- Email: [email protected]
Cytokeratin patterns of epithelial cells of the rat nasal cavity in vivo and in vitro
Toxicology Letters Toxicology Letters 88 (1996) 65-73
Cytokeratin
patterns of epithelial cells of the rat nasal cavity in vivo and in vitro W.K. Schlage*, H. Biilles, D. Friedrichs, A. Teredesai lnstitut fir hiologische Fiirschung, Fuggerstr. 3, 51149 Kbln, Germany
Abstract Nasal epithelial cells are a primary target for the actions of inhaled substances. To enable the determination of alterations in cell differentiation in the rat inhalation model, we developed a methodology to assess cytokeratin expression in rat nasal tissue. A panel of commercially available antibodies was validated for specificity to defined rat cytokeratins by immunoblotting. The development of immunohistological procedures to enhance spatial resolution enabled mapping of cytokeratin patterns in various cell types at defined regions and levels of the rat nasal cavity using serial sections and a standardized evaluation schedule. Keywords: Immunohistochemistry; tract
Western blot; Antibody
1. Introduction In previous studies, we reported on the use of cultured rat nasal epithelial (RNE) cells to determine genotoxic activity after in vitro exposure or ex vivo after inhalation exposure using sister chromatid exchanges (SCE) and chromosome aberrations as end points [1,2]. The aim of this and our future investigations is to determine nongenotoxic effects, such as the induction of differentiation changes, in RNE cells in vivo and in vitro using cytokeratin (CK) expression patterns as end points. The use of CK expression patterns as markers for the differentiation of the intermediate filament cytoskeleton of epithelial cells from human .and many vertebrate species is well established [3,4]. However, a suitable *Corresponding author.
panel; Antigen retrieval; Keratinocytes;
Respiratory
methodology to determine the CK expression pattern in rat nasal epithelia has not been developed yet; there are few published data on the CK distribution in the nasal cavity, for the rat as well as for other vertebrate species. Moreover, a welldefined antibody panel for rat CK is lacking. To achieve the aim mentioned above, two basic objectives had to be addressed: first, to validate an antibody panel for rat CK useful for immunoblotting of tissue extracts as well as for immunostaining of paraffin sections obtained from routine histology; and second, to map the normal CK distribution in rat nasal epithelia. 1.1. Validation Commercially available monoclonal antibodies to CK were screened for individual specificities to defined rat CK polypeptides using sodium
0378-4274/96/$15.00 10 1996 Elsevier Ireland Ltd. All rights resewed PII SO378-4274(96)03719-S
66
W. K Schlagc et al. I Toxicolog_v Lctrcrs 88 (I 996) 65- 73
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of cytoskeletal extracts from rat tissues with known CK composition. The CK patterns of excised epithelial samples from the rat nasal cavity and of primary cultures of RNE cells were obtained in parallel. Due to the large number of CK polypeptides and the overlapping patterns, it was necessary to enhance the spatial resolution by means of immunohistological techniques. The rat-specific CK antibodies were tested for immunohistological reactivity to rat tissues on unfixed cryosections of reference tissues and of nasal epithelia. Subsequently, immunohistochemical procedures were tailored to utilize these CK antibodies with routinely obtained, formalin-fixed and paraffin-embedded histological sections. As a result, a panel of nine antibodies suitable for paraffin sections was derived. 1.2. CK mapping Using this antibody panel, we determined the CK staining pattern in nine serial sections from two levels of the rat nasal cavity, the method of evaluation being a 4-stage individual grading for intensity as well as for distribution to discriminate and calculate local alterations of CK expression in exposed rats at high resolution. For in vitro cultured RNE cells, CK expression was determined by immunofluorescence.
2. Materials and methods
NLI
NL2
Fig. 1. The rat nasal cavity (median section through the rat head, lower jaw removed). Blue, squamous epithelium; yellow, respiratory epithelium; pink, olfactory epithelium; NLl, NL2, beginning of the defined cross-sectional levels NLl and NL2 (modified from [14]).
samples were collected, one from the respiratory (NEl) and one from the olfactory (NE2) epithelium (Fig. 1). In vitro cultured RNE cells (see below) were scraped off the culture dishes. Cytoskeletal extracts (high salt and detergents) were prepared according to [6] and separated using the Phast apparatus and Phast gradient gels with SDS buffer strips (Pharmacia, Freiburg). The proteins were transferred to PVDF membranes (Millipore) by thermoblotting. The dried blots were rehydrated and stained with various commercially available monoclonal antibodies (Table 1) according to standard procedures. Molecular weights were estimated from biotinylated low molecular weight standards (Bio-Rad) included on each blot. Details will be published elsewhere (Schlage et al., in preparation). All determinations were repeated independently at least in triplicate up to 20 times.
2.1. Electrophoresis and immunoblotting
Various epithelial tissues (foot sole epidermis, tongue mucosa, liver, small intestine), serving as reference tissues with well-documented CK composition, and nasal mucosa were obtained from adult Sprague Dawley rats. The nominal CK composition of the reference tissues was as follows: footsole epidermis, CKl, 2, 5, 6, 9, 10, 11, 14, 16; tongue, CK4, 5, 6, 13, 14, 15, 16, 17, 19; liver, CK8, 18; small intestine, CK8, 18, 19 (according to [S]). From the nasal mucosa, two
2.2. Cell culture
Epithelial cells were detached from the underlying connective tissue of the pooled NE1 and NE2 tissue fragments by enzymatic digestion (pronase, collagenase, hyaluronidase) and cultured for 5 to 6 days in serum-free medium in plastic dishes as described in [l]. For immunofluorescence experiments, the cells were seeded on plastic slides (slide flasks, Nunc).
67
W.K Schlage et al. I Toxicology Letters 88 (19%) 65-73 Table 1 Antibody panel for rat cytokeratins: specificity and suitability in immunohistochemical Name
Ks18.04 LL 002 CK-E3 Ks13.1 Ks8.7 Ks8.12 6BlO KS860 OV-TL 12/30 PCK 26 AE-I RCK 105 Ks4.62 Ks8.13 LDS68 4.1.18
c-51
Supplier
Progen BioGenex Sigma Progen P rogen Sigma Sigma Sigma BioGenex Sigma Progen Laboserv Sigma Sigma Sigma Boehringer Bio-Genex
Recommended use’
specificity Human CK”
Rat CK’
18 14 17 13, (14, 16) 8 13, (15), 16
18 14 19 13, (15) 8 15, 13 4 1, lO/ll 7 1, W, 8 lOIll, 14, 16 7 19 Pan n.d. 8 n.d.
