Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures

Histochemistry (1993) 100:303-309 Histochemistry © Springer-Verlag 1993

Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures C. Herkens 1, M. Wolff 1, J. Fandrey 1, F. Schuler 2, W. Jelkmann 1

1 Physiologisches Institut I, Rheinische Friedrich-Wilhelms-Universit/it, Nussallee 11, D-53115 Bonn, Germany 2 Institut ftir Physiologische Chemie, Rheinische Friedrich-Wilhelm-Universit/it, Nussallee 11, D-53115 Bonn, Germany

Accepted: 30 July 1993

Abstract. The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogeneous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxi- dase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus non- hypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin m R N A and by radioimmunoassay of secreted erythropoietin.

Introduction

The structure and the function of the glycoprotein hormone erythropoietin have been fairly well described (Krantz 1991; Jelkmann 1992). However, the cells that synthesize erythropoietin in kidney and liver still remain to be clearly identified. Immunohistochemical staining for erythropoietin has been accomplished in almost every part of the kidney, including the epithelium (Fisher et al. 1965; Gruber et al. 1977; Mori et al. 1985) and the mesangium (Fukushima et al. 1990) of the renal cor- puscles, tubular cells (Maxwell et al. 1990), and the en- dothelium of peritubular capillaries (Suzuki and Sasaki 1990). By in situ hybridization, erythropoietin m R N A was localized in tubules by some investigators (Maxwell et al. 1990) and in peritubular cells by others (Koury et al. 1988; Kurtz et al. 1989; Lacombe et al. 1988a, b; Bachmann et al. 1993). With regard to the liver, erythropoietin immunoreactivity was shown in Kupffer cells

Correspondence to: W. Jelkmann

of fetal mice (Gruber et al. 1977), whereas erythropoietin m R N A was demonstrated mainly in epithelial hepatic cells of anaemic mice and rats (Koury et al. 1991 ; Kurtz et al. 1989; Schuster et al. 1992).

In view of the conflicting localization of the hormone in whole organs, we chose an in vitro system to evaluate the possibility of detecting erythropoietin intracellularly by immunoperoxidase staining. We used human hepatoma cultures of the lines HepG2 and Hep3B, which are known to produce erythropoietin (Goldberg et al. 1987). Several mono- and polyclonal antibodies were tested. Control studies were carried out with the non-eryth- r0poietin producing porcine kidney cell line LLC-PK1 and a human fibroblast cell line. Hyp0xia-induced erythropoietin gene expression in the hepatoma cultures was confirmed by competitive polymerase chain reaction of erythrop0ietin m R N A in ceil extracts and by radioimmunoassay of erythropoiefin protein in the culture media. To further characterize the cells, hepatoma cultures were stained for other secretory proteins and for intermediate filament proteins.

Materials and methods

Cells grown in culture

HepG2 (ATCC no. HB 8065) and Hep3B cells (ATCC no. HB 8064) were grown in medium RPMI 1640 (Flow Laboratories, Meckenheim, Germany) supplemented with 10 % fetal bovine serum (FBS; Gibco, Eggenstein, Germany). L LC-PKa cells (ATCC no. CRL 1392) were grown in medium 199 (Biochrom, Berlin, Ger- many) with 5 % FBS and antibiotics (100 U/ml penicillin, 100 gg/ml streptomycin; Boehringer, Mannheim, Germany). Human fibroblasts (line "Bernhard", kindly provided by M. Plawky, Bonn) were grown in D-MEM medium (Biochrom) with 10 % FBS. The cultures were maintained in 75 cm 2 flasks (Falcon, Becton Dickinson, Heidelberg, Germany) at 37°C in a humidified atmosphere (5% CO2 in air; Heraeus incubators, Hanau, Germany). The cells were subcultured when confluence was reached (HepG2 at 5 x l0 s and Hep3B at 2 x l0 s cells/cmZ).

