Exp. Eye Res. (1992) 55. 203-214

Subcellular HITOSHI Departments

Localization

ISHIGOOKA,

AMY

of Ophthalmology

(Received

of bFGF in Human Epithelium In Vitro E. AOTAKI-KEEN

Houston

25 January

1991

LEONARD

AND

and Biological Chemistry, Davis, CA 95616-8635,

Retinal

M. HJELMELAND”

School of Medicine, U.S.A.

and accepted

in revised

form

Pigment

University

October

of California,

1991)

Basic fibroblast growth factor is a polypeptide mitogen with potential biological roles in angiogenesis. differentiation, and the survival of neurons. To study the expression and subcellular distribution of basic fibroblast growth factor in human retinal pigment epithelium in vitro, affinity-purified antipeptide antibodies were generated against a 15 amino acid sequence in the amino-terminus of this growth factor. Analysis of the cross reactivity and specificity of the affinity-purified antibodies demonstrated no ability to recognize acidic fibroblast growth factor and the ability to label specifically the major known forms of basic fibroblast growth factor in whole-cell lysates of retinal pigment epithelium in vitro. Examination of paraformaldehydeor glutaraldehyde-fixed pigment epithelium at the light and electron microscopic levels revealed prominent localization of basic fibroblast growth factor to the nucleus and nucleolus. In cells fixed with organic reagents, prominent cytoplasmic staining was noted in addition to the nuclear staining seen in aldehyde fixed cells. Investigation of subcellular fractions by Western blot analysis indicated cytosolic as well as nuclear localization of the basic fibroblast growth factor. These analyses, however, demonstrated that the higher molecular weight forms of basic fibroblast growth factor predominate in the nucleus. Key words:basic fibroblast growth factor: subcellular localization : human ; retinal pigment epithelium.

1. Introduction Basic fibroblast growth factor (bFGF) was the first and

most carefully studied member of a growing family of polypeptide growth factors related by sequence homology (Abraham et al., 1986). Other members of this family include acidic FGF (Mergia et al., 1989). the int2 (Brookes et al., 1989) and hst (Yoshida et al., 1987) proto-oncogenes, FGF 5 (Zhan et al., 1988), FGF 6 (Marics et al., 1989), and keratinocyte growth factor (Finch et al., 1989). Several complex functions for the FGF gene family members have been proposed, including roles in angiogenesis (Folkman and Klagsbrun, 198 7 ; Gospodarowicz, Neufeld, and Schweigerer. 1987) development (Kimelman et al., 1988; Heber et al., 1990), and maintenance of the viability and differentiated function of neurons (Morrison et al., 1986: Wagner and D’Amore, 1986; Walicke et al., 1986). Previous studies from this laboratory have explored the immunohistochemical localization of bFGF in the developing capillaries of the bovine

retina

(Hanneken

et al.,

1989)

and

in the

fibrovascular extraretinal proliferations associated with diabetic retinopathy (Hanneken et al., 1991). Several other groups have published extensively on the expression of fibroblast growth factors in the eye (D’Amore andKlagsbrun, 1984; Caruelleet al., 1989). including reports which specifically demonstrated the expression

of the bFGF gene

in bovine

and

human

retinal pigment epithelium (RPE) in vitro (Schweigerer et al., 1987; Sternfeld et al., 1989). Recently, LaVail and colleagues have examined the * For correspondence.

0014-4835/92/080203 + 12 $08.00/O

potential role of bFGF in preventing the loss of photoreceptors in dystrophic rats (Faktorovich et al., 1990). In these studies, bFGF was compared with acidic FGF (aFGF) and other unrelated growth factors and was found to be highly potent as a survival factor for photoreceptor outer segments.These observations echo earlier studies by Wagner and D’Amore (1986) and Walicke et al. (1986) which described neurite extension as one of the biological responsesof neurons to fibroblast growth factors. As an initial step in understanding the expression and subcellular distribution of bFGF by the human RPE. we elected to study these cells in vitro. The work presented here documents the preparation and affinity purification of high titer antipeptide antibodies directed against the amino-terminus of the mature form of human bFGF, and the use of these reagents in the light and electron

microscopic

level determinations

of the

subcellular distribution of bFGF.

2. Materials and Methods Antigen Preparation and immunization 01Rabbits A peptide consisting of the first 15 amino acids of basic FGF was chosen for reasons discussedin our previous article (Hanneken et al., 1989). The peptide was synthesized with an additional amino-terminal cysteine to facilitate peptide conjugation to a carrier protein. A method employing sulfo-MBS (Pierce, Rockford, IL) was utilized to prepare the conjugates. In a final volume of 2,s ml, 40 mg of Keyhole Limpet Hemocyanin (KLH, Calbiochem, Inc., La Jolla, CA) 0 1992 Academic