;1,, 10, 11 7 1, 5, 6, 8 10/l, 14, 16, 19 7 19 Pan 7, (8) 8 8
applications Suitability Cryob/paraffin cryo, cryo, cryo cryo, cry0 cry0 cryo, cry0 cryo,
paraffin paraffin paraffin
paraffin
paraffin cryo,paraffin cryo, paraffin cryo cry0 cry0 cryo, paraffin cryo, paraffin paraffin
+I+ +/+ +I+ +I+ +M+) +I+ +I+
+I+ +I+01 + n.d./+
+I0 +I0 +I0 n.d./O
n.d./(+ ) n.d./O
0, no specific reactivity; (), weak or questionable reactivity; +, specific reactivity; n.d., not determined. “As given by supplier and literature. bDetermined on cryosections and cells cultured on slides. CAs determined by immunoblotting (MS in preparation).
2.3. ImmunohistolG~gica!procedures 2.3.1. Parajin sections
Transverse sectilons at nose levels (NL) 1 and 2 (Fig. 1) were obtained from 8 adult Wistar rats
by routine histological procedures. Briefly, the procedure consisted of fixation in buffered formalin, decalcification in nitric acid in an ultrasonic bath, trimming the slices from the nose at defined levels, dehydration and embedding in paraplast, cutting sections of 5-pm thickness, mounting on adhesive slides (Superfrost Plus, Menzel-Glas, Braunschweig). The immunostaining procedure included the following principal steps: deparaffinize, unmask epitopes, inactivate endogenous peroxidase (3% hydrogen peroxide, 5 min), block nonspecific binding sites (1% swine serum, 30 min), incubate for 30 min with amibody to CK (Table 2) diluted in diluent (Cat. No. S3022, Dako, Hamburg), use
detection system with biotinylated second antibody and AEC substrate (Dako), counterstain with hematoxylin. For unmasking, we compared the effectiveness of protease treatment [7] and microwave treatment [8] in lead thiocyanate solution [9] as well as in plain water. The optimal conditions are listed in Table 2. For microwave treatment, it is necessary to interrupt heating when the bathing solution reaches the boiling point in order to prevent detachment of bone-containing and cartilaginous tissue areas from the slide: five sequential heating cycles with 30 s intermission were sufficient for antigen retrieval. It must be noted that these conditions only apply to nasal tissue fixed in buffered formaldehyde and decalcified in nitric acid. For other tissues, e.g. lungs or liver that were fixed differently and not treated with nitric acid, we found other optimal antigen retrieval requirements for some of the CK antibodies (not
68
W. K. Schlage et al. I Toxicology Letters 88 (I 9%) 65 73
Table 2 Antibody panel for paraffin sections from rat nasal cavity: unmasking and antibody dilution Antibody
Epitope unmasking
Dilution
Remark
negative control _ _ _
Treatment
Time (min)
Nonimmune serum
various’
to/15
1:lOO
Ks18.04 LL 002 CK-E3 Ks13.1 Ks8.12 6B10 Ks8.60
protease, 1 g/l microwave, lead protease, 1 g/l protease, 1 g/l protease, 1 g/l microwave, water microwave, water
15 IO 15 15 I5 10 10
I:200 1:lOO 1:300 1:lO I:30 I:100 I:600
OV-TL12/30 PCK 26
protease, 1 g/l various”
15 IO/15
I:200 1:300
affinity purified _ positive controP
“According to corresponding antibody treatment. bPerforms equally well with microwave or protease treatment.
shown). Negative (nonimmune IgG) and positive a (mAb PCK26) control slides were included in each staining series. 2.3.2. Cryosections Cryosections (5-7 pm thick) from reference tissues were obtained from rats by standard procedures. Cryosections of the nasal cavity were obtained from the undecalcified head by a method modified from [lo] using a C-type knife with a tempered steel edge. Staining was performed according to standard procedures using the same detection system as for paraffin sections. 2.3.3. In vitro cultured RNE cells These cells were rinsed in PBS, fixed with cold methanol and immunofluorescently stained according to standard procedures. Endogenous peroxidase blocking was most effective using a mixture of 10% acetic acid and 90% methanol at -20°C for lo-15 s. 2.4. Histological evaluation The epithelial staining pattern of the transverse sections at NLl and NL2 was evaluated separ-
b
Fig. 2. Mapping of CK staining on transverse sections through the rat nasal cavity at NLl (a) and NL2 (b). The boxes approximately indicate the evaluation areas. (a) NLl stained with Ks8.12 (CK15, 13). A, squamous epithelium; B, vomeronasal organ; C-E, respiratory epithelium; F, submucosal glands; G, nasolacrimal duct. (b) NL2 stained with Ks18.04 (CK18). A, squamous epithelium; B, vomeronasal organ; C-F, respiratory epithelium; G, submucosal glands; H, olfactory epithelium; I, Bowman’s glands; J, nasolacrimal duct.
ately at the representative areas as shown in Fig. 2. Within each area, the staining was assessed separately for each distinguishable cell type, e.g. basal, ciliated, and goblet cells within the respir-
W.K. Schlage et al. I Toxicology Letters 88 (19%) 65-73
69
Table 3 Example of detailed evaluation: individual scores (mean of 8 rats) obtained for the respiratory epithelium at NLl Epithelium
Ciliated
Cell type
Basal
Ciliated
Low cuboidal
Basal
nonciliated
Antibody
Ks18.04 CK-E3 Ks8.12 LLO02 Ks18.04 Ks8.12 CK-E3 LLO02 Ks18.04 CK-E3 Ks8.12 LLO02 Ks18.04 Ks8.12 CK-E3 LLO02 OV/TL12/30
CK
Area
18 19 15,13 14 18 15, 13 19 14 18 19 15,13 14 18 15, 13 19 14 7
C Int./Distr.
D Int./Distr.
E Int./Distr.
3.012.0 2.511.8 3.012.8 1.8/1.8 3.OJ2.0 0.3jO.l 3.012.3 O/O n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
3.012.0 1.7/1.1 2.412.3 1.411.3 2.811.9 0.3/0.1 3.012.1 O.l/O.l 3.012.0 1.310.9 2.612.1 1.5/1.6 3.011.9 1.310.8 2.311.4 O.l/O.l O/O
3.OJ2.0 1.6/1.1 2.912.6 1.2/1.8 3.Of2.0 O/O 3.012.1 0.210.4 3.012.0 l.lJO.6 3.012.6 1.0/1.2 3.011.9 1.9/1.1 2.311.0 O/O 0.510.3
Individual scores: (intensity) 0, negative; 1, weak (only detectable at higher magnification, e.g. x 40 lens); 2, distinct (already detectable at lower magnification, still transparent); 3, intense (opaque, red to brown staining); (distribution, related to percentage of the cell type in the observation area) 0, none; 1, focal (up to _ 15 %); 2, patchy (_ 15%-85%); 3, diffuse (* 85%- 100%). n.a., not applicable. The negative results obtained for antibodiesOV/TL 12/30, 6B10, Ks8.60, and Ks13.1 are not listed.
atory epithelium. For each cell type, the staining intensity and relative distribution was individually scored using the 4-stage scores as defined in the legend to Table 3. All histological evaluations were reviewed by a veterinary pathologist.