For immunohistochemistry, cells were plated on 4 cm 2 coverslips placed in sterilized glass dishes (9 cm in diameter; Schott,

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Table 1. List of primary antibodies

Designation Specification Source Dilution (PAP)

aEpoI MonAb, anti-rhuEpo, IgG Boehringer, Mannheim 1 : 100-1:20 aEpoII MonAb, anti-rhuEpo, IgG Medac, Hamburg 1 : 10 aEpoIII MonAb, anti-rhuEpo, horseradish-peroxidase-conjugated Medac, Hamburg 1:20 aEpoIV PolyAb, anti-rhuEpo, IgG Boehringer, Mannheim 1 : 100 aEpoV PolyAb, anti-rhuEpo, serum This laboratory 1 : 400 aEpoVI PolyAb, anti-rhuEpo, partially purified Dr. Rich, Ulm 1 : 50 aEpoVII PolyAb, anti-Epo-peptide (aa 1-18), serum This laboratory 1:500 aEpoVIII PolyAb, anti-Epo-peptide (aa 42-56), serum This laboratory 1 : 500 aEpoIX PolyAb, anti-Epo-peptide (aa 83 97), serum This laboratory 1:500 aEpoX PolyAb, anti-Epo-peptide (aa 153-166), serum This laboratory 1 : 500 LE-41 MonAb, anti-cytokeratin Lane (1982) - aAlb PolyAb, anti-human-albumin Dakopatts, Glostrup 1 : 500-1 : 700 actAT PolyAb, anti-human-ct-l-antitrypsin Dakopatts, Glostrup 1 : 50 aaFP PolyAb, anti-human-ct-fetoprotein Dakopatts, Glostrup 1:2000-1:1200

MonAb, Monoclonal mouse antibodies; PolyAb, polyclonal rabbit antibodies; rhuEpo, recombinant human erythropoietin; aa, amino acids of Epo sequence

Mainz, Germany). The medium volume was 32 ml for HepG2 and Hep3B cultures, and 20 ml for LLC-PK 1 and fibroblast cultures. To study effects of PO2 changes on erythropoietin production, the culture dishes were kept in an acrylic incubation chamber, which was gassed with humidified mixtures (W6sthoff gas mixing pumps, Bochum, Germany) of either air and 5% CO2 ("hypoxia', see below), or 95% 02 and 5% CO2 ("non-hypoxia') at 37 ° C. Media for the assay of erythropoietin, lactate and glucose were collected after 24 h incubation periods. For staining, the dishes were rinsed twice with ice cold phosphate-buffered saline (PBS, pH 7.4), followed by the removal of the coverslips. These were then fixed with warmed Kayser's gelatin (Merck, Darmstadt, Germany) on glass slides.

Immunohistochemistry

Mounted cover slides were fixed for 4 min at - 20 ° C in acetone for staining of erythropoietin and cytokeratin filaments, or 30 rain at 4 ° C in Bouiffs fixative for the staining of albumin, (x-fetoprotein and ~-1-antitrypsin. Objects were washed 3 x 5 min in ice cold PBS. Endogenous peroxidase activity was blocked by treatment with 3 % H20 2 in 25% methanol (in PBS) for 9 min at room temperature. Slides were then incubated for 1 h at 4 ° C with neutral protein solutions in PBS (2% bovine serum albumin, 5% goat or rabbit non-immune serum; Dakopatts, Glostrup, Denmark) to reduce unspecific binding. The next step was the incubation of the slides with primary antibodies, the appropriate concentrations of which were determined in dilution series.

The primary antibodies used in this study and their respective sources are listed in Table 1. Rabbit antibodies available in our own laboratory obtained earlier by immunization with recombinant human erythropoietin (Cilag, Sulzbach, Germany) or synthetic peptides corresponding to part of the erythropoietin molecule (Biochrom) in combination with bacterial adjuvants (TDM; Sebak, Aidenbach, Germany). For immunization, the peptides were coupled to haemocyanin from keyhole limpets. Peptide-binding activity of the antisera and of isolated IgG-fractions was confirmed by enzyme-linked immunoassay.

