rod photoreceptors from postmitotic precursors is promoted by mammalian ciliary neurotrophic factor (CNTF; Fuhrmann et al., 1995). Using the cell ELISA protocol we now demonstrate that growth-promoting activity (GPA), the presumed chick CNTF homologue, which was purified and cloned from chick eye tissue (Nishi and Berg, 1981; Barbin et al., 1984; Leung et al., 1992) has identical effects and that a corresponding stimulating activity is present in the retina and pigment epithelium.
2. Materials and methods
2.1. Cell culture Cultures were prepared from embryonic chick retinae (stage 33–34 according to Hamburger and Hamilton, 1951) as previously described (Fuhrmann et al., 1995). Briefly, retinae were carefully dissected free of pigment epithelium and then incubated for 10 min in Ca2 + / Mg2 + -free Hank’s balanced salt solution (HBSS). The tissue was treated with 0.25% trypsin (Sigma) in HBSS for 25 min at 37°C followed by inactivation of the enzyme with culture medium containing 5% heat-inactivated fetal calf serum (FCS). Retinae were washed once with culture medium containing 1% FCS and then dissociated by gentle trituration through a flame-narrowed glass pipette. Cells were seeded on poly-L-lysine (0.1 mg/ml) coated microtiter plates (96-well, Becton Dickinson) at a density of 10 ×104 cells per well if not indicated otherwise. For light microscopic studies, cells were grown on poly-L-lysine coated coverslips (14 mm diameter placed in a 24-multiwell plate) at a density of 10 or 20× 104 cells/cm2. Culture medium consisted of
Dulbecco’s modified Eagles medium (DMEM) supplemented with 2 mM glutamine, 100 U/ml penicilline, 100 U/ml streptomycin and 1% FCS. Cultures were grown for 1–3 days at 37°C in 5% CO2/95% air. Factors and cell extracts were added prior to seeding of the cells. Recombinant rat CNTF was kindly supplied by Regeneron Pharmaceuticals and recombinant GPA by Drs R. Nishi and H. Rohrer. For the preparation of cell extracts, retina, tectum, and remaining brain were dissected from 15 day-old embryonic chick (stage 41). The tissues were homogenized in ice-cold deionized water, centrifuged for 1 h at 100 000× g and stored frozen at − 80°C before use. Protein content was determined according to Lowry et al. (1951).
2.2. Cell ELISA Cells were washed once in DMEM, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.35, for 20 min and carefully washed with PB. Membrane permeabilization and blocking of nonspecific binding was done by treatment with 1% Triton X-100 and 10% normal goat serum (NGS) in PB for 30 min followed by rinsing in PBS (Serva) with 0.05% Tween20 (PBS/Tween). Cells were incubated for 1 h in 60 ml/well with the primary monoclonal antibodies in PB containing 1% bovine serum albumin (BSA) and 1% NGS: anti-MAP2, 1:100 000; rho-4D2, 1:400. The antiopsin antibody (rho-4D2) has been raised against bovine rhodopsin and was kindly provided by R.S. Molday (for characterization of the antibody see Molday, 1989). The antibody against MAP2 was purchased from Sigma. Cultures were then incubated with biotinylated goat anti-mouse antibody (Biotrend; 1:350) for 1 h followed by a 20 min incubation in streptavidin-conjugated peroxidase (Amersham; 1:1000), both diluted in PB/1% BSA. Each incubation step was followed by three washes in PBS/Tween. Finally, the plates were developed with 0.1% o-phenylendiamine (OPD), 0.03% H2O2 in 100 mM sodium citrate (pH 5.0; 100 ml per well). The conversion of OPD to its oxidized product was stopped after 20 min by addition of 50 ml 12.5% H2SO4. Product formation was determined by measuring the optical density at 492 nm using a microplate reader (Titertek). Data were corrected for blank values obtained in the absence of the first antibody.
2.3. Immunocytochemistry Cultures grown on coverslips for three days were fixed for 30 min and washed in PB. After permeabilization and blocking of nonspecific binding (as described above), cells were incubated with anti-MAP2 (1:500) or rho-4D2 antibody (1:50) in PB containing 1% NGS overnight at 4°C. Visualization of the antibodies was performed by successive treatment of cells with biotiny-
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Fig. 1. Immunolabeling of dissociated cultures of embryonic chick retina (E8) by cell type-specific antibodies. Cultures were grown for 3 days in vitro at a cell density of 20×104 cells/well. (A) Fluorescence micrograph demonstrating MAP2-positive cells. (B) Same visual field as in A shown with phase contrast optics. (C) Immunfluorescence staining with anti-opsin antibody (rho-4D2). (D) Phase contrast micrograph showing the same visual field as in C. Scale bar, 40 mm.
lated goat anti-mouse antibody and fluoresceine isothiocyanate (FITC)-conjugated avidin. Cultures were mounted in potassium iodide/glycerol and viewed with a Zeiss Axiovert equipped with epifluorescence optics. To determine the proportion of cells expressing MAP2 and opsin, the total number of cells and the number of immunopositive cells were counted along one diameter of each coverslip. A minimum of 2300 cells per coverslip was evaluated in this way. Determinations were performed in triplicate and each experiment was repeated at least twice.
3. Results For the standardization of the cell ELISA protocol, we used two different antibodies: anti-MAP2 antibody which recognizes cone photoreceptors and a small subpopulation of amacrine cells in the intact retina (Tucker and Matus, 1987; not shown) and anti-opsin antibody which specifically labels rod photoreceptors (Fuhrmann et al., 1995). Fig. 1 shows chick retinal cultures immunofluorescence-stained for MAP2 and opsin after 3 days of culture demonstrating the marked difference in the number of cells labeled with the two antibodies.
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The anti-MAP2 antibody recognized 15 – 16% of all cells in different experiments and 1.6 – 4.4% of all cells were opsin-positive. To determine the appropriate conditions for the cell ELISA, cultures of different seeding densities were incubated after 3 days in vitro with serial dilutions of anti-MAP2, anti-opsin and control antibodies. With anti-MAP2 antibody, saturating sigmoid dose response curves with half maximal signals at dilutions of 5 –10 × 105 were observed at all cell densities tested (3, 5 and 10× 104 cells/well corresponding to 4.8, 8 and 16× 103 MAP2-positive cells/well, respectively) and the magnitude of the signals was proportional to the cell density (Fig. 2A). With anti-opsin antibody a saturable dose response curve was obtained only at the highest seeding density used (10 × 104 cells/well) which corresponded to approximately 3×103 opsin-positive cells/well. The
Fig. 2. Titration of antibody binding in chick retinal cultures determined by cell ELISA. Cultures were grown for 3 days in vitro at different cell densities. Antibody binding of MAP2 (A) and of rho-4D2 (B) are demonstrated at cell densities of 3× 104 (), 5 ×104 () and 10× 104 ( ) cells/well. Values represent means9 S.D. (N = 3).
Fig. 3. Quantification of opsin and MAP2 expression in retinal cultures by cell ELISA grown under different conditions and for different time periods. Cultures were grown either for 1 day (1 DIV) or 3 days (3 DIV) without treatment (open bars) or with 5 ng/ml CNTF (hatched bars). Values represent means9 S.D. (n = 6− 12).
number of opsin-positive cells present at lower cell densities seemed to be too low to be accurately determined by the cell ELISA. Antigens which are not expressed by cultured retinal cells as determined by immunofluorescence (e.g. parvalbumin, 200 kD neurofilament protein) elicited no specific titrable signal in the cell ELISA (not shown) demonstrating the specificity of the signals observed with the anti-MAP2 and anti-opsin antibodies. To further assess the reliability of the cell ELISA, we used the same protocol in comparison with cell counts after immunofluorescence staining to study the in vitro development of rod photoreceptors and MAP2-positive cells in the presence and absence of recombinant rat CNTF. In agreement with results from cell counting (Fuhrmann et al., 1995), the cell ELISA indicated that no opsin-positive rods were present after 1 DIV, but started to differentiate between 1 and 3 DIV (Fig. 3). As also shown previously by immunocytochemistry, the expression of opsin immunoreactivity was markedly enhanced in the presence of 5 ng/ml CNTF (by 150– 300% in different experiments) reflecting an increase in the number of differentiating rod photoreceptors. Expression of MAP2 was clearly detectable at earlier stages (1 DIV) and was not influenced by CNTF (Fig. 3). Again, this corresponded with data from immunocytochemical experiments. However, the significant increase in MAP2 expression between 1 and 3 DIV measured by cell ELISA did not correlate with changes in the number of MAP2-positive cells determined by immunocytochemistry (1 DIV: 13.9%9 2.0; 3 DIV: 16.2%91.8). Rather, immunofluorescence experiments revealed an increase in the staining intensity of the labeled cells suggesting a higher MAP2 expression per cell (not shown).
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Non-retinal chick eye tissue has been shown to contain relatively high levels of a CNTF-like activity (Adler et al., 1979). The protein GPA which presumedly represents the chick CNTF and was more recently cloned from the same tissue shares only about 50% amino acid identity with mammalian CNTF (Leung et al., 1992). We used the newly established cell ELISA protocol to investigate, whether GPA can mimick the effects on cultured photoreceptors observed with mammalian CNTF (Fuhrmann et al., 1995; Kirsch et al., 1996) and whether a corresponding endogenous activity is present in the chick retina. As shown in Fig. 4, GPA promoted the expression of opsin in a dose-dependent manner. Half-maximal activity was observed at a concentration of 50 pg/ml corresponding to 2.3 pM. Very similar results were obtained by counting immunolabeled cells in parallel control experiments. The EC50 value was virtually identical to that determined for GPA (data not shown) in the ciliary ganglion cell survival assay (Barbin et al., 1984) and closely correlated with the EC50 (2.6 pM) measured for the CNTF effect on opsin expression in retinal cultures (Fuhrmann et al., 1995). The production of endogenous GPA-like, rod-promoting activity in chick tissue was studied by growing the retinal cultures in the presence of soluble extracts (17 – 100 mg protein/ml) prepared from E15 retina, pigment epithelium, tectum and brain. As shown in Fig. 5, extracts from embryonic retina and pigment epithelium stimulated the expression of opsin by 200–300%, whereas other extracts did not contain detectable levels of activity. 4. Discussion The results presented in this study show that the cell ELISA technique is suited to study cell type-specific
Fig. 4. Dose response curve for the stimulating effect of GPA on opsin expression in chick retinal cultures measured by cell ELISA (EC50: 2.3 pM). Chick retinal cultures were grown for 3 days under the concentrations indicated. Values represent means9 S.D. of 4 determinations.
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Fig. 5. Influence of nervous tissue extracts of embryonic chick (E15) on opsin expression determined by cell ELISA. Retinal cultures were grown for 3 days without additives (CON), in the presence of 5 ng/ml CNTF (CNTF), 18 mg/ml retina extract (R), 17 mg/ml pigment epithelium extract (PE), 100 mg/ml tectum extract (T) or 100 mg/ml remaining brain extract (B). Values represent means 9 S.D. of 3–4 determinations.