3. Results and discussion 3.1. Validation of antibody panel
First, we used SDS-PAGE and immunoblotting to screen commercially available monoclonal antibodies to CK -most frequently directed against human or bovine CK polypeptides for their specificity to rat CK. This was necessary because in the literature such CK antibodies cross-reacting with rat CK were often defined only for human or for murine CK, and thus it was presumed that their specificity for rat CK was -equivalent. As the molecular weights of rat
CK frequently differ from those of their human counterparts, we used the reference tissues to extrapolate the CK polypeptides. We thereby obtained a panel of 15 antibodies with validated specificities to rat CK (Table 1). In general, the specificities were indeed the same as for the CK donor species. Exceptions from the nominal specificities were found, e.g. for the frequently used antibody AEl which does not recognize rat CK19 (also reported by [l l]), for CK-E3 specific for human CK17 but which recognizes rat CK19 instead of CK17 [12,13], and for K&.12 which does not recognize rat CK16. Due to the limitations of our methodology (reference tissues and one-dimensional electrophoresis) it was not possible to distinguish between CK5 and 6, CKlO and 11, or to identify CK3, 9, 12, and 17. From the antibody series defined by Western blotting (Table l), a selection of antibodies was found to be useful for immunohistological application with formalin-fixed, paraffin-embedded
W.K. Schlugeet (11.I ToxicologyLetters 88 (19%) 65-73
70
Table 4 General CK expression profiles in the rat nasal epithelium Region
Ventral meatus Respiratory
Olfactory
Cell type/ localization
Basal Suprabasal Basal Ciliated, cuboidal Gland Gland duct Basal zone Mid/apical zone Gland
Antibody (CK) KS860 (l/10/l 1)
6BlO (4)
OV/TL (7)
Ksl3.1 (13/15)
Ks8.12 (15/13)
LLO02 (14)
Ksl8.04 (18)
CK-E3 (19)
0 + 0 0 0 0 0 0 0
0 0 0 0
0 0 0 (+) 0 + 0 0 0
0 + 0 (+) (+)
+ +
+ +
0 0 +
I’+) 0 (+) (+) 0 0
0’ P+)
0 0 + + + + + + +
0 0 0 0 0
I’+) 0 0
(1) 0
I’+) (+) 0 0 0
+, distinct to strong staining intensity with patchy to diffuse distribution; (+), weak to distinct staining intensity with focal to patchy distribution; 0, no staining observed.
sections (Tables 1, 2) a crucial requirement being the antigen unmasking. In order to ascertain the specificity for rat CK also in immunohistological applications, the antibodies were tested for suitability with cryosections of unfixed reference tissues and tissues of the rat nasal cavity (Table 1). Although it was possible to obtain cross-sections of the undecalcified rat nasal cavity, this was not useful to obtain the high number of good quality serial sections required. For the selected antibodies, the same structures recognized in paraffin sections were also recognized in cryosections. With regard to rat CKS, we could not find an antibody which was suitable for paraffin sections of the rat nasal cavity. The antibody Ks8.7, however, showed weak staining in paraffin sections of soft tissues that had not been treated with nitric acid after fixation. In the nasal cavity, a pronounced nuclear staining (possibly due to cross-reaction with nuclear lamins) was observed, primarily in the submucosal glands, when staining with this and another antibody (LDS68). Due to the lack of monospecificity, it was not possible to distinguish histologically between CKl/lO/ll (Ks8.60), CK13/15 (Ks8.12, Ks13.1), and CKl/ 5/6/8 (PCK26); the last antibody behaved as a “Pan-CK”, staining all epithelia in the rat nasal
cavity, and therefore served as a positive control in each staining procedure. The antibodies Ks8.12 and Ks13.1 both recognize rat CK13 and 15, however, it appears that the CK15 staining intensity is more pronounced for Ks13.1 and the CK13 staining intensity is more pronounced for CK8.12 (Table 4). 3.2. CK expression patterns obtained by Western blotting
For the in vivo situation, differences were found between NE1 and NE2. NE2 contained CKl, 5/6, 14, 15, 16, and NE1 expressed in addition CK4, 8, 10, 13, 18, 19 (Table 5). For the in vitro situation, RNE cells contained all the CK from NE1 and NE2 with the exception of CK8 and 18, but they additionally expressed CK7. The enzymatically isolated cells from NE1 and NE2 were combined for culturing, because the cloning efficiencies of the separate cell fractions were too poor. From the large number of different CK polypeptides expressed in these epithelia and the overlapping patterns, it became obvious that more spatial resolution was required for comparisons. Therefore, the immunohistological methodology was developed.
W.K Schlugr et al. I Toxicology Letters 88 (19%) 65-73
3.3. CK expression in cross-sections of the rat
71
a
nasal cavity Simply mapping the epithelial staining distribution (Fig. 2) was not sufficient as the staining patterns along a certain type of epithelium (respiratory, olfactory, squamous, glandular) appeared differentially with regard to intensity, distribution, and cell type in the defined areas (Fig. 2). Hence, we developed the 4-stage individual scoring for intensity, and distribution of cellular staining (Table 3) at the defined areas (Fig. 2). Examples for the histological staining of respiratory and olfactory epithelial cells are shown in Fig. 3. The detailed results of the evaluation were expressed as mean scores (intensity/distribution) for each antibody and cell type at all the sites listed (an example is given in Table 3). For the purpose of this paper, the general CK expression patterns for the different cell types in the three main epithelial regions of the rat nasal cavity are summarized in Table 4. As expected, the markers for cornified stratified epithelia CKl/lO/ll were exclusively expressed in the suprabasal cell layers of the squamous epithelium of the ventral meatus, whereas the “simple epithelial” CK18 and 19 were completely absent.
Table 5 CK expression patterns determined in cytoskeletal extracts from rat nasal epithelia and RNE cells CK
NE1 1 4 516 7 8 IO/l 1 13 14 15 18 19
In vitro
In vivo
(+G ++ 0 (‘t) + ++ + I’+)
NE2
RNE
+ 0 ++ 0 0 0 0 ++ + 0 0
(+) (+) ++ ++ 0 (+) ++ ++ ++ 0 ++
+ +, 75%-100% of all blots positive; +, 34%-74% of all blots positive; (+), lo%-33% of all blots positive; 0, less than 10% of all blots positive.
Fig. 3. Examples of CK staining in various epithelia (paraffin sections). (a) Respiratory epithelium, Ks18.04 (CK18), ciliated and basal cells positive; (b) olfactory epithelium, Ks18.04 (CK18), positive cells in basal, apical, and mid zone.
The typical basal cell marker, CK14, was expressed in all basal cells, in the suprabasal cells of the squamous epithelium of the ventral meatus, and to a lesser degree in cells of the mid and apical zones of the olfactory epithelium. CK7 was only expressed in the gland ducts and sometimes in ciliated cells of the respiratory epithelium. CK13 and/or 15 was found in almost all cell types with the exception of gland cells and cells from the mid and apical zones of the olfactory epithelium. CK18 was present in all cell types of the respiratory and olfactory epithelia. In contrast, the expression of CK19 was confined to the respiratory epithelium (basal, ciliated, and cuboidal cells) and sometimes in the glands and gland ducts. The staining for CK4 was always negative in all nasal epithelia; however, it was strongly positive in the stratified epithelium of the hard palate which is included in the crosssections at NLl and NL2.
W.K. Schlage et al. I Toxicology Lelters 88 (19%) 65-73
12
Table 6 CK expression in RNE cells cultured in vitro for 6 days Antibody
CK
CK-positive cells (%)
Ks8.60 6B10 OV/TL 12/30 Ks8.7 Ks8.12 LLO02 Ks18.04 CK-E3
1/10/l 1 4 I 8 13/15 14 18 19
0 17.5 13.7 25.5 0.7 99.1 48.2 33.0
For each determination, approximately 200 to 1000 cells were counted for immunofluorescent staining of cytoplasmic filaments.
3.4. CK expression in RNE cells cultured in vitro The results of the differential counting of 15 to 20 image fields per culture slide are given in Table 6. The basal cell-related CK14 was expressed in practically all cells. In addition, half of the cell population expressed CK 18 (Fig. 4a). The corresponding basic cytokeratin, CK8, was found in only -25% of the cells counted (Fig. 4b). Probably, different affinities of the two antibodies in the immunofluorescent staining may have caused this difference. The squamous markers CKl/lO/ll for cornified epithelia were not found; CK4 for noncornified squamous epithelia was found in N 18% of the cells. CK7, typical of glandular differentiation, was detected in N 14% of the cells. Unexpectedly, the expression of CK13/15, abundant in the in vivo situation, was found in less than 1% of the cells. 3.5. Comparison of results from tissue extracts and immunohistology There were some discrepancies with regard to the histological CK profiles in the respiratory and olfactory epithelia (Table 4) and the corresponding Western blot results for NE1 and NE2 (Table 5). The occurrence of CKl and 10 in the extracts of NE1 and NE2 indicates that squamous cells from the ventral meatus epithelium were included in these samples which ideally should be composed of only respiratory
Fig. 4. Immunofluorescent staining of RNE cells for CK (green), nuclei counterstained (blue). (a) Ks18.04 (CK18); (b) K&7 (CK8).
or olfactory epithelia, respectively. This cannot be avoided during dissection due to the close proximity of these epithelia (Fig. 1); the CK4 signal in NE1 is probably due to contaminating cells from the hard palate. The failure to detect CK18 in NE2 and also in cultured RNE cells may result from the relative insensitivity of the antibody Ks18.04 to rat CK18 on immunoblots, which was found also with reference tissue from rat liver. In cultured RNE cells immunofluorescently stained with this antibody, N 50% of the cells are positive (Table 6, Fig. 4a). When comparing results obtained with the same antibody by different immunochemical and immunohistological methods, the different modes of denaturation, e.g. detergent extraction, alcohol fixation, or even formaldehyde fixation plus antigen retrieval must be taken into account; this may be even more important if an antibody
W.K Schlage et al. I Toxicology Letters 88 (1996) 65-73
exhibits only cross-reactivity to the homologous protein of a different species. Similarly, there were differences between the immunofluorescence and blotting results with regard to the expression of CK8 as well as CK13/ 15. Whether the cause is different antibody affinities or individual differences in the CK profiles of each in vitro culture population will have to be investigated in the future, e.g. by comparing immunofluorescence and blotting profiles from the same cell pool. Due to the low cloning efficiency of RNE cells (1% or less of the cells seeded), heterogeneities of the in vitro cell populations are very likely.
4. Concluding remarks We have developed the tools for routine CK determination which now enable us to obtain complex, spatially resolved CK expression patterns for representative, defined sites of the rat nasal cavity and from in vitro cultured RNE cells. Future investigation will focus on the comparison between the unexposed and exposed in vivo situation or in vitro cultures to detect exposure-related differentiation changes.
Acknowledgments
This work was sponsored by Philip Morris (USA). The authors wish to thank Dr. K. Behrens and Mr. D. Hanselmann for critically reviewing the manuscript and MS M. Kuhn, MS M. Willecke, MS A. Keimes, and MS L. Kopplin for excellent technical assistance.
References [1] Bachmayer, D. and Schlage, W.K. (1989) Cytogenetic assays using shortterm culture of primary rat nasal and tracheal epithelial (RNE, RTE) cells. Eur. J. Cell. Biol. 48, 4.
73
[2] Bachmayer, D. and Schlage, W.K. (1990) SCE analysis in primary rat nasal and tracheal epithelial (RNE, RTE) cells following in vivo or in vitro exposure to inhalable materials. Abstracts of EEMS Annual Meeting, York, 22-27 July 1990, p. 188. [3] O’Guin, W.M., Galvin, S., Schermer, A. and Sun, T.T. (1987) Patterns of keratin expression define distinct pathways of epithelial development and differentiation. Curr. Top. Dev. Biol. 22, 97-125. c41 Kartenbeck, J. and Franke, W.W. (1993) Cytokeratins. In: T. Kreis and R. Vale (Eds.), Guidebook to the Cytoskeletal and Motor Proteins. Oxford University Press, Oxford, New York, Tokyo, pp. 145-148. PI Mall, R., Franke, W.W., Schiller, D.L., Geiger, B. and Krepler, R. (I 982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31, 1l-24. iI61Achtstaetter, T., Hatzfeld, M., Quinlan, R.A., Parmelee, D.C. and Franke, W.W. (1986) Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. Methods Enzymol. 134, 355-371. c71Battifora, H. and Kopinski, M. (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation. J. Histochem. Cytochem. 34, 1095-l 100. PI Boon, M.E. and Kok, L.P. (1994) Microwaves for immunohistochemistry. Micron 25, 15l- 170. c91Shi, S.R., Key, M.E. and Kalra, K.L. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39, 741-748. El01 McElroy, H.H., Shih, M.-S. and Parfitt, A.M. (1993) Producing frozen sections of calcified bone. Biotech. Histochem. 68, 50-55. Cl11Lichtner, R.B., Julian, J.A., Glasser, S.R. and Nicolson, G.L. (1989) Characterization of cytokeratins expressed in metastatic rat mammary adenocarcinoma cells. Cancer Res. 49, 104-111. Cl21Troyanovsky, SM., Guelstein, V.I., Tchipysheva, T.A., Krutosvkikh, V.A. and Bannikov, G.A. (1989) Patterns of expression of keratin 17 in human epithelia: dependency on cell position, J. Cell Sci. 93, 419-426. Cl31Troyanovsky, S.M., Krutosvkikh, V.A. and Bannikov, G.A. (1986) Preparation and properties of monoclonal antibodies to individual prekeratins of simple rat epithelium. Bull. Exp. Biol. Med. 101, 824-827. Cl41Hebel, R. and Stromberg, M.W. (1976) Respiratory system. In: Anatomy of the Laboratory Rat. The Williams & Wilkins Company, Baltimore, MD, pp. 55-61.
Cytokeratin
patterns of epithelial cells of the rat nasal cavity in vivo and in vitro W.K. Schlage*, H. Biilles, D. Friedrichs, A. Teredesai lnstitut fir hiologische Fiirschung, Fuggerstr. 3, 51149 Kbln, Germany
Abstract Nasal epithelial cells are a primary target for the actions of inhaled substances. To enable the determination of alterations in cell differentiation in the rat inhalation model, we developed a methodology to assess cytokeratin expression in rat nasal tissue. A panel of commercially available antibodies was validated for specificity to defined rat cytokeratins by immunoblotting. The development of immunohistological procedures to enhance spatial resolution enabled mapping of cytokeratin patterns in various cell types at defined regions and levels of the rat nasal cavity using serial sections and a standardized evaluation schedule. Keywords: Immunohistochemistry; tract
Western blot; Antibody
1. Introduction In previous studies, we reported on the use of cultured rat nasal epithelial (RNE) cells to determine genotoxic activity after in vitro exposure or ex vivo after inhalation exposure using sister chromatid exchanges (SCE) and chromosome aberrations as end points [1,2]. The aim of this and our future investigations is to determine nongenotoxic effects, such as the induction of differentiation changes, in RNE cells in vivo and in vitro using cytokeratin (CK) expression patterns as end points. The use of CK expression patterns as markers for the differentiation of the intermediate filament cytoskeleton of epithelial cells from human .and many vertebrate species is well established [3,4]. However, a suitable *Corresponding author.
panel; Antigen retrieval; Keratinocytes;
Respiratory
methodology to determine the CK expression pattern in rat nasal epithelia has not been developed yet; there are few published data on the CK distribution in the nasal cavity, for the rat as well as for other vertebrate species. Moreover, a welldefined antibody panel for rat CK is lacking. To achieve the aim mentioned above, two basic objectives had to be addressed: first, to validate an antibody panel for rat CK useful for immunoblotting of tissue extracts as well as for immunostaining of paraffin sections obtained from routine histology; and second, to map the normal CK distribution in rat nasal epithelia. 1.1. Validation Commercially available monoclonal antibodies to CK were screened for individual specificities to defined rat CK polypeptides using sodium
0378-4274/96/$15.00 10 1996 Elsevier Ireland Ltd. All rights resewed PII SO378-4274(96)03719-S
66
W. K Schlagc et al. I Toxicolog_v Lctrcrs 88 (I 996) 65- 73
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of cytoskeletal extracts from rat tissues with known CK composition. The CK patterns of excised epithelial samples from the rat nasal cavity and of primary cultures of RNE cells were obtained in parallel. Due to the large number of CK polypeptides and the overlapping patterns, it was necessary to enhance the spatial resolution by means of immunohistological techniques. The rat-specific CK antibodies were tested for immunohistological reactivity to rat tissues on unfixed cryosections of reference tissues and of nasal epithelia. Subsequently, immunohistochemical procedures were tailored to utilize these CK antibodies with routinely obtained, formalin-fixed and paraffin-embedded histological sections. As a result, a panel of nine antibodies suitable for paraffin sections was derived. 1.2. CK mapping Using this antibody panel, we determined the CK staining pattern in nine serial sections from two levels of the rat nasal cavity, the method of evaluation being a 4-stage individual grading for intensity as well as for distribution to discriminate and calculate local alterations of CK expression in exposed rats at high resolution. For in vitro cultured RNE cells, CK expression was determined by immunofluorescence.
2. Materials and methods
NLI
NL2
Fig. 1. The rat nasal cavity (median section through the rat head, lower jaw removed). Blue, squamous epithelium; yellow, respiratory epithelium; pink, olfactory epithelium; NLl, NL2, beginning of the defined cross-sectional levels NLl and NL2 (modified from [14]).
samples were collected, one from the respiratory (NEl) and one from the olfactory (NE2) epithelium (Fig. 1). In vitro cultured RNE cells (see below) were scraped off the culture dishes. Cytoskeletal extracts (high salt and detergents) were prepared according to [6] and separated using the Phast apparatus and Phast gradient gels with SDS buffer strips (Pharmacia, Freiburg). The proteins were transferred to PVDF membranes (Millipore) by thermoblotting. The dried blots were rehydrated and stained with various commercially available monoclonal antibodies (Table 1) according to standard procedures. Molecular weights were estimated from biotinylated low molecular weight standards (Bio-Rad) included on each blot. Details will be published elsewhere (Schlage et al., in preparation). All determinations were repeated independently at least in triplicate up to 20 times.
2.1. Electrophoresis and immunoblotting
Various epithelial tissues (foot sole epidermis, tongue mucosa, liver, small intestine), serving as reference tissues with well-documented CK composition, and nasal mucosa were obtained from adult Sprague Dawley rats. The nominal CK composition of the reference tissues was as follows: footsole epidermis, CKl, 2, 5, 6, 9, 10, 11, 14, 16; tongue, CK4, 5, 6, 13, 14, 15, 16, 17, 19; liver, CK8, 18; small intestine, CK8, 18, 19 (according to [S]). From the nasal mucosa, two
2.2. Cell culture
Epithelial cells were detached from the underlying connective tissue of the pooled NE1 and NE2 tissue fragments by enzymatic digestion (pronase, collagenase, hyaluronidase) and cultured for 5 to 6 days in serum-free medium in plastic dishes as described in [l]. For immunofluorescence experiments, the cells were seeded on plastic slides (slide flasks, Nunc).
67
W.K Schlage et al. I Toxicology Letters 88 (19%) 65-73 Table 1 Antibody panel for rat cytokeratins: specificity and suitability in immunohistochemical Name
Ks18.04 LL 002 CK-E3 Ks13.1 Ks8.7 Ks8.12 6BlO KS860 OV-TL 12/30 PCK 26 AE-I RCK 105 Ks4.62 Ks8.13 LDS68 4.1.18
c-51
Supplier
Progen BioGenex Sigma Progen P rogen Sigma Sigma Sigma BioGenex Sigma Progen Laboserv Sigma Sigma Sigma Boehringer Bio-Genex
Recommended use’
specificity Human CK”
Rat CK’
18 14 17 13, (14, 16) 8 13, (15), 16
18 14 19 13, (15) 8 15, 13 4 1, lO/ll 7 1, W, 8 lOIll, 14, 16 7 19 Pan n.d. 8 n.d.
;1,, 10, 11 7 1, 5, 6, 8 10/l, 14, 16, 19 7 19 Pan 7, (8) 8 8
applications Suitability Cryob/paraffin cryo, cryo, cryo cryo, cry0 cry0 cryo, cry0 cryo,
paraffin paraffin paraffin
paraffin
paraffin cryo,paraffin cryo, paraffin cryo cry0 cry0 cryo, paraffin cryo, paraffin paraffin
+I+ +/+ +I+ +I+ +M+) +I+ +I+
+I+ +I+01 + n.d./+
+I0 +I0 +I0 n.d./O
n.d./(+ ) n.d./O
0, no specific reactivity; (), weak or questionable reactivity; +, specific reactivity; n.d., not determined. “As given by supplier and literature. bDetermined on cryosections and cells cultured on slides. CAs determined by immunoblotting (MS in preparation).
2.3. ImmunohistolG~gica!procedures 2.3.1. Parajin sections
Transverse sectilons at nose levels (NL) 1 and 2 (Fig. 1) were obtained from 8 adult Wistar rats
by routine histological procedures. Briefly, the procedure consisted of fixation in buffered formalin, decalcification in nitric acid in an ultrasonic bath, trimming the slices from the nose at defined levels, dehydration and embedding in paraplast, cutting sections of 5-pm thickness, mounting on adhesive slides (Superfrost Plus, Menzel-Glas, Braunschweig). The immunostaining procedure included the following principal steps: deparaffinize, unmask epitopes, inactivate endogenous peroxidase (3% hydrogen peroxide, 5 min), block nonspecific binding sites (1% swine serum, 30 min), incubate for 30 min with amibody to CK (Table 2) diluted in diluent (Cat. No. S3022, Dako, Hamburg), use
detection system with biotinylated second antibody and AEC substrate (Dako), counterstain with hematoxylin. For unmasking, we compared the effectiveness of protease treatment [7] and microwave treatment [8] in lead thiocyanate solution [9] as well as in plain water. The optimal conditions are listed in Table 2. For microwave treatment, it is necessary to interrupt heating when the bathing solution reaches the boiling point in order to prevent detachment of bone-containing and cartilaginous tissue areas from the slide: five sequential heating cycles with 30 s intermission were sufficient for antigen retrieval. It must be noted that these conditions only apply to nasal tissue fixed in buffered formaldehyde and decalcified in nitric acid. For other tissues, e.g. lungs or liver that were fixed differently and not treated with nitric acid, we found other optimal antigen retrieval requirements for some of the CK antibodies (not
68
W. K. Schlage et al. I Toxicology Letters 88 (I 9%) 65 73
Table 2 Antibody panel for paraffin sections from rat nasal cavity: unmasking and antibody dilution Antibody
Epitope unmasking
Dilution
Remark
negative control _ _ _
Treatment
Time (min)
Nonimmune serum
various’
to/15
1:lOO
Ks18.04 LL 002 CK-E3 Ks13.1 Ks8.12 6B10 Ks8.60
protease, 1 g/l microwave, lead protease, 1 g/l protease, 1 g/l protease, 1 g/l microwave, water microwave, water
15 IO 15 15 I5 10 10
I:200 1:lOO 1:300 1:lO I:30 I:100 I:600
OV-TL12/30 PCK 26
protease, 1 g/l various”
15 IO/15
I:200 1:300
affinity purified _ positive controP
“According to corresponding antibody treatment. bPerforms equally well with microwave or protease treatment.
shown). Negative (nonimmune IgG) and positive a (mAb PCK26) control slides were included in each staining series. 2.3.2. Cryosections Cryosections (5-7 pm thick) from reference tissues were obtained from rats by standard procedures. Cryosections of the nasal cavity were obtained from the undecalcified head by a method modified from [lo] using a C-type knife with a tempered steel edge. Staining was performed according to standard procedures using the same detection system as for paraffin sections. 2.3.3. In vitro cultured RNE cells These cells were rinsed in PBS, fixed with cold methanol and immunofluorescently stained according to standard procedures. Endogenous peroxidase blocking was most effective using a mixture of 10% acetic acid and 90% methanol at -20°C for lo-15 s. 2.4. Histological evaluation The epithelial staining pattern of the transverse sections at NLl and NL2 was evaluated separ-
b
Fig. 2. Mapping of CK staining on transverse sections through the rat nasal cavity at NLl (a) and NL2 (b). The boxes approximately indicate the evaluation areas. (a) NLl stained with Ks8.12 (CK15, 13). A, squamous epithelium; B, vomeronasal organ; C-E, respiratory epithelium; F, submucosal glands; G, nasolacrimal duct. (b) NL2 stained with Ks18.04 (CK18). A, squamous epithelium; B, vomeronasal organ; C-F, respiratory epithelium; G, submucosal glands; H, olfactory epithelium; I, Bowman’s glands; J, nasolacrimal duct.
ately at the representative areas as shown in Fig. 2. Within each area, the staining was assessed separately for each distinguishable cell type, e.g. basal, ciliated, and goblet cells within the respir-
W.K. Schlage et al. I Toxicology Letters 88 (19%) 65-73
69
Table 3 Example of detailed evaluation: individual scores (mean of 8 rats) obtained for the respiratory epithelium at NLl Epithelium
Ciliated
Cell type
Basal
Ciliated
Low cuboidal
Basal
nonciliated
Antibody
Ks18.04 CK-E3 Ks8.12 LLO02 Ks18.04 Ks8.12 CK-E3 LLO02 Ks18.04 CK-E3 Ks8.12 LLO02 Ks18.04 Ks8.12 CK-E3 LLO02 OV/TL12/30
CK
Area
18 19 15,13 14 18 15, 13 19 14 18 19 15,13 14 18 15, 13 19 14 7
C Int./Distr.
D Int./Distr.
E Int./Distr.
3.012.0 2.511.8 3.012.8 1.8/1.8 3.OJ2.0 0.3jO.l 3.012.3 O/O n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
3.012.0 1.7/1.1 2.412.3 1.411.3 2.811.9 0.3/0.1 3.012.1 O.l/O.l 3.012.0 1.310.9 2.612.1 1.5/1.6 3.011.9 1.310.8 2.311.4 O.l/O.l O/O
3.OJ2.0 1.6/1.1 2.912.6 1.2/1.8 3.Of2.0 O/O 3.012.1 0.210.4 3.012.0 l.lJO.6 3.012.6 1.0/1.2 3.011.9 1.9/1.1 2.311.0 O/O 0.510.3
Individual scores: (intensity) 0, negative; 1, weak (only detectable at higher magnification, e.g. x 40 lens); 2, distinct (already detectable at lower magnification, still transparent); 3, intense (opaque, red to brown staining); (distribution, related to percentage of the cell type in the observation area) 0, none; 1, focal (up to _ 15 %); 2, patchy (_ 15%-85%); 3, diffuse (* 85%- 100%). n.a., not applicable. The negative results obtained for antibodiesOV/TL 12/30, 6B10, Ks8.60, and Ks13.1 are not listed.
atory epithelium. For each cell type, the staining intensity and relative distribution was individually scored using the 4-stage scores as defined in the legend to Table 3. All histological evaluations were reviewed by a veterinary pathologist.