The slides were stained by means of the peroxidase-antiperoxidase method (PAP; Sternberger 1979), the streptavidin-biotin-complex technique (StaBC; Hsu et al. 1981) or indirect immunofluorescence. In the PAP-system, the slides were incubated for 1 h at 4°C with goat anti-rabbit serum or rabbit anti-mouse serum (1:100; Dakopatts) followed for 1 h at 4 ° C by rabbit/mouse-PAP- complex (1:150; Dakopatts). In the StaBC-system the slides were incubated for 20 min at 4 ° C with biotinylated goat anti-rabbit or

rabbit anti-mouse antibody (1:300; Jackson Immunoresearch, West Grove, Pa., USA) followed for 20 rain at 4°C by streptavidin- peroxidase-complex (1:500; Jackson Immunoresearch). For indirect immunofluorescence, slides were incubated for 1 h at 4 ° C with ftuorescein isothiocyanate (FITC)-coupled rabbit anti-mouse IgG1 (1:60; Dunn, Asbach, Germany). Between all incubation steps, ceils were washed three times with ice cold PBS. Peroxidase was visualized by the addition of 3,3-diaminobenzidine-tetrahyd- rochloride (0.5 mg/ml in PBS containing 0.06% H202) for 8 rain at 4 ° C. The slides were then counterstained with Mayer's haemalaun (Merck, Darmstadt, Germany), and finally the cover slides were mounted in Entellan (Merck).

All staining procedures were controlled by replacing the primary antibody by PBS, rabbit or goat non-immune serum, or by first blocking the primary antibody by incubation with recombinant human erythropoietin (Boehringer, Mannheim), human albumin, u-fetoprotein, or human c~-l-antitrypsin (Sigma, Mfinchen, Ger- many). LLC-PK1 and fibroblasts "Bernhard" served as control cells devoid of erythropoietin gene expression. Peroxidase-stained objects were examined and photographed with an Axiovert 405 M microscope (Zeiss, Oberkochem, Germany). Fluorescence micro- graphs were taken using a Zeiss Standard 16 microscope fitted with a 450-490 nm band pass filter.

Imrnunoblotting of HepG2 extracts

HepG2 cells were harvested at confluence from six wells each of 24 mm in diameter. Cellular protein was pelleted and delipidated by the method of Wessel and F1/igge (1984). Protein was resuspended in sample buffer and boiled for 5 rain. Separation was achieved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) on a 12.5 % (w/v) polyacrylamide gel according to Laemmli (1970). After electrophoresis protein was transferred to nitrocellulose using the method described by Towbin et al. (1979). Non- specific binding of the primary antibody to nitrocellulose was prevented by incubation for 1 h at room temperature with blocking buffer (1% bovine serum albumin in PBS/0.05% Tween 20). After blocking the blots were incubated overnight at 4 ° C with the primary antibody diluted 1:1000 in PBS/0.05% Tween 20. For detection of primary antibody the Western blot was incubated with biotinylated goat anti-rabbit IgG (1:1000 diluted in PBS/0.05% Tween 20) for 2 h at room temperature. After washing with PBS/ 0.05 % Tween 20, the Western blot was incubated with streptavidin- coupled horseradish peroxidase (1:1000 in PBS/0.05% Tween 20) followed by development of the Western blot with chloronaphthol. Controls omitting the first antibody did not show any positive reaction with cellular proteins.

Assay of erythropoietin

Erythropoietin in the culture media was measured in duplicate by the radioimmunoassay described earlier (Fandrey et al. 1990). The assay system included 125I-labelled recombinant human erythropoietin (11-33 TBq/mmol; Amersham Buchler, Braunschweig, Germany), rabbit antiserum against recombinant human erythropoietin and human urinary erythropoietin standard. Antibody- bound and free [12SI]erythropoietin molecules were separated by precipitation with polyethylene glycol (PEG 6000; Merck). For determination of the erythropoietin binding capacity of the various anti-erythropoietin antibodies, these were incubated in serial dilutions with 125I-labelled erythropoietin (0.6 fmol/vial) for 48 h at 4 ° C followed by polyethylene glycol precipitation. Erythropoietin bioactivity in hepatoma-conditioned medium was determined by bioassay in exhypoxic polycythaemic mice (Jelkmann and Bauer 1981).