changes in mixed neuronal cultures. In view of the considerable heterogeneity of CNS cultures it was particularly important to demonstrate that this simple and rapid technique can quantitatively assess alterations in cell number or in the expression of differentiation markers in distinct cell types comprising only a minor subpopulation of the cultured cells. Previous cell ELISA protocols frequently included enzymatic detachment of cells and plating into microtiter plates prior to incubation with antibodies (Gaffar et al., 1989; Zhou et al., 1996; Sedgwick and Czerninsky, 1992). This method is more laborious and requires the availability of large numbers of cells. In addition, cell loss during replating and interference of the enzymatic treatment with antibody binding may reduce the sensitivity and reliability of the assay. For applying the cell ELISA technique to neuronal cultures it was important to fix and process the cells directly on the culture plates (Wewetzer et al., 1996) in order to preserve the neuronal processes, because changes in neurite formation or expression of neuritic proteins may contribute to the cellular responses to exogenous factors. Using high-abundant proteins (e.g. neurofilament proteins) as neuronal markers, cell ELISAs have been applied to quantify overall cell survival and/or neurite formation in neuronal monolayer cultures (Doherty et al., 1984; Ip et al., 1991). We now show that, by optimizing the protocol, the technique can be used for more sensitive and specific characterizations of developmental processes in neuronal cultures. By directly comparing results obtained by the cell ELISA technique with cell countings after immunofluorescence staining we could demonstrate that subpopulations comprising only 2–4% of the total cell number can be reliably detected as shown with the rod-specific anti-opsin antibody. For this marker anti-
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gen, the limit of sensitivity was reached at about 2000 immunoreactive cells per well. With other immunocytochemical markers which are expressed at higher levels per cell the sensitivity should be even higher. As shown by varying cell densities and by measuring opsin expression after different culture periods, cell ELISA signals were directly proportional to the number of immunoreactive cells. This was confirmed by studying the effect of CNTF which is known to increase the number of opsin-immunoreactive cells without detectably affecting the total number of cells in the cultures (Fuhrmann et al., 1995). The stimulatory effect of CNTF on opsin expression and the lack of an effect on MAP2 expression closely correlated with the results obtained by counting immunofluorescence-labeled cells demonstrating again that minor changes in cell numbers (2–4%) can be specifically monitored with the cell ELISA. It is trivial to say that changes in the cell ELISA signal do not necessarily indicate changes in the number of cells expressing a particular marker antigen, but could equally reflect an altered production of the antigen by the individual cell. This was the case with MAP2-positive cells which did not change in cell number during a 3 day culture period, whereas their specific cell ELISA signal increased substantially during this period. Although the direct interpretation of the cell ELISA data is hampered by this ambiguity, it does not diminish the usefulness of the technique. Rather, it is an advantage of the cell ELISA technique as compared to immunolabeling combined with cell counting that it allows to quantify changes in marker expression caused by either alterations in cell number or by changes in neuronal differentiation. Thus this protocol is particularly suited for the rapid screening of cellular responses in mixed CNS cultures. We have used the optimized cell ELISA protocol to investigate if chick retinal tissue contains a CNTF-like activity which could be related to the effects on retinal development observed in retinal cultures after addition of mammalian CNTF (Hofmann, 1988; Fuhrmann et al., 1995). Previous studies suggest that the recently cloned protein GPA represents the chick CNTF (Heller et al., 1993; 1995). Furthermore, in accordance with the observed in vitro effects of CNTF on retinal neurons, the expression of the CNTF receptor a-subunit in the developing chick retina (Heller et al., 1995) indicates a role of chick CNTF in retinal development. However, no GPA expression in the chick retina was found by Northern blot analysis (Leung et al., 1992). We now demonstrate that GPA can mimick the CNTF effect on chick photoreceptors very potently (EC50, 2.3 pM) stimulating opsin expression in chick retinal cultures. In addition, we show that a GPA-like activity is specifically expressed in the developing retina at the time of photoreceptor differentiation. Although the active molecule in the retinal extracts remains to be identified,
our results support the conclusion that GPA or a closely related factor is involved in the regulation of retinal development. In sum, our cell ELISA protocol proved to be useful for the characterization of exogenous effects on lowpercentage subpopulations in mixed CNS cultures and should thus be of general interest as a large-scale screening assay.
Acknowledgements The authors wish to thank M. Winter for the photographic work. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 505/A4.
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