3. Results and discussion 3.1. Validation of antibody panel
First, we used SDS-PAGE and immunoblotting to screen commercially available monoclonal antibodies to CK -most frequently directed against human or bovine CK polypeptides for their specificity to rat CK. This was necessary because in the literature such CK antibodies cross-reacting with rat CK were often defined only for human or for murine CK, and thus it was presumed that their specificity for rat CK was -equivalent. As the molecular weights of rat
CK frequently differ from those of their human counterparts, we used the reference tissues to extrapolate the CK polypeptides. We thereby obtained a panel of 15 antibodies with validated specificities to rat CK (Table 1). In general, the specificities were indeed the same as for the CK donor species. Exceptions from the nominal specificities were found, e.g. for the frequently used antibody AEl which does not recognize rat CK19 (also reported by [l l]), for CK-E3 specific for human CK17 but which recognizes rat CK19 instead of CK17 [12,13], and for K&.12 which does not recognize rat CK16. Due to the limitations of our methodology (reference tissues and one-dimensional electrophoresis) it was not possible to distinguish between CK5 and 6, CKlO and 11, or to identify CK3, 9, 12, and 17. From the antibody series defined by Western blotting (Table l), a selection of antibodies was found to be useful for immunohistological application with formalin-fixed, paraffin-embedded
W.K. Schlugeet (11.I ToxicologyLetters 88 (19%) 65-73
70
Table 4 General CK expression profiles in the rat nasal epithelium Region
Ventral meatus Respiratory
Olfactory
Cell type/ localization
Basal Suprabasal Basal Ciliated, cuboidal Gland Gland duct Basal zone Mid/apical zone Gland
Antibody (CK) KS860 (l/10/l 1)
6BlO (4)
OV/TL (7)
Ksl3.1 (13/15)
Ks8.12 (15/13)
LLO02 (14)
Ksl8.04 (18)
CK-E3 (19)
0 + 0 0 0 0 0 0 0
0 0 0 0
0 0 0 (+) 0 + 0 0 0
0 + 0 (+) (+)
+ +
+ +
0 0 +
I’+) 0 (+) (+) 0 0
0’ P+)
0 0 + + + + + + +
0 0 0 0 0
I’+) 0 0
(1) 0
I’+) (+) 0 0 0
+, distinct to strong staining intensity with patchy to diffuse distribution; (+), weak to distinct staining intensity with focal to patchy distribution; 0, no staining observed.
sections (Tables 1, 2) a crucial requirement being the antigen unmasking. In order to ascertain the specificity for rat CK also in immunohistological applications, the antibodies were tested for suitability with cryosections of unfixed reference tissues and tissues of the rat nasal cavity (Table 1). Although it was possible to obtain cross-sections of the undecalcified rat nasal cavity, this was not useful to obtain the high number of good quality serial sections required. For the selected antibodies, the same structures recognized in paraffin sections were also recognized in cryosections. With regard to rat CKS, we could not find an antibody which was suitable for paraffin sections of the rat nasal cavity. The antibody Ks8.7, however, showed weak staining in paraffin sections of soft tissues that had not been treated with nitric acid after fixation. In the nasal cavity, a pronounced nuclear staining (possibly due to cross-reaction with nuclear lamins) was observed, primarily in the submucosal glands, when staining with this and another antibody (LDS68). Due to the lack of monospecificity, it was not possible to distinguish histologically between CKl/lO/ll (Ks8.60), CK13/15 (Ks8.12, Ks13.1), and CKl/ 5/6/8 (PCK26); the last antibody behaved as a “Pan-CK”, staining all epithelia in the rat nasal
cavity, and therefore served as a positive control in each staining procedure. The antibodies Ks8.12 and Ks13.1 both recognize rat CK13 and 15, however, it appears that the CK15 staining intensity is more pronounced for Ks13.1 and the CK13 staining intensity is more pronounced for CK8.12 (Table 4). 3.2. CK expression patterns obtained by Western blotting
For the in vivo situation, differences were found between NE1 and NE2. NE2 contained CKl, 5/6, 14, 15, 16, and NE1 expressed in addition CK4, 8, 10, 13, 18, 19 (Table 5). For the in vitro situation, RNE cells contained all the CK from NE1 and NE2 with the exception of CK8 and 18, but they additionally expressed CK7. The enzymatically isolated cells from NE1 and NE2 were combined for culturing, because the cloning efficiencies of the separate cell fractions were too poor. From the large number of different CK polypeptides expressed in these epithelia and the overlapping patterns, it became obvious that more spatial resolution was required for comparisons. Therefore, the immunohistological methodology was developed.
W.K Schlugr et al. I Toxicology Letters 88 (19%) 65-73
3.3. CK expression in cross-sections of the rat
71
a
nasal cavity Simply mapping the epithelial staining distribution (Fig. 2) was not sufficient as the staining patterns along a certain type of epithelium (respiratory, olfactory, squamous, glandular) appeared differentially with regard to intensity, distribution, and cell type in the defined areas (Fig. 2). Hence, we developed the 4-stage individual scoring for intensity, and distribution of cellular staining (Table 3) at the defined areas (Fig. 2). Examples for the histological staining of respiratory and olfactory epithelial cells are shown in Fig. 3. The detailed results of the evaluation were expressed as mean scores (intensity/distribution) for each antibody and cell type at all the sites listed (an example is given in Table 3). For the purpose of this paper, the general CK expression patterns for the different cell types in the three main epithelial regions of the rat nasal cavity are summarized in Table 4. As expected, the markers for cornified stratified epithelia CKl/lO/ll were exclusively expressed in the suprabasal cell layers of the squamous epithelium of the ventral meatus, whereas the “simple epithelial” CK18 and 19 were completely absent.
Table 5 CK expression patterns determined in cytoskeletal extracts from rat nasal epithelia and RNE cells CK
NE1 1 4 516 7 8 IO/l 1 13 14 15 18 19
In vitro
In vivo
(+G ++ 0 (‘t) + ++ + I’+)
NE2
RNE
+ 0 ++ 0 0 0 0 ++ + 0 0
(+) (+) ++ ++ 0 (+) ++ ++ ++ 0 ++
+ +, 75%-100% of all blots positive; +, 34%-74% of all blots positive; (+), lo%-33% of all blots positive; 0, less than 10% of all blots positive.
Fig. 3. Examples of CK staining in various epithelia (paraffin sections). (a) Respiratory epithelium, Ks18.04 (CK18), ciliated and basal cells positive; (b) olfactory epithelium, Ks18.04 (CK18), positive cells in basal, apical, and mid zone.
The typical basal cell marker, CK14, was expressed in all basal cells, in the suprabasal cells of the squamous epithelium of the ventral meatus, and to a lesser degree in cells of the mid and apical zones of the olfactory epithelium. CK7 was only expressed in the gland ducts and sometimes in ciliated cells of the respiratory epithelium. CK13 and/or 15 was found in almost all cell types with the exception of gland cells and cells from the mid and apical zones of the olfactory epithelium. CK18 was present in all cell types of the respiratory and olfactory epithelia. In contrast, the expression of CK19 was confined to the respiratory epithelium (basal, ciliated, and cuboidal cells) and sometimes in the glands and gland ducts. The staining for CK4 was always negative in all nasal epithelia; however, it was strongly positive in the stratified epithelium of the hard palate which is included in the crosssections at NLl and NL2.
W.K. Schlage et al. I Toxicology Lelters 88 (19%) 65-73
12
Table 6 CK expression in RNE cells cultured in vitro for 6 days Antibody
CK
CK-positive cells (%)
Ks8.60 6B10 OV/TL 12/30 Ks8.7 Ks8.12 LLO02 Ks18.04 CK-E3
1/10/l 1 4 I 8 13/15 14 18 19
0 17.5 13.7 25.5 0.7 99.1 48.2 33.0
For each determination, approximately 200 to 1000 cells were counted for immunofluorescent staining of cytoplasmic filaments.
3.4. CK expression in RNE cells cultured in vitro The results of the differential counting of 15 to 20 image fields per culture slide are given in Table 6. The basal cell-related CK14 was expressed in practically all cells. In addition, half of the cell population expressed CK 18 (Fig. 4a). The corresponding basic cytokeratin, CK8, was found in only -25% of the cells counted (Fig. 4b). Probably, different affinities of the two antibodies in the immunofluorescent staining may have caused this difference. The squamous markers CKl/lO/ll for cornified epithelia were not found; CK4 for noncornified squamous epithelia was found in N 18% of the cells. CK7, typical of glandular differentiation, was detected in N 14% of the cells. Unexpectedly, the expression of CK13/15, abundant in the in vivo situation, was found in less than 1% of the cells. 3.5. Comparison of results from tissue extracts and immunohistology There were some discrepancies with regard to the histological CK profiles in the respiratory and olfactory epithelia (Table 4) and the corresponding Western blot results for NE1 and NE2 (Table 5). The occurrence of CKl and 10 in the extracts of NE1 and NE2 indicates that squamous cells from the ventral meatus epithelium were included in these samples which ideally should be composed of only respiratory
Fig. 4. Immunofluorescent staining of RNE cells for CK (green), nuclei counterstained (blue). (a) Ks18.04 (CK18); (b) K&7 (CK8).
or olfactory epithelia, respectively. This cannot be avoided during dissection due to the close proximity of these epithelia (Fig. 1); the CK4 signal in NE1 is probably due to contaminating cells from the hard palate. The failure to detect CK18 in NE2 and also in cultured RNE cells may result from the relative insensitivity of the antibody Ks18.04 to rat CK18 on immunoblots, which was found also with reference tissue from rat liver. In cultured RNE cells immunofluorescently stained with this antibody, N 50% of the cells are positive (Table 6, Fig. 4a). When comparing results obtained with the same antibody by different immunochemical and immunohistological methods, the different modes of denaturation, e.g. detergent extraction, alcohol fixation, or even formaldehyde fixation plus antigen retrieval must be taken into account; this may be even more important if an antibody
W.K Schlage et al. I Toxicology Letters 88 (1996) 65-73
exhibits only cross-reactivity to the homologous protein of a different species. Similarly, there were differences between the immunofluorescence and blotting results with regard to the expression of CK8 as well as CK13/ 15. Whether the cause is different antibody affinities or individual differences in the CK profiles of each in vitro culture population will have to be investigated in the future, e.g. by comparing immunofluorescence and blotting profiles from the same cell pool. Due to the low cloning efficiency of RNE cells (1% or less of the cells seeded), heterogeneities of the in vitro cell populations are very likely.
4. Concluding remarks We have developed the tools for routine CK determination which now enable us to obtain complex, spatially resolved CK expression patterns for representative, defined sites of the rat nasal cavity and from in vitro cultured RNE cells. Future investigation will focus on the comparison between the unexposed and exposed in vivo situation or in vitro cultures to detect exposure-related differentiation changes.
Acknowledgments
This work was sponsored by Philip Morris (USA). The authors wish to thank Dr. K. Behrens and Mr. D. Hanselmann for critically reviewing the manuscript and MS M. Kuhn, MS M. Willecke, MS A. Keimes, and MS L. Kopplin for excellent technical assistance.
References [1] Bachmayer, D. and Schlage, W.K. (1989) Cytogenetic assays using shortterm culture of primary rat nasal and tracheal epithelial (RNE, RTE) cells. Eur. J. Cell. Biol. 48, 4.
73
[2] Bachmayer, D. and Schlage, W.K. (1990) SCE analysis in primary rat nasal and tracheal epithelial (RNE, RTE) cells following in vivo or in vitro exposure to inhalable materials. Abstracts of EEMS Annual Meeting, York, 22-27 July 1990, p. 188. [3] O’Guin, W.M., Galvin, S., Schermer, A. and Sun, T.T. (1987) Patterns of keratin expression define distinct pathways of epithelial development and differentiation. Curr. Top. Dev. Biol. 22, 97-125. c41 Kartenbeck, J. and Franke, W.W. (1993) Cytokeratins. In: T. Kreis and R. Vale (Eds.), Guidebook to the Cytoskeletal and Motor Proteins. Oxford University Press, Oxford, New York, Tokyo, pp. 145-148. PI Mall, R., Franke, W.W., Schiller, D.L., Geiger, B. and Krepler, R. (I 982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31, 1l-24. iI61Achtstaetter, T., Hatzfeld, M., Quinlan, R.A., Parmelee, D.C. and Franke, W.W. (1986) Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. Methods Enzymol. 134, 355-371. c71Battifora, H. and Kopinski, M. (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation. J. Histochem. Cytochem. 34, 1095-l 100. PI Boon, M.E. and Kok, L.P. (1994) Microwaves for immunohistochemistry. Micron 25, 15l- 170. c91Shi, S.R., Key, M.E. and Kalra, K.L. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39, 741-748. El01 McElroy, H.H., Shih, M.-S. and Parfitt, A.M. (1993) Producing frozen sections of calcified bone. Biotech. Histochem. 68, 50-55. Cl11Lichtner, R.B., Julian, J.A., Glasser, S.R. and Nicolson, G.L. (1989) Characterization of cytokeratins expressed in metastatic rat mammary adenocarcinoma cells. Cancer Res. 49, 104-111. Cl21Troyanovsky, SM., Guelstein, V.I., Tchipysheva, T.A., Krutosvkikh, V.A. and Bannikov, G.A. (1989) Patterns of expression of keratin 17 in human epithelia: dependency on cell position, J. Cell Sci. 93, 419-426. Cl31Troyanovsky, S.M., Krutosvkikh, V.A. and Bannikov, G.A. (1986) Preparation and properties of monoclonal antibodies to individual prekeratins of simple rat epithelium. Bull. Exp. Biol. Med. 101, 824-827. Cl41Hebel, R. and Stromberg, M.W. (1976) Respiratory system. In: Anatomy of the Laboratory Rat. The Williams & Wilkins Company, Baltimore, MD, pp. 55-61.