Determination of erythropoietin mRNA

Competitive polymerase chain reaction was performed as described recently (Fandrey and Bunn 1993).

Determination of protein, glucose and lactate

For the assay of total cellular protein, the cultures were washed with PBS and lysed with SDS-NaOH (5 g/1 SDS in 0.1 tool/1 NaOH). Cell protein was determined according to Lowry et al. (1951) by means of a micro determination kit (Sigma). For the determination of glucose and lactate in the culture media, photometric assays were carried out using commercial kits (Sigma).

Determination of pericellular PO 2

Pericelluiar PO 2 was measured with polarographic O2-sensitive solid state catheter probes (Neocath; Biomedical Sensors, High Wycombe, Bucks, UK) placed in the monolayer (Wolff et al. 1993). The electrical signals were processed by microcomputer (LICOX PO2; GMS, Kiel, Germany).

Statistics

Results are given as the mean + SD. Student's t-test was used to determine the significance of the difference between two means values at P< 0.05.

Results

Comparison of anti-erythropoietin antibodies

Radioimmunological studies showed that all antibodies and sera raised against intact erythropoietin (Table !) had the capacity significantly to bind recombinant human~25I-labelled erythropoietin, while none of the antisera directed towards synthetic erythropoietin fragments bound the intact hormone. In the immunohistochemical studies with hepatoma cells only one of the three monoclonal antibodies (aEpoI, dilutions 1:20 1:100) proved to be suitable for staining of erythropoietin. Sufficient signals were obtained with both the PAP- and the StaBC- methods in hypoxic Hep3B cells (Fig. la) and with the PAP-method in HepG2 cells. Labelling was most promi-

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nent in the perinuclear region, while the peripheral cyto- plasm was more faintly immunostained. In addition, the staining intensity differed from cell to cell. The coloured enzymic reaction was missing when aEpoI was sub- stituted by PBS or non-immune serum. In addition, no positive staining was obtained with aEpoI applied to the non-erythropoietin producing cell cultures, LLC-PK1 and fibroblasts.

With all of the polyclonal antibodies, positive staining (PAP and StaBC) of hypoxic Hep3B cells was demonstrated, while one (aEpoVI) was inactive on HepG2 cells. aEpoIV (1:100) and aEpoV (1:400) also produced more strongly coloured signals in Hep3B compared to HepG2 cells. Clusters of labelled cells were surrounded by areas showing little immunostaining (Fig. lb). Control experiments with PBS, non-immune serum and rabbit IgG were clearly negative in the PAP procedure, but not always in the StaBC procedure. However, aEpoIV and aEpoV produced some colour reaction of LLC-PK1 cells and, to a lesser extent, of fibroblasts. Control experiments with pre-absorbed aEpoIV lowered the staining intensity in Hep3B and HepG2 preparations but not in LLC-PK1 cells or fibroblasts. None of the antisera raised against synthetic erythropoietin fragments were suitable for detection of the hormone in hepatoma cells.

Immunoblottin9

Figure 2 shows the Western blot of an extract from a hypoxic HepG2 culture. The primary antibody was rabbit IgG to pure human recombinant erythropoietin (aEpolV). Two bands were visible, a minor band at 34 kDa and a major one at 30 kDa. Additional blotting studies with recombinant human erythropoietin revealed a single band at 34 kDa.