Press Limited

H. ISHIGOOKA

204

was dissolved in 10 mM phosphate buffer, pH 7.2. with 7.46 mg of sulfo-MBS. The reaction was stirred for 30 min at room temperature, after which the reaction mixture was applied to a PD-10 column (Pharmacia. Uppsala, Sweden) and eluted with 3.5 ml of 50 mM phosphate buffer, pH 6.0. The column flow through was collected, and 1.75 ml of the eluate was mixed with 20 mg of the peptide. The pH of the peptide solution was neutralized (pH 7.0-7.5) with 1 N NaOH, stirred at room temperature for 2 hr and, subsequently, at 4°C overnight. The reaction medium was then dialyzed against 150 mM NaCl(2 x 100 volumes) followed by extensive dialysis against water. The final material was collected by lyophilization, and amino acid analysis was performed to determine the percentage composition of peptide in the conjugate. Analysis yielded a value of 50% by weight of the final complex conjugate as the fraction related to peptide. Immunization of rabbits followed the procedures outlined by Vaitukaitis (198 1) and Hurn and Chantler (1980). To immunize five rabbits, 0.5 mg of the conjugate was dispersed in 5 ml of phosphate-buffered saline (PBS) and emulsified with 5 ml of Freund’s Complete Adjuvant containing 5 mg of heat-killed Tubercle bacillus (Difco, Detroit, MI). Emulsification was accomplished with two glass syringes with luer lock tips connected together with a three-way valve. For the immunization, rabbits were anesthetized with 0.05 mg Innovar kg-’ body weight, and the backs of the animals were shaved. After collecting 20 ml of whole blood (per rabbit) for pre-immune serum, 40 separate intradermal injections (50 ,~l each) were made on the back of each animal. Six weeks after the initial immunization, titers were evaluated at 34week intervals by immunoblot analysis. When the titers began to fall (5-6 months), all animals were boosted with 2 ml of emulsion containing 25 pg ml-’ of the conjugate in a 1: 1 mixture of PBS and Freund’s Incomplete Adjuvant. Booster injections were given in four separate intramuscular sites on the limbs (0,2 5 m1 per site) and 20 separate intradermal sites on the back as before (50 ~1 per site). Titers were evaluated 2-3 weeks after the booster injection, and final bleeds were obtained when the serum antibody had reached a maximum titer. Preparation of Afinity

Resins

Affinity resin was prepared by coupling recombinant human bFGF (Amgen Biologicals. Thousand Oaks, CA) or recombinant human aFGF (generous gift of Dr Michael Jaye, Rhone-Poulenc Rorer, King of Prussia, PA) to Ai%Gel 10 (Bio-Rad Laboratories, Richmond, CA) according to manufacturer’s recommendations. Briefly, 0.2 mg of bFGF dissolved in 0.34 ml of 20 mM sodium citrate, pH 5.0, and 100 mM NaCl or 0.4 mg of aFGF in O-2 ml of 50 mM phosphate buffer, pH 7.4, was added to 1 ml of washed resin. A slurry with a final volume of 1.5 ml was obtained by the addition of water and was gently agitated at 4°C for 4 hr. To block

unreacted sites, 0.1 ml pH 8.0, was added to the agitated for an additional washed extensively with stored in this buffer with AfJinity Purijcation

ET AL.

of 1 M ethanolamine HCl, reaction medium and gently hour. The resins were then 25 mM Hepes, pH 7.0, and 0.2% (w/v) sodium azide.

and lmmunoabsorption

Before each cycle of affinity purification or immunoabsorption, affinity resin was thoroughly washed with successive application of: ten volumes of 6 M Guanidine-HCl with 10 mM CHAPS: 1 S-20 volumes of 50 mM Tris-HCl (TB), pH 7.5; ten volumes of elution buffer (100 mM glycine-HCl, pH 2.5, containing 4 M urea and 1 mg ml-’ bovine serum albumin) ; and 20 volumes of TB. For affinity purification, the affinity resin was then blocked with 1 ml normal rabbit serum (Vector Laboratories, Burlingame. CA) for 20 min and washed with ten volumes of 50 mM Tris-HCl, pH 7.5, with 150 mM NaCl (TBS). Crude antiserum (5 ml) was diluted with equal volume of TBS. added to 1 ml of prepared affinity resin and incubated overnight with gentle agitation at 4°C. Resin with bound antibody was then collected by centrifugation and transferred to a small column. The resin was washed with 20-30 volumes of 50 mM Tris-HCl, pH 7.5, with 500 mM NaCl and 0.01% (by volume) Noniodet P-40 (NP-40), followed by 20-30 volumes of TBS with 0.01 Y0 NP-40 and finally with TBS. This wash was continued until the absorbance of the eluate at 280 nm was 0,005 or less (usually 30 volumes). Bound antibody was then harvested with elution buffer at a flow rate of (I.25 ml min’ in 1 ml fractions. Fractions were dialyzed four times against 100 volumes of TBS with 002% (w/v) sodium azide. Fractions were titered by immunoblot analysis, and fractions with high titer were pooled, dispensed into 100 ,~l aliquots, and stored at - 80°C for further use. The IgG content of the pooled fractions was determined by immunoblot analysis comparing serial dilutions of the pooled fractions to serial dilutions of purified rabbit IgG standards. Quantitation was performed using a Bio-Dot SF microfiltration apparatus (Bio-Rad Laboratories) and a densitometer (Model 620 : Bio-Rad Laboratories). For immunoabsorption experiments. 0 3 5 ml of affinity purified antibody was diluted to a final volume of 3.5 ml with TBS containing 3 % bovine serum albumin and 0.02% sodium azide and gently agitated with 1 ml of prepared affinity resin overnight at 4°C. The supernatant was recovered by centrifugation and stored at 4°C. Immunoblot Analysis Immunoblot analysis was performed using the BioDot microfiltration apparatus from Bio-Rad Laboratories. Nitrocellulose membrane with 0.2 ,um pores (Schleicher and Schuell Inc.. Keene, NH) was soaked in 20 IIIM Tris-HCl (pH 7.5) with 0.5 M NaCl (TBhS) and placed in the dot blot manifold as instructed by the manufacturer. Basic FGF (R and D Systems,

LOCALIZATION

OF

bFGF

IN

RPE

CELLS

Minneapolis, MN) at a variety of concentrations was prepared in TBhS containing 10 ,ug ml-l bovine serum albumin (BSA) and applied to the membrane, taking care not to exceed a load of 80 ,ug total protein cm2. Membrane was then removed from the manifold and blocked with 10% non-fat dry milk dissolved in TBhS with 0.05 y0 Tween 20 (TBhS/Tween) for 1 hr at room temperature. Following a brief wash in TBhS/Tween, the membrane was cut into strips representing columns and incubated with a primary antibody (crude immune serum or affinity purified antibody) diluted in 3% non-fat dry milk in TBhS/Tween overnight at 4°C with gentle agitation. Strips were then washed with TBhS/Tween and incubated with 0.2 ,ug ml-’ alkaline phosphatase conjugated goat anti-rabbit IgG (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) in TBhS/Tween containing 3% non-fat dry milk, for 1 hr at room temperature. Strips were washed again with TBhS/Tween, then with TBhS, followed by color development with BCIP/NBT (Kirkegaard and Perry Laboratories, Inc.) as instructed by the manufacturer.

205

weight markers (Bio-Rad Laboratories) were added at 2 ~1 (diluted to final volume with 1 x sample buffer) per lane. Samples of 1 S-20 ~1 each were loaded on a 15% SDS polyacrylamide gel in a Mini Protean electrophoresis unit (Bio-Rad Laboratories) and run according to the manufacturer’s instructions. After electrophoresis, proteins were transferred to 0.2 pm pore sized nitrocellulose membrane with a Mini TransBlot apparatus (Bio-Rad Laboratories) according to manufacturer’s instructions. The membrane was then processed for immunochemical detection as in the Immunoblot analysis, with an additional TBhS wash before the blocking step to remove any traces of methanol from the transfer step. Primary Culture Of Human Retinal Pigment Epithelium

Subcellular fractions of human RPE in vitro were prepared according to Renko et al. (1990). Human RPE cells in 150 cm2 tissue culture flasks were treated for 10 min with 1 mM cycloheximide and harvested by trypsinization. Cells were washed three times with Hank’s balanced salt solution (HBSS, GIBCO Life Technologies, Inc., Long Island, NY) containing 3 pug ml-’ aprotinin. Approximately 4.2 x 10’ cells were suspended in a final volume of 1.3 5 ml of 10 mM Tris-HCl, pH 7.5, containing aprotinin (3 ,ug ml-l), leupeptin (1 pugml-‘), and pepstatin A (10 ,ug ml-l) and homogenized in a tight-fitting Dounce homogenizer. Sucrose was added to a final concentration of 2 50 mM. The suspension was centrifuged at 700 g for 10 min. The resulting pellet was collected as the nuclear fraction. The supernatant was then centrifuged at 100 000 g for 90 min, yielding a high-speed pellet (membrane fraction) and a supernatant (cytosolic fraction). Subcellular fractions were diluted directly with SDS sample buffer. Each sample was boiled for 4 min and subjected to electrophoresis and Western blot analysis. Protein concentration was determined by the Bio-Rad DC Protein Assay (Bio-Rad Laboratories) according to the manufacturer’s instructions.

Post mortem eyes were obtained from the Lions Eye and Tissue Bank, Sacramento, CA. Eyes were washed with calcium/magnesium-free Hank’s balanced salt solution (CMF HBSS, Gibco, Inc.) containing 0.1 mg ml-’ streptomycin and 100 U ml-’ penicillin (Gibco, Inc.). The anterior segment was then removed by hemisection of the globe at the ora serrata. The retina was detached from the pigment epithelium and dissected away from the optic disc. The eyecup was rinsed with CMF HBSS and placed in the bottom of a 50 ml centrifuge tube, scleral side down. The eyecup was then filled with dispersal solution [0.25 ‘X, (w/v) trypsin and 2% (by volume) chicken serum in CMF HBSS] and allowed to incubate for 30 min at 37°C. The retinal pigment epithelium (RPE) along with the dispersal solution was then removed from the eyecup by gentle pipetting, and transferred to a tube with 3 ml of culture medium. The culture medium consisted of Dulbecco’s Modified Eagle Medium (DMEM)/Nutrient Mixture F12 with Hepes buffer (Gibco, Inc.) containing 20% fetal bovine serum (Hazelton Biologicals, Inc., Lenexa, KS), 0,348 % additional sodium bicarbonate, 1% (by volume) 200 mM L-glutamine solution (Gibco, Inc.), 0.1 mg ml-’ streptomycin, and 100 U ml-’ penicillin. Cells were centrifuged at 800 g, resuspended in 16 ml of culture medium and plated in four 25 cm2 flasks. Cultures were incubated for 2 days at 37°C in 10% CO,, after which the medium was replaced. Cultures were fed weekly until confluence. Routine passage of cultures was accomplished by dissociation in 0.05 y0 (w/v) trypsin and 0.02 ‘% (w/v) ethylenediaminetetraacetic acid, tetrasodium salt, in CMF HBSS, followed by replating at a split ratio of 1: 4.

Electrophoresis and Western Blot Analysis

lmmunohistochemistry

Electrophoresis and Western blot analysis were accomplished with whole-cell lysates and subcellular fractions of cultured human RPE cells. As a positive control, 6-7.5 ng of human recombinant bFGF (R and D Systems) with 300-375 kg of carrier protein (human serum albumin) was used per lane. Low molecular weight markers (Bio-Rad Laboratories) were used at 7 is1 per lane. Biotinylated low molecular

Cells to be studied were plated into 24-well tissue culture plates (Costar, Cambridge, MA) with 12 mm diameter glass coverslips (Bellco Glass, Inc., Vineland, NJ) which had been washed with 95% ethanol and autoclaved before use. After 3 days in culture, cells were rinsed in HBSS (GIBCO, Inc.) and fixed. After washing off the fixative with TBS. endogenous peroxidase activity was quenched by a 5-min in-

Subcellular Fractionation

H. ISHIGOOKA

206

cubation with 3% hydrogen peroxide in water followed by washes (3 x 5 min) in TBS. Non-specific antibody binding sites were blocked by a 15-min incubation with 6% (w/v) non-fat dry milk and 5% (by volume) normal goat serum (Vector Laboratories, Inc.) in TBS. Cells were washed (2 x 5 min) with TBS and incubated overnight with primary antibody diluted in 3% non-fat dry milk in TBS at 4°C in a humidified chamber. Cells were washed again with TBS (4 x 5 min) and incubated in biotinylated goat anti-rabbit IgG (Vector Laboratories, Inc.) diluted 1: 200 (7.5 ,ug ml-‘) in 3% (w/v) non-fat dry milk/ TBS for 1 hr at room temperature. This was followed by successivewashes in TBS (4 x 5 min) after which the cells were incubated with avidin :biotinylated horseradish peroxidase complex (Vector Laboratories, Inc.) for 45 min. Cells were washed with TBS (4 x 5 min) and developed for 5 min with diaminobenzidine solution (0.5 mg ml-’ DAB) as suggestedby Vector Laboratories. Coverslips were rinsed in water, dehydrated through alcohol and xylene, and mounted for light microscopy.

ET AL.

sciences,Inc., Warrington, PA) and 100% ethanol for 1 hr, followed by 100% LR White monomer for an additional hour at room temperature. After this, specimens were polymerized for 24 hr at 52°C. Polymerized blocks were then removed from the original culture slides. exposing the basal surface of the cultured cells. This block surface was then placed upright, and an additional layer of LR White was polymerized on the upper surface for another 24 hr at 52°C. in order to embed the cell layer completely. Ultrathin sections were cut on an tlltrotome III (LKB Instruments, Stockholm, Sweden) and placed on nickel grids. Sections were blocked with 1.5% (by volume) normal goat serum and 0.5 ‘X0(w/v) BSA in phosphatebuffered saline (PBS) containing 0.01% (w/v) sodium azide for 30 min. Sections were then incubated with affinity-purified anti-[cys-‘]bFGF( 1-l 5) diluted in PBS containing 0.5% (w/v) BSA (PBS/BSA), overnight at 4°C. Sections were washed with PBS/BSA (3 x 5 min) and incubated with 15 nm colloidal gold-labeled goat anti-rabbit IgG (Amersham Corporation. Arlington v-vu

Immunoelectron

Microscopy

Cell cultures were rinsed in HBSS and fixed in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) or 2 % buffered paraformaldehyde containing either 0.1 or 0.5% glutaraldehyde for 10 min at room temperature. Fixed cells were washed in 0.1 M phosphate buffer, pH 7.4, and dehydrated through a graded seriesof alcohols to 7 5 % ethanol, stained for 1 hr with 2% uranyl acetate in 75% ethanol, followed by further dehydration to 100 % ethanol. Specimenswere then infiltrated with a 1: 1 mixture of LR White monomer solution (PolyBasic

7

FGF (ng)

382

Immune

384

385

7

388

7

391

382

Preimmune

384

385388

I

0.05

-

0.04

-

0.03

-

0.02

-

O.Ol-

I 2 Basic FGF (ngl

2

50 391

s

I 3 3

I 4 4

I 5 5

I 6 6

I 7 7

1 8 8

1 9 9

1 IO 10

W

F

c

:

**@*err

P

‘o***il*

z*

0

100

5+1)ae

e

50 I

“”

IO 0.5 5 0.1

0.05

0.5 0.1

0

0

FIG. 1. Immunoblot analysis of immune and pre-immune sera raised against [cys-‘]bFGF( l-l 5) conjugated to KLH. For immunoblot analysis. bFGF was dotted on nitrocellulose in the final amounts indicated for each row, ranging from 100 to 0 ng. The last row was dotted with the diluent containing BSA as a control. Individual strips were incubated with immune or pre-immune sera at a final dilution of 1: 2 50, and visualized with indirect alkaline phosphatase immunochemistry as described in Materials and Methods. Lanes S and P represent controls where either the secondary or primary antibody was deleted from the protocol. respectively.

Frc. 2. Elution profile of the affinity purification of immune serum from rabbit 388 and immunoblot analysis of individual fractions. A. Tracing at 280 nm of the affinity purification elution profile of rabbit 388 immune serum chromatographed on an affinity resin synthesized with recombinant human bFGF. Fractions 2 to 10 are indicated, each consisting of a volume of 1 ml. B, Immunoblot analysis of individual fractions of the column effluent. The immunoblot analysis was performed as in Fig. 1. with individual column fractions being used at final dilutions of 1 : 100. Lanes W, F, and C represent whole immune serum, column flow through, and a primary antibody deletion control, respectively. Whole serum and column flow through were both tested at a final serum dilution of 1 :200.

LOCALIZATION

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bFGF

IN

RPE

207

CELLS

Heights, IL) diluted 1: 10 in PBS/BSA for 1 hr at room temperature. This was followed by successive washes in PBS (3 x 5 min) and ultrapurified water (3 x 5 min), and then by air drying. Sections were double stained with uranyl acetate and lead citrate and observed on a Philips EM-410 electron microscope. For control studies, primary antibody was replaced by an equivalent concentration of non-immune rabbit IgG (Dako Corporation, Carpinteria, CA). As a separate control,

affinity-purified rabbit polyclonal antibody was absorbed with recombinant bFGF coupled to AffiGel 10 and used at the same dilution as the primary antibody, 3. Results A group of five rabbits was immunized with a synthetic antigen made from a peptide consisting of the first 15 amino acids of bFGF coupled to KLH. After

i

FIG. 3. The effect of affinity purification of rabbit 388 antiserum on the immunohistochemical localization of bFGF in human RPE. Human RPE were cultured on 12-mm glass coverslips, fixed in methanol at -20°C for 15 min and processed for immunohistochemistry. A, Light micrograph of cells stained with whole serum at a dilution of 1: 100, an effective IgG concentration of 120 pg ml-‘. B, The non-absorbed or flow-through fraction from the affinity purification was used at a dilution of 1 : 100, equivalent to an IgG concentration of 86 fig ml-‘. C, Affinity-purified antibodies were used at a concentration of 0.2 7 i&g ml-l. The final dilution of the affinity purified antibodies used in (C) corrects the concentrating effect of affinity purification and yields a dilution factor of 1: 100 based on the original volume of crude serum which was purified. D, Cells stained with affinity-purified antibodies which had been subjected to solid phase immunoabsorption on a bFGF resin as described in Materials and Methods. E, Cells stained with affinity-purified antibodies absorbed on a resin coupled with aFGF. F. Normal rabbit IgG control at 0.3 pg ml-‘. Magnification bar represents 200 itrn and refers to all panels.

208

H. ISHIGOOKA

ET AL.

FIG. 4. The effect of fixation on the subcellulardistribution of bFGF by immunohistochemistry.Human RPE cellswere cultured on 12-mmglasscoverslipsandprocessed for immunohistochemistryafter the indicatedfixation protocols. A, Cellsfixed in methanol at -2O’C for 15 min. B, Cells fixed in 4% buffered paraformaldehydeat 4’C for 15 min followed by permeabiltzation with 0.1% Triton X-100 in TBS for 10 min at room temperature. C, Cells fixed in 2% buffered paraformaldehyde/O.l% glutaraldehydeat 4°C for 15 min followedby permeabilizationwith 0.1% Triton X-100 in TBS for 10 min at room temperature.D, Cellsfixed in 2 % bufferedparaformaldehyde/OS% glutaraldehydeat 4°C for 15 min followed by permeabilizationwith 0.1‘j$Triton X-100 in TBSfor 10 min at room temperature.Affinity-purified antibodieswere usedat a concentration of 0.3 ,ugml-l. Magnification bar represents200 pm and refersto all panels.

titers began to fall, all animals

were boosted and evaluated for antibody production against recombinant human bFGF. Figure 1 illustrates an immunoblot analysis of both the immune and the pre-immune sera of the five rabbits that were immunized. Each column contains decreasing amounts of bFGF dotted on the paper as indicated in the figure. An individual serum was reacted with one such strip to determine the titer of the antibody directed at the peptide. Two very good sera are apparent in the figure. Rabbits 384 and 388 show reactions down to 0.1 ng of bFGF dotted on the paper. The worst rabbit serum (rabbit 385) could detect only 10 ng of bFGF, some 100 times less sensitive than the best serum. Pre-immune sera show

little reactivity to bFGF. Lanes marked S and P are control strips where either the secondary or primary antibody was deleted from the analysis. In a separate control experiment (data not shown), these sera were shown not to cross-react with recombinant human aFGF by immunoblot analysis. In order to obtain reagents with the highest degree

of specificity and lowest background for immunohisto-

chemical techniques, it was necessary to affinity purify even the best serum of the five rabbits. An affinity matrix was synthesized using AffiGel 10, and a method previously published by Mains and Eipper (19 76) for the affinity purification of an antipeptide antibody was adapted for our specific needs. Figure 2 iIlustrates the elution profile of a typical affinity purification as well as the titer of the column fractions by immunoblot analysis. Figure 2(A) is a trace at 280 nm of the column effluent, showing a sharp peak after elution buffer was pumped through the column. Analysis of the fractions [Fig. 2(B)] demonstrates that the bulk of the antibody reactive against bFGF is associated with the peak eluted with 4 M urea and 0.1 M glycine, pH 2-5. Essentially no immunoreactivity for bFGF was

seen in the unbound or flow-through fraction of the column. Lanes W, F, and C on the immunoblot indicate whole serum, flow through, and a primary

antibody deletion control, respectively. In Fig. 3, various crude serum and purified antibody

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209

Molecular weight

Molacwlor weight

(kDa1

I

2

3

4

5

6

(kDa)

7

2

I

3

4

5

6

7

10680-

32.527*5-

FIG. 5. Western blot analysis of whole-cell lysates from human RPE in vitro. Samples of whole-cell lysates or recombinant human bFGF were subjected to SDSpolyacrylamide gel electrophoresis on 15 % T gels. transferred to nitrocellulose, and visualized with indirect alkaline phosphatase immunochemistry as described in Materials and Methods. Lane 1 contains 1 pg of biotinylated molecular weight markers. Lane 2 contains 6 ng of recombinant human bFGF and lane 3 contains 110 ,ug of RPE cell lysate. Lanes 2 and 3 were incubated with 0.22 pg ml-’ affinitypurified antibodies in 3 % non-fat dry milk and 0.05 % Tween 20 in TBS overnight at 4°C as the primary antibody. Lane 4 contains 6 ng of recombinant human bFGF and lane 5 contains 110 pg of RPE cell lysate. Lanes 4 and 5 were exposed to an original concentration of 0.22 yg ml-’ affinitypurified antibodies which had been absorbed on bFGF coupled to AffiGel 10. Lanes 6 and 7 contain 6 ng of recombinant human bFGF and 110 fig of RPE cell lysate. Lanes 6 and 7 were exposed to an original concentration of 0.22 pugml-’ of affinity-purified antibodies which had been absorbed against aFGF coupled to AffiGel 10.

fractions were evaluated for their ability to label human RPE cultures by standard immunohistochemical techniques. Figures 3(A), (B), and(C) illustrate staining with crude serum, the non-absorbed or flowthrough fraction of the affinity purification, and the bound fraction from the affinity purification, respectively. Both cytoplasmic and nuclear staining are apparent in all three panels, and the apparent result of affinity purification is a slight enhancement of cytoplasmic staining relative to nuclear staining. To illustrate the specificity of the affinity-purified antibodies for bFGF, immunoabsorption controls are shown in Figs 3(D) and (E). Affinity-purified antibody from rabbit 388 was absorbed against affinity resin conjugated with either bFGF or aFGF, and the resulting

supernatants were utilized for immunohistochemistry on human RPE cells. Figure 3(D) demonstrates that bFGF affinity

resin completely

blocks the staining

RPE cells, while Fig. 3(E) demonstrates

of

that aFGF

coupled resin only slightly diminishes the overall intensity of the stain. Figure 3(F) is a normal rabbit

IgG control. In Fig. 4, the effect of various fixation protocols on the staining of human RPE with affinity-purified antibodies against bFGF is illustrated. A number of different protocols were initially examined, including

methanol l‘l

(- 20°C for 15 min), acetone (- 20°C for

FIG. 6. Western blot analysis of whole-cell lysates and subcellular fractions of cultured human RPE. Whole-cell lysates and subcellular fractions from human RPE cultures were prepared, electrophoresed on 15% SDS gel, and transferred to nitrocellulose. Samples of cellular fractions were prepared to retain standard volume relationships of these fractions in a typical cell. Nuclear fraction equals 17 % of total volume, membrane fraction equals lo%, and the cytosolic fraction equals 73%. The blot was blocked and then incubated in affinity-purified antiserum from rabbit 388 at 0.3 pg ml-l. Lane 1 is a set of low molecular weight markers. Corresponding molecular weights are indicated on the side. Lane 2 is 7.5 ng of human recombinant bFGF. Lane 3 is whole-cell lysate, lane 4 is the cytosolic fraction, and lane 5 is the nuclear fraction. Lane 6 is the high-speed pellet or membrane fraction and lane 7 is the whole-cell lysate not incubated with the primary antibody. The blot was visualized with indirect alkaline phosphatase immunochemistry.

15 min), ethanol (- 20°C for 15 min), ethanol with 5 % acetic acid ( - 20°C for 15 min), 4% buffered paraformaldehyde (4°C for 15 min), 2% buffered paraformaldehyde containing 0.1% glutaraldehyde (4°C for 15 min), and 2 % buffered paraformaldehyde

containing 0.5 % glutaraldehyde (4°C for 15 min). In general, all organic fixatives gave very similar results, and only one example of immunostaining with methanol-fixed

cells is given in Fig. 4(A). Figure 4 (B)

illustrates immunohistochemistry 4 % buffered paraformaldehyde. is markedly

reduced in comparison

on cells fixed with Cytoplasmic staining with Fig. 4(A), and

in addition, nucleolar staining is now apparent within a prominently stained nucleus. Figures 4(C) and (D) illustrate the effects of fixation with paraformaldehyde and low concentrations of glutaraldehyde. The immunohistochemical

staining in these panels shows

a progressively weaker nuclear and nucleolar stain as the glutaraldehyde content increases, In order to examine the specificity of our affinitypurified antibodies for bFGF, Western blot analysis was

performed on whole cell lysates from human RPE in tissue culture. Figure 5 presents the results of a typical IIER

55

H. ISHIGOOKA

ET AL

FIG. 7. Immunogold labeling of human RPE cells in vitro for the subcellular distribution of bFGF. Human KPE cells were passaged and maintained in culture for 3 days and fixed in 4 % buffered paraformaldehyde before processing for post-embedding immunogold electron microscopy. A, Affinity-purified antibodies from rabbit 388 were used at a concentration of 0.11 ,ug ml-‘. B, Affinity-purified antibodies from rabbit 388 at a final concentration of 0.11 pugml-’ were absorbed on bFGF coupled to AffiGel 10. C, Non-immune rabbit IgG at a final concentration of 0.11 yg ml-’ was used as the primary antibody. Magnification bars represent 1 pm. N. Nucleus; arrow indicates the nucleolus.

analysis. Using affinity-purified antibodies from rabbit 388, whole-cell lysates from RPE (lane 3) show a pattern of three major bands at approximately 27, 24, and 18.5 kDa. The intermediate molecular weight band at 24 kDa in some blots was resolved into a closely spaced doublet. Lane 4 contains 6 ng of recombinant human bFGF as a standard, which migrates with the lowest molecular weight band in lane 3. In lanes 4 and 5 samples of recombinant human bFGF and RPE whole-cell lysate were developed with affinity-purified antibody which was

subsequently absorbed on bFGF affinity resin before use on this blot. Solid phase absorption of antibodies effectively removes all immunoreactive bands in lanes 4 and 5. In lanes 6 and 7, the bFGF standard and RPE whole cell lysates were subjected to affinity-purified antibody immunoabsorbed with affinity resin coupled with recombinant human aFGF, and it is apparent that solid-phase absorption with aFGF does not block the recognition of bFGF bands by our affinity-purified antibodies. Figure 6 presents the results of a Western blot

LOCALIZATION

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bFGF

IN RPE

CELLS

FIG. 8. Immunogoldlabelingof human RPEin vitro for the subcellulardistribution of bFGF after mild glutaraldehyde fixation (2 % buffered paraformaldehydecontaining 0.1% glutaraldehyde). Human RPE cells were passagedand maintainedin culture for 3 days beforebeing processed for post-embedding immunogoldelectron microscopy.Affinitypurified antibodiesfrom rabbit 388 were used at a liual concentration of 0.45 ,ugml-‘. Magnification bar represents 1 pm. N, Nucleus: M. mitochondria.

analysis of subcellular

fractions of human RPE in vitro. Lane 1 is a set of prestained low molecular weight markers. Lane 2 is 7.5 ng of recombinant human bFGF. Lane 3 is whole-cell lysate of human RPE cells. Lanes 4 to 7 are subcellular fractions of human RPE cells including the cytosolic fraction (4), nuclear fraction (5), and high-speed pellet or membrane fraction (6). Lane 7 is a sample of whole-cell

lysate processed without the primary antibody as a control. Three major bands, the middle of which was actually a closely spaced doublet, are apparent in all

fractions except for the nuclear fraction which lacks the lowest molecular weight form of bFGF. These four bands are likely to be the same as the species at 17.8, 22.5, 23.1, and 24.2 kDa reported by Florkiewicz and Sommer (1989). To confirm the nuclear and nucleolar localization of bFGF in human RPE cells in vitro, RPE cultures were

examined by post-embedding immunoelectron microscopy according to the method of Erickson, Anderson and Fisher (1987). Cultures were fixed in

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4% buffered paraformaldehyde, embedded in LR White, sectioned, and exposed to affinity-purified antibodies from rabbit 388. Staining was visualized with 15 nm colloidal gold labelled second antibody. Figure 7 demonstrates the subcellular distribution of bFGF in RPE cells by this technique. In Fig. 7(A), labeling can be seen most prominently in the nucleolus and nucleus, but definite labeling of cytoplasm is also present. In Fig. 7(B), solid-phase immunoabsorption was used to block the affinity-purified antibodies with bFGF, and only sparse background labeling is evident. Figure 7(C) is a control where primary antibody was replaced with an equivalent concentration of nonimmune IgG, and again, only very low background labeling is evident. Because the images in Fig. 7 give evidence of poor fixation and do not illustrate many features typical of differentiated RPE cells, better fixation and longer-term cultures were examined. Figure 8 presents specimens which were fixed in 2 % buffered paraformaldehyde containing 0.1% glutaraldehyde. Again the nucleus and cytoplasm are labeled, but most of the mitochondria are not labeled. In Fig. 9, cultures were examined which had been maintained in stationary culture for an extended period of time (greater than 1 year). Under our culture conditions, such cultures regularly pigment and produce extracellular matrix. The specimens for Fig. 9 were fixed in 2 % buffered paraformaldehyde containing 0.5 % glutaraldehyde in order to improve fixation as much as possiblewhile maintaining the antigenicity of bFGF. In Fig. 9(A), labeling can again be observed in the

nucleolus, nucleus, and cytoplasm, although the intensity of the nucleolar labeling appears to be diminished in comparison with Fig. 7. No labeling can be seen over most phagosomes or melanin granules. Figure 9(B) shows the apical portion of an RPE cell which also gives evidence of only slight labeling on apical microvilli. In Fig. 9(C), the basal portion of an RPE cell including its associated basement membrane is shown. Little evidence of labeling is present on the basement membrane.

4. Discussion These studies present our data regarding the production and rigorous characterization of affinitypurified antibodies to a 15-amino acid peptide at the amino-terminus of the mature form of human basic FGF. Our previous study of developing capillaries in the bovine retina utilized antibodies produced in the goat by a similar method (Hanneken et al., 1989). This study demonstrates the variability of responsesin

individual

animals, and the variability

of non-specific

antibodies found in the pre-immune sera which necessitatesaffinity purification. Theseantibodies were also demonstrated to be specific for basic and not acidic FGF. Our evidence both by Western blot analysis and light and electron microscopy indicates that bFGF

is found both in the cytosolic and nuclear fractions of 14-2

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H. ISHIGOOKA

ET AL

FIG. 9. Immunogold labeling of long-term human RPE cells in vitro for the subcellular distribution of bFGF after moderate glutaraldehyde fixation (2% buffered paraformaldehyde containing 0.5% glutaraldehyde). Human RPE cells that were maintained in stationary culture for long term (greater than 1 year) were fixed in and processedfor post-embedding immunogoldelectronmicroscopy.Affinity-purified antibodiesfrom rabbit 388 wereusedat a final concentrationof 0.9 pg ml-‘. A, Portion of the nucleus,nucleolus.and the cytoplasmcontaining pigmentgranulesand phagosomes. B. The apical portion of an RPEcell. C, The basalaspectof an RPEcell with its associatedbasementmembrane.Magnification barsrepresentI L/m. N. Nucleus: BM, basementmembrane; arrow indicatesthe nucleolus. human RPE. Fixation protocols, however, directly affect the apparent distribution of bFGF by microscopic methods. In our hands, fixation with aldehydes yields specimenswhich have pronounced nuclear and nucleolar labeling, but which lack the cytoplasmic labeling of specimensfixed with organic solvents. This observation forces a note of caution with respect to our electron microscopic localizations. At the EM level, we detected labeling in the nucleus, nucleolus and cytoplasm, but could find only sparse labeling in basement membrane and apical microvilli, and no

apparent labeling of mitochondria, phagosomes, or melanin granules. The widespread observation of bFGF associated with extracellular matrix (CordonCardo et al., 1990; Gonzales et al., 1990). including our own previous studies (Hanneken et al.. 1989, 199 l), indicates the possibility that aldehyde fixation may selectively alter the apparent localization of bFGF by microscopic techniques. Our Western blot analysis of subcellular fractions substantiates the presence of the three higher molecular weight forms of bFGF in the nucleus. Recently. several other groups have

LOCALIZATION

OF

bFGF

IN

RPE

CELLS

published reports of the appearance of fibroblast growth factors in the nucleus. Amalric and colleagues (Bouche et al., 1987; Baldin et al., 1990) have observed that exogenous bFGF is taken up by bovine aortic endothelium and is transported to the nucleus and nucleolus. Renko et al. (1990) have also observed bFGF localization to the nucleus of 3T3 fibroblasts transfected with an expression construct containing the bFGF gene and SK-Hep-1 cells expressing constitutive levels of bFGF. Studies on aFGF by Maciag and colleagues (Imamura et al., 1990) have also recently explored the significance of nuclear localization for aFGF. Not only do our results on the nuclear and nucleolar localization of bFGF appear to agree with the studies cited above, the Western analysis of the cellular forms of bFGF also appears to be very similar. The studies by Florkiewicz and Sommer (1989) and Renko et al. (1990) include Western analyses in which three main bands of immunoreactive bFGF are present in wholecell lysates of transfected 3T3 cells as well as SK-Hep1 cells. These three bands are in fact the species at 17.8, 22.5. 23.1, and 24.2 kDa where the middle two bands are not resolved due to their very similar molecular weights. In our analysis, all four bands are resolved on the Western blot, due, in all probability, to the fact that we used a 15% resolving gel instead of the 12 % gel used by Florkiewicz and Sommer (1989) and Renko et al. (1990). The four cellular forms of bFGF apparently arise from initiation of translation at three alternative codons upstream from the nominal translation start site (Florkiewicz and Sommer, 1989 ; Prats et al., 1989). The functional significance of the nuclear localization of bFGF is currently unclear. Amalric has proposed that bFGF may play some role in the regulation of rRNA transcription (Bouche et al., 1987). and Maciag suggests that the mitogenic activity of aFGF may in some way be linked to its nuclear localization (Imamura et al., 1990). In addition to these speculations, it is necessary to remember that the studies presented here were performed on relatively undifferentiated cells. The subcellular distribution of bFGF in well-differentiated pigment epithelium either in vitro or in vivo awaits further study. Acknowledgements This work was supported in part by a grant from the National Eye Institute (EY06473). the Juvenile Diabetes Foundation (390362), and by an unrestricted grant from Research to Prevent Blindness, Inc., NY.

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Subcellular localization of bFGF in human retinal pigment epithelium in vitro.

Basic fibroblast growth factor is a polypeptide mitogen with potential biological roles in angiogenesis, differentiation, and the survival of neurons...
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