02-dependence of erythropoietin production

Due to the diffusion-limited 0 2 supply, cellular hypoxia develops when confluent hepatoma cultures in glass or polystyrene dishes are incubated in an atmosphere of air with 5% CO2 (Wolff et al. 1993). Hypoxia-induced production of erythropoietin and lactate as well as glucose consumption were lowered when 02 instead of air was used for incubation (Table 2).

Figure 3 demonstrates that PO2-dependent changes occurred in the level of erythropoietin mRNA. In HepG2 cells erythropoietin m R N A levels rose from 0.3 amol/gg total R N A in non-hypoxic cells to 1.7 amol/~tg total RNA in hypoxic cells. As seen for the protein (Table 2), the amplitude of erythropoietin m R N A production was much larger in Hep3B cells Which were stimulated 10- fold by hypoxia (non-hypoxic 0.07 am01/gg total R N A versus 0.7 amol/gg total R.NA in hypoxic Hep3B cells).

By immunohistochemistry, reduced staining for erythropoietin was seen when HepG2 or Hep3B cells were incubated in 95% 02 instead of 20% 02 (Fig. lc and d). According to microelectrode measurements, the pericellular PO2 was 250 4- 7 mm Hg (mean of six experiments)

Fig. la-h. Immunostaining of human hepatoma cell cultures. a Hep3B; monoclonal antibody, aEpoI; peroxidase-antiperoxidase (PAP). b HepG2; polyclonal antibody, aEpoIV; PAP. c and d Hep3B incubated in 95% or 20% Oa, respectively; aEpoI; PAP.

e Hep3B; anti-albumin; PAP. f Hep3B; anti-ct-l-antitrypsin; StaBC. g Hep3B; anti-a-fetoprotein; StaBC. h HepG2; anti- cytokeratin; immunoftuorescence detection, a, b and e--h x 330, e and d x 520

kDa 1 2

66-

4 5 - 3 6 -

2 9 - 2 4 -

Fig. 2. Western blot of HepG2 extract following sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and incubation with rabbit IgG to recombinant human erythropoietin (lane 2) ; molecular weight markers (lane 1)

HepG2 Hep3B

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in HepG2 monolayers grown on cover slides in glass dishes (six slides per dish) during incubation in 95% 02.

Immunocytochemistry of other secretory proteins and cytokeratin

Both HepG2 and Hep3B cells showed clear colour responses when stained for albumin (Fig. le), a-l-antitrypsin (Fig. lf) or a-fetoprotein (Fig. lg). There was signifi- cant cell-to-cell variation in the signal intensity, with islands of strongly stained cells being surrounded by poorly reacting cells (see e.g. Fig. lg). When the two hepatoma cell lines were compared under similar incubation conditions, a stronger colour reaction was exhibited by Hep3B for albumin and a-l-antitrypsin, and by HepG2 for a-fetoprotein. In control experiments with PBS, normal rabbit serum or pre-absorbed antisera either no (albumin) or significantly reduced (a-l-antitrypsin) staining was observed. Cytokeratin was visualized by immunofluorescence in HepG2 cells incubated with the monoclonal antibody, LE-41 (Fig. lh; Hep3B was not examined). Replacement of the primary antibody com- pletely abolished the fluorescence response.

H N H N Fig. 3. Ethidium bromide stained agarose gel of erythropoietin (Epo) cDNA amplified by polymerase chain reaction (PCR) following reverse transcription of erythropoietin mRNA. Lane 1 from left displays the 100 bp ladder as molecular weight marker. Lanes 2 and 3 from left represent Epo mRNA levels in hypoxic(H) and non- hypoxic(N) HepG2 cells, while lanes 4 and 5 are from hypoxic and non-hypoxic Hep3B cells, respectively. Lane 6 from left is a negative control for the PCR reaction

Discussion

This is the first immunohistochemical study showing positive staining for erythropoietin in homogeneous cell cultures. The experiments were carried out with HepG2 and Hep3B cells, which are capable of controlled erythropoietin gene expression (Goldberg et al. 1987; Ueno et al. 1989). Earlier attempts to demonstrate erythropoietin immunoreactivity in cultured murine eryth- roleukaemia cells were not successful (Lacombe et al. 1988b). The difficulties encountered in the immunostaining of erythropoietin can be partly explained by the fact that the hormone is present intracellularly only in small amounts. Some erythropoietin can be extracted from blood-depleted kidneys of hypoxic animals but not from the liver (Jelkmann and Bauer 1981). Erythropoietin mRNA is synthesized de novo following the induction of hypoxia (Schooley and Mahlmann 1972; Goldberg et al. 1987; Fandrey and Bunn 1993).

Table 2. Production of immunoreactive erythropoietin a and lactate, and consumption of glucose in HepG2 and Hep3B cultures during 24 h of hypoxic (20% 02) and non-hypoxic (95% O2) incubation

Cell line 02 Erythropoietin production Lactate formation conc. (U/mg protein) (mmol/mg protein)

Glucose consumption (mmol/mg protein)

HepG2 20% 0.31 _+0.09 32-t-9 20_+4 95% 0.09_+0.02* 12_+2" 12_+1"

Hep3B 20% 0.59_+0.05 24_+2 14_+1 95% 0.03_+0.03* 12_+2" 8_+1"

Data are means -+ SD of five experiments, using confluent monolayers in polystyrene vials (0.5 ml medium/cm 2) a Comparative determinations of erythropoietin by in vivo bioassay showed the activity in HepG2 culture-conditioned medium was

only 26% compared to the immunoreactivity (Hep3B culture- conditioned media were not measured by bioassay) * Significantly different (P<0.05) compared to values at 20% 02

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Because antibodies directed against recombinant human erythropoietin were used in the present studies, it is important to note that native and recombinant human erythropoietins are identical with regard to amino acid sequence, position of disulphide bonds, glycosylation sites and secondary structure (cf. Jelkmann 1992). Radioimmunological studies have shown cross-reactivity between the native hormone and the recombinant product (Egrie et al. 1987).

Of the three monoclonal antibodies tested, only one yielded sufficiently strong colour signals with the PAP- and the StaBC-methods in hepatoma cells. The three polyclonal antibodies, raised in rabbits, were partially deficient in specificity. Antisera raised against synthetic erythropoietin-fragments were not suitable for staining of the hormone in hepatoma cultures. This finding is of interest with regard to the antibody directed towards the peptide spanning residues 153-166 (aEpoX). The car- boxy-terminal arginyl residue in position 166 is thought to occur only intracellularly and to be cleaved from the translation product immediately before secretion (Imai et al. 1990; Recny et al. 1987).

When hypoxic and non-hypoxic hepatoma cultures were compared, staining for erythropoietin was more intense in hypoxic Hep3B than in hypoxic HepG2 cells. As shown in Table 2, this difference corresponds to the higher rate of secretion of the hormone in Hep3B compared to HepG2 cultures. The levels of erythropoietin mRNA and of immunoreactive erythropoietin in the culture medium decreased when the 02 supply was increased by incubation in 02 instead of air. It is notewor- thy that immunoperoxidase staining for erythropoietin was not negative in all hepatoma cells incubated in 95% Oz, indicating that some cells were still producing the hormone despite non-hypoxic culture conditions. This observation is in agreement with the low base-line level of erythropoietin mRNA (mean of 0.6 molecules per cell) recently detected in hyperoxic Hep3B cells by means of a competitive polymerase chain reaction (Fandrey and Bunn 1993). Moreover, immunoreactive erythropoietin was always measurable in the culture media of Hep3B and HepG2 cells maintained in 95% O2 (Table 2).

Our Western blot analysis of erythropoietin immunoreactivity in HepG2 cell extracts revealed two bands at 34 kDa and 30 kDa. Studies carried out earlier showed that HepG2 cells release two forms of erythropoietin that differ in their sugar moiety: Ueno et al. (1989) reported two bands of immunoreactive erythropoietin with an apparent molecular mass of 36 kDa and 34 kDa on Western blots with HepG2-conditioned medium. We measured the bioactivity of secreted erythropoietin in HepG2 medium and found it to be only 26% of the activity determined by radioimmunoassay, which con- firms the assumption by Ueno et al. (1989) that the lower molecular weight form is incompletely glycosylated and thus likely to be inactive in vivo. We propose that immu- noblotting following SDS-gel electrophoresis of HepG2 and Hep3B cell extracts may provide a useful tool in studies of the process of erythropoietin glycosylation.

Although the kidneys are the main site of erythropoietin synthesis in the adult organism, as yet no renal

cell culture system has been established that is capable of controlled erythropoietin gene expression. Thus the two human hepatoma cell lines HepG2 and Hep3B are presently considered the most appropriate cell culture model for study of the molecular mechanisms of erythropoietin production (Goldberg et al. 1987; Ueno et al. 1989; Fandrey and Bunn 1993; Wolff et al. 1993).

The cell lines HepG2 and Hep3B were established several years ago from liver turnout biopsies of two children (Aden et al. 1979). The biopsies revealed well- differentiated hepatocellular epithelial tissue with a trabecular pattern; also, by electron microscopy cultured Hep3B cells were demonstrated to share morphological features with liver cell carcinomas (Aden et al. 1979). However the exact nature of the cells of origin of the two hepatoma lines has been questioned. In fact the possibility has been considered that they might be mesenchym- ally derived (Koury 1989). Support for an epithelial origin was provided in the present study by the demonstration of cytokeratin filaments while vimentin filaments were missing. In addition, HepG2 and Hep3B cells secrete hepatic proteins (Knowles et al. 1980). By immunohistochemistry, Bouma et al. (1989) have recently shown albumin, ~-antitrypsin and et-fetoprotein in cultured HepG2 cells. In the present study these proteins were also demonstrated in Hep3B cells, giving rise to islands of strongly stained cells surrounded by poorly coloured cells; Bouma et al. (1989), by contrast, have reported a uniform staining of their cultures. In addition, these authors observed a perinuclear dot-labelling, which was not seen in the present experiments.

With regard to 1Lhe renal localization of erythropoietin by immunoperoxidase staining, preliminary experiments carried out here were less conclusive (photographs not shown). Incubation of cryostat sections from kidneys of mice exposed to hypobaric hypoxia with polyclonal antibody (aEpoIV) resulted in staining of the renal cor- puscles; this staining was abolished when pre-absorbed antibody was used. In kidneys from anaemic mice, however, positive staining was restricted to the tubular cells. This discrepancy could be due to the lack of specificity of the polyclonal antibodies used. Unfortunately, none of the present monoclonal antibodies mediated staining of hypoxic or anaemic murine kidney sections. There is only one report of the successful application of the StaBC-method with a monoclonal antibody towards human erythropoietin for localization of the hormone in renal tissue. In anaemic mouse kidney, Suzuki and Sasaki (1990) have demonstrated immunostaining in the en- dothelium of peritubular capillaries, while the renal cor- puscles and the tubules were negative.

Acknowledgements. Thanks are due to Natalie Broscheid for ex- cellent technical assistance and to Ursula Isenberg for secretarial help. We also wish to thank the following individuals and in- stitutions for generous gifts: Prof. C.A. Baldamus, K61n, for Hep3B cells; M. Plawky, Bonn, for fibroblasts "Bernhard"; Dr. C. Vieb- ahn, Bonn, for anti-intermediate filament antibodies; Dr. I. Rich, Ulm, for anti-erythropoietin antibody; Dr. P. Hirth, Boehringer, Mannheim, for recombinant human erythropoietin and anti-erythropoietin antibody; and Dr. S. Saadat, Medac, Hamburg, for anti- erythropoietin antibody. Part of this study was financially support-

309

ed by Sebak GmbH, Aidenbach, and the Bundesministerium fiir Forschung und Technologic (BMFT-01ZR8702).

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Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures