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Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin*
Differentiation
Differentiation (1989) 40:207-214
Ontogeny and Neoplasia 0 Springer-Verlag 1989
Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin * Michael Kasper **, Uwe Karsten ’,Peter Stosiek ’, and Roland Moll Institute of Pathology, District Hospital Gorlitz, DDR-8900 Gorlitz, German Democratic Republic Department of Immunology, Central Institute of Molecular Biology, Academy of Science of the German Democratic Republic, DDR-1115 Berlin-Buch, German Democratic Republic Institute of Pathology, University of Mainz, Langenbeckstrasse 1, D-6500Mainz, Federal Republic of Germany
Abstract. We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible “neoexpression” during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.
Introduction According to the established and accepted patterns of intermediate filament (IF) distribution [46], cytokeratins are expressed in epithelia, whereas vimentin is restricted to mesenchymal cells. In recent years, however, the simultaneous expression of both types of IFs has been detected in a number of specialized tissues [ l , 14, 15, 19-22, 24-29, 32, 33, 48, 53, 601. Van Muijen et al. [58] have interpreted the simultaneous occurrence of vimentin and cytokeratins as being a transient phenomenon restricted to certain ontogenetic stages or, in adult tissues, to reactive proliferation. Our own data suggest that the coexpression of vimentin and cytokeratins may be determined by functional factors [ 191. We have found both IF types in nonglandular epithelia, in cells bordering cavities filled with low-protein body fluids, and in cells localized in proteoglycan-rich extracellular matrix. At present, it is not clear whether all of these apparently isolated cases are linked by their having a functional or regulatory principle in common. In pursuing our
* Dedicated to the orthodontist Dr. Georg Klammt, Gorlitz
** To whom offprint requests should be sent
working hypothesis that the coexpression of vimentin and cytokeratin may, in some way, be related to secretory function, we examined the distribution of IFs in the enamel organ of human fetuses, which consists of epithelial cells involved in the production and secretion of enamel-forming substances [38, 391. Methods Tissues. Primary tooth germs (n, 11) were obtained from human fetuses (abortion material; 2 x loth, 1 x 16th and 1 ~ 2 7 t hweek of gestation, maxilla and mandible in each case and 2 x 18th and 1 x 26th week of gestation (mandible alone)) and from a 9-month-old child (autopsy material). After embedding in a special mounting medium (Jung, Heidelberg, FRG), the tooth germs were frozen and stored in liquid nitrogen. Formalin-fixed tissue of human maxillae (n, 2) obtained from a 40th-gestational-week fetus and a 2-day-old neonate were decalcified, embedded in paraffin, and sectioned. To avoid the loss of tissue sections during protease digestion using 0.1% Pronase (Serva, Heidelberg, FRG) in phosphate-buffered saline (PBS), pH 7.4, for 15 min at 37” C, we precoated the slides with a 1 YOaqueous solution of Cementite glue (Merz and Beuteli, Niederwangen, Switzerland). The sections were deparaffinized using xylene and ethanol prior to protease digestion and immunostaining. Antibodies. The primary antibodies used in the present study are listed in Table 1. In addition to monoclonal antibodies, we used polyclonal guinea-pig antisera against vimentin [9] and rabbit antisera against keratin (Prof. J. Schulz, Biochemical Institute, Berlin, German Democratic Republic).
Indirect-immunojluorescence microscopy. Indirect-immunofluorescence microscopy of frozen, unfixed, 5 l m thick sections was performed as previously described [23]. For immunofluorescence, the secondary antibodies used were goat antibodies coupled with fluorescein isothiocyanate (FITC) or tetramethylrhodamin isothiocyanate (TRITC) to guinea-pig, rabbit, or mouse immunoglobulins (Dianova, Hamburg, FRG; SIFIN, Berlin, GDR). For double-immunofluorescence microscopy, two primary antibodies raised in different animal species were applied simultaneously. After washing in PBS, the fluorochrome-labeled secondary antibodies (diluted 1 :20) were applied together.
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Table 1. Monoclonal mouse antibodies used
Antibody
Antigens recognized
Reference and/or source
A45- B/ B3 lu-s RCK 102 6BlO
Broad range of cytokcratins Broad range of cytokeratins Cytokeratins 5 and 8 Cytokeratin 4
AE14 RCK 105
Cytokeratin 5 Cytokeratin 7
C18
Cytokeratin 7
CK7 K, 8.1.42 c10 RKSE 60 Kk 8.60 K , 18.174 207 c 04 RGE 53 BA 16 BA 17 A53-BIA2 K, 19.2.10.3 RV 202 v 9 VIM-9 VIM-38 4 D E- B-S G-A-5
Cytokeratin 7 Cytokeratin 8 Cytokeratin 8 Cytokeratin 10 Cytokeratins 10 and 11 Cytokeratin 18 Cytokeratin 13 Cytokeratin 18 Cytokeratin 18 Cytokeratin 19 Cytokeratin 19 Cytokeratin 19 Cytokeratin 19 Vimentin Vimentin Vimcntin Vimen tin Desmin Glial fibrillary acidic protein (GFAP)
~7,231 [lo, 671 Provided by Hoffmann-LaRoche (Basel, Switzerland) 1521 [65] Provided by Dr. W.W. Franke (German Cancer Research Center, Heidelberg, FRG) [35] Provided by Dr. T.T. Sun (New York University, NY) [52] Provided by Dr. F.C.S. Ramaekers (University of Nijmegen, The Netherlands) Providcd by Dr. J. Bartek (Research Institute of Clinical and Experimental Oncology, Brno, Czechoslovakia) [63] Obtained from Boehringer (Mannheim, FRG)
PI
Dr. Bartek [511 [I 61 [43] Progen Biotechnics (Heidelberg, FRG) [65] Provided by Dr. W.W. Franke Dr. Bartek
POI 121 (21 1181 [43] Progen Provided by Dr. Ramaekers [47] Boehringer Viramed (Martinsried, FRG) Progen Boehringcr Boehringer
Table 2. Immunohistochemical staining of human fetal enamcl organs and oral epithelia by monoclonal antibodies directed against different IF proteins
Tissue
CK CK 5 + 8 CK 5" CK 4" CK 13' CK 10" CK 8" CK 18 CK 19 CK 7 (broad-range an ti bodies)
External enamel epithelium
Dental lamina
+ + + +
+ + + +
Oral epithelium: Basal cells Suprabasal cells
+ +
+ +
Stellate reticulum Jnternal enamel epithelium
+= +
-
-d
-
-f
+ c
-
-
+
-
-I+
-
+/-h
-
-I+
-
+I-
+'
+
-
-/+m
+ +
-
+
-a
-j
-Y
-I+
-
+ +
+/-
-
+
-/+
-n
-f
+= +'
Vimentinb
-/+
+'
-
-/+k
-/+
-
-
-
"
-
CK, cytokeratin; + , positive; - , negative; + / - and - / + , focal patterns ( + / - , positive cells predominate; -I+, negative cells predominate) a In 18th and 27th gestational week not tested; In 26th and 27th gestational week all epithelial cells were negative; In 10th gestational week, weak staining; In 16th and 26th gestational week, few cells were positive; In 10th gestational week, only few cells were weakly positive; In 10th gestational week, some cells werc positive; In 10th gestational week, weakly positive; In 10th gestational week, uniformly positive; In 26th and 27th gestational week, only some cells were positive; In 10th gestational week, positive; ' Basal portions of cells were positive (few cells in the 10th gestational weck; many cells in the 16h and 18th gestational week); 'Some parabasal cells were negative; Only some upper cells were positive; " In 10th gestational week, some superficial cells (periderm-like layer) were positive
Negative controls were performed by replacing the primary antibody with PBS. Furthermore, the antibodies against desmin and GFAP produced consistently negative results with the enamel organ and thus served as additional negative controls.
Immunoperoxidase techniques. Cryostat sections or deparaffinized and protease-treated paraffin sections were incubated with 1YOhydrogen peroxide in methanol for 30 min in order to block endogenous peroxidase activity. This was followed by washing in PBS and preincubation with normal
209
C Fig. 1 a-d. Distribution of cytokeratins and vimentin in early fetal enamel-organ anlage (10-week-old human fetus); immunoperoxidase staining. a Antibody AE14 against cytokeratin 5 produced weak staining of the external enamel epithelium ( E E Q and the internal enamel epithelium (IEE; prospective ameloblasts), but stronger staining of the stellate reticulum (SR). The dental lamina (DL) was strongly positive. Inset: antibody 207 against cytokeratin 13 stained some cells of the stellate reticulum. b Antibody K, 8.1.42 decorated all epithelial elements of the enamel organ anlage. c Antibody RCK 105 against cytokeratin 7 prominently stained the stellate reticulum. d Antibody VIM-9 against vimentin exhibits a strong positive reaction in the stellate reticulum. Note the additional decoration of some cells of the external enamel epithelium (arrowheads) as well as the staining of the basal portions of individual cells of the intcrnal enamel epithelium (arrow). Bur, 50 pm
210
goat serum (1% in PBS) in order to reduce background staining. The sections were then incubated with the primary antibody (undiluted supernatant or commercially available antibodies at the appropriate dilution) for 45 min at room temperature. After three washes in PBS (10 min each), the sections were incubated with goat anti-mouse Ig (SIFIN ; diluted 1 :40) for 30 min. After three washes in PBS (10 min each), monoclonal antibody BL-POD (Sektion Biowissenschaften, Karl-Marx-Universitat Leipzig, GDR) directed against peroxidase was added at a dilution of 1 : 100. This was followed by three washes in PBS (10 min each) and incubation with a 0.001% peroxidase solution in PBS (Arzneimittelwerk Dresden, GDR) for 30 min. After five 5-min washes in PBS, the peroxidase activity was visualized by the addition of a freshly prepared solution of 3,3-diaminobenzidine tetrahydrochloride (Chemapol, Prague, Czechoslovakia) for 8 min. Finally, hematoxylin was used for counterstaining. Alternatively, an indirect immunoperoxidase technique was applied to cryostat sections [8]. Gel electrophoresis.The preparation of cytoskeletal material by the microdissection of defined tissue structures in frozen sections, the soluble-protein extraction procedure, and the analysis of cytoskeletal proteins by two-dimensional gel electrophoresis were all performed as described previously [411.
Results
Table 2 summarizes the immunohistochemical results obtained using frozen sections. All of the monoclonal anti-
bodies directed against a broad range of cytokeratins stained epithelial cells throughout the human fetal enamel organ. However, different patterns were obtained when antibodies specific for individual cytokeratin polypeptides (Table 1) were applied. The antibody AE14 against cytokeratin 5 stained most epithelial cells including the dental lamina (Figs. 1a, 2a), whereas cytokeratin 13 as detected by antibody 207 was found to be restricted to a few cells of the enamel organ (Fig. l a , inset). Incubation with the antibodies against cytokeratin 10 (RKSE 60, Kk 8.60) produced a positive reaction in the suprabasal cells of fetal oral stratified squamous epithelium but not in the enamel organ (not shown). Whereas cytokeratin 8 was detected in most cells of the enamel organ (Fig. 1 b), the exception being the internal enamel epithelium at later stages, cytokeratin 18 was only weakly expressed or appeared to be absent at this site (Table 2; for the antibodies, see Table 1). On the other hand, cytokeratin 19 was detectable in most epithelial cells of the enamel organ (Fig. 2b) and dental lamina. Cytokeratin 7 was consistently detected in the stellate reticulum, in certain cells of the dental lamina, and also, in 16- and 18-week-old fetuses, in the external epithelium of the enamel organ (Figs. l c , 2c). This cytokeratin was not detectable in the oral stratified squamous epithelium but was observed in the inner cells of the peg-like epithelial outgrowths, located immediately beneath the oral surface epithelium, which might have been salivary-gland germs (not shown). Cytokeratin 4 was not found in the enamel organ (Fig. 2d), except for a few cells of the external enamel epithelium of week 26 that were positive, but was detected in the upper layers of the oral squamous epithelium (not shown).
Fig. 2s-e. Distribution of cytokeratins and vimentin in fetal enamel-organ anlage at a more advanced stage (1 6-week-old-human fetus; immunoperoxidase staining) during which enamel production begins (not illustrated). a, b Antibodies AE14 against cytokeratin 5 (a) and BA 16 against cytokeratin 19 (b) stained most cells of the enamel organ (EEE, external enamel epithelium; SR,stellate reticulum; IEE, internal enamel epithelium). c Antibody RCK 105 against cytokeratin 7 decorated the externat enamel epithelium and the stellate reticulum. d Negative reaction obtained with antibody 6B10 against cytokeratin 4. e Antibody V9 against vimentin stained the basal portions of cells of the internal enamel epithelium as well as stellate-reticulum cells. Bars, 50 pm
21 1
’
TNEPHG
S
SD
Discussion
._ **
J1
.*: ”*
Fig. 3. Cytokeratin-polypeptidepattern of human fetal enamel-organ anlage (10-week-old fetus) as revealed by two-dimensional gel electrophoresis of cytoskeletal material obtained by microdissection (NEPHG, direction of the first dimension using nonequilibrium pH gradient electrophoresis; SDS, direction of the seconddimension electrophoresis in the presence of sodium dodecyl sulfate; silver staining). Cytokeratin polypeptides present are indicated by their numbers, according to the previously published catalog of human cytokeratins [41]. V , vimentin; A , actin; P, 3-phosphoglycerokinase from yeast, which was added as an internal marker protein
In formalin-fixed tissues of older tooth germs (newborn children), antibody lu-5, which recognizes a cytokeratin epitope resistant to treatment with formaldehyde, stained the resting “atrophic” epithelial cells, including the secretory ameloblasts (not shown). The antibodies against vimentin stained not only the stromal elements but also the stellate reticulum of the enamel organ (Figs. Id, 2e). The coexpression of cytokeratins and vimentin was not only apparent from serial sections but could also be confirmed by double-labeling immunofluorescence microscopy (not shown). Vimentin immunostaining was also observed in some cells of the external enamel epithelium and in the internal epithelium; in the latter, the positive reaction was restricted to the basal portions of the cells (Fig. 2e, Table 2). The dental lamina was always negative for vimentin (not shown). During later stages of tooth development (26th and 27th gestational week and in neonates), the epithelial cells still expressed cytokeratins, but no vimentin expression was detectable (not shown). The cells of the enamel organ were always negative with antibodies against desmin and glial fibrillary acidic protein (GFAP; not shown). Negative results were also obtained when the primary antibody was replaced by PBS. Gel electrophoresis was performed on a cytoskeletal preparation obtained by microdissecting the enamel organ from frozen sections through the upper jaw of a 10-weekold fetus. Silver staining of the two-dimensional gel revealed cytokeratins 5, 14, and 17 (stratified epithelial type) to be the major components, while only minor amounts of the simple-epithelium-typecytokeratins 7, 8 and 19 were present (Fig. 3). In addition, a sizeable quantity of vimentin was detected in this preparation.
While studies on molecular aspects of the differentiation of tooth-forming tissue have mainly been concerned with molecules functionally related to biomineralization (e.g. [59]), relatively little work has been done on the expression of intermediate filaments proteins, which is related to differentiation processes in a more-general way [ 5 , 34, 36, 441. We selected the developing human enamel organ for investigation, because, according to our working hypothesis, it seemed a likely candidate for a tissue expressing both cytokeratins and vimentin. Although the results justified our expectation, all of the available data concerning the coexpression of cytokeratins and vimentin could be explained within the framework of several hypotheses: 1. This coexpression may occur as a transient event during ontogenetic development. Many fetal tissues such as the collecting tubules of the kidneys as well as rat and human Sertoli cells and some inner ear epithelia coexpress cytokeratins and vimentin [15, 481, our own unpublished data); in some other cases, desmin is also present [31, 661. The data obtained in the present study show that, in the enamel organ, the coexpression of cytokeratins and vimentin is restricted to a brief ontogenetic phase, the so-called bell stage. 2. The expression of vimentin in epithelial cells is a typical feature of proliferative (including reactive-proliferative) situations, e.g., in proliferative mesothelial cells [54], in proliferative endometrial glands [6, 371, and in regenerating kidney-tubule epithelia [13]. Our findings for the proliferative stage of stellate reticulum development could also be interpreted along these lines. 3. Vimentin expression in epithelial cells may be connected with the loss of cell-to-cell contact [3] or with reduced epithelial-cell density in vivo [49]. This may be applicable to stellate-reticulum cells. 4. The coexpression of cytokeratins and vimentin appears to be associated with the secretory and/or resorptive function(s) of nonglandular epithelia bordering cavities filled with low-protein body fluids as well as with cells submerged in such fluids [19, 24281. The stellate reticulum is an atypical epithelium submerged in a proteoglycan-rich matrix [57] and, in this respect, is comparable to, for example, the stellate cells of Wharton’s jelly in the umbilical cord, which also simultaneously express cytokeratins, vimentin, and also desmin [26]. Furthermore, complex secretion processes are involved in enamel formation [55]. Which of these possible explanations is applicable to the coexpression of cytokeratins and vimentin in the human enamel organ must remain an open question. It is also noteworthy that several of the cytokeratins coexpressed with vimentin in the enamel organ belong to the “ stratified-epithelial type” (see below). This unusual combination appears to also occur occasionally in the corneal (271 and the fetal tongue epithelium [66]. Species differences are another factor that has to be considered. Lesot et al. [34] have analyzed the localization of prekeratin and vimentin in the mouse enamel organ, but were unable to find any vimentin reactivity in the enamel epithelia. Other investigators have described heterogeneity of cytokeratins during rat odontogenesis [5, 441. It is interesting to note that ameloblastomas express various cytokeratins [41] as well as focal concentrations of vimentin [A. Similarly,coexpression of cytokeratins and vimentin has recently been found in certain cells of adenomatoid odontogenetic tumors [61]. These findings
212
confirm the general observation that tumor cells containing more than one IF protein often have normal precursor cells that exhibit similar coexpression (for reviews, see [19, 37, 401. Thus, it appears that such coexpressions are, in many cases, not random, but are, to some extent, intrinsic and cell-type-related. The findings of a slight staining for cytokeratins, in addition to strong staining for vimentin, in fibromatous cells of some ameloblastic fibromas [62] cannot be explained on the basis of the present results. The complex combinations of the individual cytokeratin polypeptides expressed in the epithelial cells of the human enamel organ are unusual. Using two-dimensional gel electrophoresis, we found cytokeratin polypeptides 5, 14, and 17 [41] to be the major components. On the basis of their distribution in the various epithelial cell types [38, 641, cytokeratins 5, 14 and 17 are often regarded as markers of stratification or of keratinocytes [4,45]; in fact, cytokeratins 5 and 14, and 5, 14, and 17 are the main constituent polypeptides of epidermal basal keratinocytes (11, 42, 581 and cutaneous basal-cell carcinomas (undoubtedly keratinocyte-type tumors [30,41, 42]), respectively. Similarly, isolated basal cells of young cultures of rabbit corneal epithelial cells have been found to primarily contain keratins 5 and 14 [56]. The detection of this basal-keratinocyte-typical set of cytokeratins in cells of the enamel organ indicates the maintenance of a squamous epithelial differentiation component during development, even in stellate-reticulum cells that have lost a great deal of their squamous-epithelialtype coherence. This finding corresponds to ultrastructural demonstrations of the presence of bundles of tonofilaments and well-developed desmosomes connecting the epithelial cells to each other in the fetal enamel organ, including the stellate reticulum [38. 391. In the human enamel epithelia, cytokeratins 10 and 1 1 , and cytokeratins 4 and 13, which are markers of maturing layers of cornifying and noncornifying stratified squamous epithelia, respectively [4], are absent or very sparsely distributed. This indicates that, in the enamel organ, no terminal squamous epithelial differentiation occurs, this being in accordance with the available ultrastructural data [38, 391. Among the cytokeratins of the simple-epithelium type, cytokeratins 8 and 19, which are present in cells of the fetal oral epithelium, are apparently retained during the formation of the enamel organ. Perhaps our most interesting finding for the human enamel organ is our detection of the expression of cytokeratin 7, since during all of the ontogenetic stages so far studied, the squamous oral surface epithelia are negative for this cytokeratin, as are most other stratified squamous epithelia of the human body [52], with the squamous nests of the pituitary gland representing an exception [19]. If it is correct to assume that cytokeratin 7 is also absent in the oral epithelium at the very early stage during which oral epithelial cells enter the enamel organ lineage, this cytokeratin must be “switched on” in the sense of a “neoexpression”. Thus, we consider that this increase in simple-epithelium-type cytokeratins represents a characteristic differentiation step in the development of the enamel organ. Interestingly, a complex cytokeratin pattern similar to those observed by us in fetal odontogenic epithelia has recently been reported for the proliferating epithelium within periapical granulomas that is derived from remnants of those fetal epithelia [12], suggesting that this fetal phenotype may, under certain circumstances, be
reexpressed in adults. However, tests for vimentin have not been performed in that study. In conclusion, we were able to show that the patterns of IF expression in the enamel organ of human fetuses are unusually complex, particularly in the external enamel epithelium and in the stellate reticulum. The full functional significance of these patterns and their alterations remains to be elucidated. Acknowledgemenfs. We are grateful to Dr. J. Bartek (Research Institute of Clinical and Experimental Oncology, Brno, Czechoslovakia), Dr. W.W. Franke (German Cancer Research Center, Heidelberg, FRG), Dr. F.C.S. Ramaekers (University of Nijmegen, Nijmegen, The Netherlands), and Dr. T.-T. Sun (New York University, NY) for their generous gifts of antibodies. The excellent technical assistance of Mrs. M. Haase (Gorlitz) and Mrs. C. Zech, J. Jacobi, H. Breitbach, and K. Essling (Mainz) is gratefully acknowledged. We thank Mrs. C. Biirkner for her careful typing of the manuscript. This work was supported by grants from the Ministry of Health of the German Democratic Republic (to M.K.) and the Deutsche Forschungsgemeinschaft (to R.M.).
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52. Ramaekers F, Huysmans A, Schaart G, Moesker 0, Vooijs P (1987) Tissue distribution of keratin 7 as monitored by a monoclonal antibody. Exp Cell Res 170:235-249 53. Regauer S, Franke WW, Virtanen I (1985) Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: Expression of epidermal cytokeratins in cells of a simple epithelium. J Cell Biol 100:997-1009 54. Rheinwald JG, OConnell TM, Connell ND, Ryback SM, Allen-Hoffmann BL, LaRocca PJ, Wu YJ, Rehwoldt SM (1984) Expression of specific keratin subsets and vimentin in normal human epithelial cells: A function of cell type and conditions of growth during serial culture. In: Levine AJ, Van de Woude GF, Topp WC, Watson J D (eds) Cancer cells. 1 . The transformed phenotype. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 217-227 55. Robinson C, Kirkham J, Hallsworth AS (1988) Volume distribution and concentration of protein, mineral and water in developing bovine enamel. Arch Oral Biol 33 : 159-1 62 56. Schermer A, Calvin S, Sun T-T (1986) Differentiation-related expression of a major 64 K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 103:49-62 57. Schroeder HE (1982) Orale Strukturbiologie. Georg Thieme, Stuttgart New York 58. Skerrow D, Skerrow CJ (1983) Tonofilament differentiation in human epidermis; isolation and polypeptide chain composltion of keratinocyte subpopulations. Exp Cell Res 143: 27-35 59. Slavkin HC, Bessem C, Bringas P Jr, Zeichner-David M, Nanci A, Snead ML (1988) Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis. Differentiation 37 :2 6 3 9 60.Stosiek P, Goertchen R (1986) Immunpathologischer Beitrag zum malignen diffusen Mesotheliom. Zentralbl Allg Pathol 1321119-128
61. Tatemoto Y, Tdnaka T, Okada Y, Mori M (1988) Adenomatoid odontogenic tumour: co-expression of keratin and vimentin. Virchows Arch A (Path Anat) 413: 341-347 62. Tatemoto Y, Yamamoto N, Onojima M, Okada Y, Mori M (1988) Ameloblastic fibroma: growth potentiality of odontogenic epithelium and coexpression of intermediate filament proteins of intermediate filament proteins in fibromatous cells. J Oral Pathol 17: 168-174 63. Tolle H-G, Weber K, Osborn M (1985) Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allows insight into the composition of particular 10 nm filaments. Eur J Cell Biol 38 :234-244 64. Tseng SCG, Jarvinen MJ, Nelson WG, Huang J-W, Woodcock-Mitchell J, Sun T-T (1982) Correlation of specific keratins with different types of epithelial differentiation: Monoclonal antibody studies. Cell 30: 361-372 65. Van Muijen GNP, Ruiter DJ, Franke WW, Achtstatter T, Haasnoot WHB, Ponec M, Warnaar SO (1986) Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas as demonstrated by monoclonal antibodies specific for cytokeratins nos. 4 and 13. Exp Cell Res 162 :97-113 66. Van Muijen GNP, Ruiter DJ, Warnaar SO (1987) Coexpression of intermediate filament polypeptides in human fetal and adult tissues. Lab Invest 57: 359-369 67. Von Overbeck I, Stahli C, Gudat F, Carmann H, Lautenschlager C, Dorrmiiller C, Takacs B, Miggiani V, Staehelin T. Heitz P (1 985) Immunohistochemical characterization of an anti-epithelial monoclonal antibody (mab lu-5).Virchows Arch (A) 407: 1-12
Accepted in revised form April 29, 1989
Differentiation (1989) 40:207-214
Ontogeny and Neoplasia 0 Springer-Verlag 1989
Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin * Michael Kasper **, Uwe Karsten ’,Peter Stosiek ’, and Roland Moll Institute of Pathology, District Hospital Gorlitz, DDR-8900 Gorlitz, German Democratic Republic Department of Immunology, Central Institute of Molecular Biology, Academy of Science of the German Democratic Republic, DDR-1115 Berlin-Buch, German Democratic Republic Institute of Pathology, University of Mainz, Langenbeckstrasse 1, D-6500Mainz, Federal Republic of Germany
Abstract. We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible “neoexpression” during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.
Introduction According to the established and accepted patterns of intermediate filament (IF) distribution [46], cytokeratins are expressed in epithelia, whereas vimentin is restricted to mesenchymal cells. In recent years, however, the simultaneous expression of both types of IFs has been detected in a number of specialized tissues [ l , 14, 15, 19-22, 24-29, 32, 33, 48, 53, 601. Van Muijen et al. [58] have interpreted the simultaneous occurrence of vimentin and cytokeratins as being a transient phenomenon restricted to certain ontogenetic stages or, in adult tissues, to reactive proliferation. Our own data suggest that the coexpression of vimentin and cytokeratins may be determined by functional factors [ 191. We have found both IF types in nonglandular epithelia, in cells bordering cavities filled with low-protein body fluids, and in cells localized in proteoglycan-rich extracellular matrix. At present, it is not clear whether all of these apparently isolated cases are linked by their having a functional or regulatory principle in common. In pursuing our
* Dedicated to the orthodontist Dr. Georg Klammt, Gorlitz
** To whom offprint requests should be sent
working hypothesis that the coexpression of vimentin and cytokeratin may, in some way, be related to secretory function, we examined the distribution of IFs in the enamel organ of human fetuses, which consists of epithelial cells involved in the production and secretion of enamel-forming substances [38, 391. Methods Tissues. Primary tooth germs (n, 11) were obtained from human fetuses (abortion material; 2 x loth, 1 x 16th and 1 ~ 2 7 t hweek of gestation, maxilla and mandible in each case and 2 x 18th and 1 x 26th week of gestation (mandible alone)) and from a 9-month-old child (autopsy material). After embedding in a special mounting medium (Jung, Heidelberg, FRG), the tooth germs were frozen and stored in liquid nitrogen. Formalin-fixed tissue of human maxillae (n, 2) obtained from a 40th-gestational-week fetus and a 2-day-old neonate were decalcified, embedded in paraffin, and sectioned. To avoid the loss of tissue sections during protease digestion using 0.1% Pronase (Serva, Heidelberg, FRG) in phosphate-buffered saline (PBS), pH 7.4, for 15 min at 37” C, we precoated the slides with a 1 YOaqueous solution of Cementite glue (Merz and Beuteli, Niederwangen, Switzerland). The sections were deparaffinized using xylene and ethanol prior to protease digestion and immunostaining. Antibodies. The primary antibodies used in the present study are listed in Table 1. In addition to monoclonal antibodies, we used polyclonal guinea-pig antisera against vimentin [9] and rabbit antisera against keratin (Prof. J. Schulz, Biochemical Institute, Berlin, German Democratic Republic).
Indirect-immunojluorescence microscopy. Indirect-immunofluorescence microscopy of frozen, unfixed, 5 l m thick sections was performed as previously described [23]. For immunofluorescence, the secondary antibodies used were goat antibodies coupled with fluorescein isothiocyanate (FITC) or tetramethylrhodamin isothiocyanate (TRITC) to guinea-pig, rabbit, or mouse immunoglobulins (Dianova, Hamburg, FRG; SIFIN, Berlin, GDR). For double-immunofluorescence microscopy, two primary antibodies raised in different animal species were applied simultaneously. After washing in PBS, the fluorochrome-labeled secondary antibodies (diluted 1 :20) were applied together.
208
Table 1. Monoclonal mouse antibodies used
Antibody
Antigens recognized
Reference and/or source
A45- B/ B3 lu-s RCK 102 6BlO
Broad range of cytokcratins Broad range of cytokeratins Cytokeratins 5 and 8 Cytokeratin 4
AE14 RCK 105
Cytokeratin 5 Cytokeratin 7
C18
Cytokeratin 7
CK7 K, 8.1.42 c10 RKSE 60 Kk 8.60 K , 18.174 207 c 04 RGE 53 BA 16 BA 17 A53-BIA2 K, 19.2.10.3 RV 202 v 9 VIM-9 VIM-38 4 D E- B-S G-A-5
Cytokeratin 7 Cytokeratin 8 Cytokeratin 8 Cytokeratin 10 Cytokeratins 10 and 11 Cytokeratin 18 Cytokeratin 13 Cytokeratin 18 Cytokeratin 18 Cytokeratin 19 Cytokeratin 19 Cytokeratin 19 Cytokeratin 19 Vimentin Vimentin Vimcntin Vimen tin Desmin Glial fibrillary acidic protein (GFAP)
~7,231 [lo, 671 Provided by Hoffmann-LaRoche (Basel, Switzerland) 1521 [65] Provided by Dr. W.W. Franke (German Cancer Research Center, Heidelberg, FRG) [35] Provided by Dr. T.T. Sun (New York University, NY) [52] Provided by Dr. F.C.S. Ramaekers (University of Nijmegen, The Netherlands) Providcd by Dr. J. Bartek (Research Institute of Clinical and Experimental Oncology, Brno, Czechoslovakia) [63] Obtained from Boehringer (Mannheim, FRG)
PI
Dr. Bartek [511 [I 61 [43] Progen Biotechnics (Heidelberg, FRG) [65] Provided by Dr. W.W. Franke Dr. Bartek
POI 121 (21 1181 [43] Progen Provided by Dr. Ramaekers [47] Boehringer Viramed (Martinsried, FRG) Progen Boehringcr Boehringer
Table 2. Immunohistochemical staining of human fetal enamcl organs and oral epithelia by monoclonal antibodies directed against different IF proteins
Tissue
CK CK 5 + 8 CK 5" CK 4" CK 13' CK 10" CK 8" CK 18 CK 19 CK 7 (broad-range an ti bodies)
External enamel epithelium
Dental lamina
+ + + +
+ + + +
Oral epithelium: Basal cells Suprabasal cells
+ +
+ +
Stellate reticulum Jnternal enamel epithelium
+= +
-
-d
-
-f
+ c
-
-
+
-
-I+
-
+/-h
-
-I+
-
+I-
+'
+
-
-/+m
+ +
-
+
-a
-j
-Y
-I+
-
+ +
+/-
-
+
-/+
-n
-f
+= +'
Vimentinb
-/+
+'
-
-/+k
-/+
-
-
-
"
-
CK, cytokeratin; + , positive; - , negative; + / - and - / + , focal patterns ( + / - , positive cells predominate; -I+, negative cells predominate) a In 18th and 27th gestational week not tested; In 26th and 27th gestational week all epithelial cells were negative; In 10th gestational week, weak staining; In 16th and 26th gestational week, few cells were positive; In 10th gestational week, only few cells were weakly positive; In 10th gestational week, some cells werc positive; In 10th gestational week, weakly positive; In 10th gestational week, uniformly positive; In 26th and 27th gestational week, only some cells were positive; In 10th gestational week, positive; ' Basal portions of cells were positive (few cells in the 10th gestational weck; many cells in the 16h and 18th gestational week); 'Some parabasal cells were negative; Only some upper cells were positive; " In 10th gestational week, some superficial cells (periderm-like layer) were positive
Negative controls were performed by replacing the primary antibody with PBS. Furthermore, the antibodies against desmin and GFAP produced consistently negative results with the enamel organ and thus served as additional negative controls.
Immunoperoxidase techniques. Cryostat sections or deparaffinized and protease-treated paraffin sections were incubated with 1YOhydrogen peroxide in methanol for 30 min in order to block endogenous peroxidase activity. This was followed by washing in PBS and preincubation with normal
209
C Fig. 1 a-d. Distribution of cytokeratins and vimentin in early fetal enamel-organ anlage (10-week-old human fetus); immunoperoxidase staining. a Antibody AE14 against cytokeratin 5 produced weak staining of the external enamel epithelium ( E E Q and the internal enamel epithelium (IEE; prospective ameloblasts), but stronger staining of the stellate reticulum (SR). The dental lamina (DL) was strongly positive. Inset: antibody 207 against cytokeratin 13 stained some cells of the stellate reticulum. b Antibody K, 8.1.42 decorated all epithelial elements of the enamel organ anlage. c Antibody RCK 105 against cytokeratin 7 prominently stained the stellate reticulum. d Antibody VIM-9 against vimentin exhibits a strong positive reaction in the stellate reticulum. Note the additional decoration of some cells of the external enamel epithelium (arrowheads) as well as the staining of the basal portions of individual cells of the intcrnal enamel epithelium (arrow). Bur, 50 pm
210
goat serum (1% in PBS) in order to reduce background staining. The sections were then incubated with the primary antibody (undiluted supernatant or commercially available antibodies at the appropriate dilution) for 45 min at room temperature. After three washes in PBS (10 min each), the sections were incubated with goat anti-mouse Ig (SIFIN ; diluted 1 :40) for 30 min. After three washes in PBS (10 min each), monoclonal antibody BL-POD (Sektion Biowissenschaften, Karl-Marx-Universitat Leipzig, GDR) directed against peroxidase was added at a dilution of 1 : 100. This was followed by three washes in PBS (10 min each) and incubation with a 0.001% peroxidase solution in PBS (Arzneimittelwerk Dresden, GDR) for 30 min. After five 5-min washes in PBS, the peroxidase activity was visualized by the addition of a freshly prepared solution of 3,3-diaminobenzidine tetrahydrochloride (Chemapol, Prague, Czechoslovakia) for 8 min. Finally, hematoxylin was used for counterstaining. Alternatively, an indirect immunoperoxidase technique was applied to cryostat sections [8]. Gel electrophoresis.The preparation of cytoskeletal material by the microdissection of defined tissue structures in frozen sections, the soluble-protein extraction procedure, and the analysis of cytoskeletal proteins by two-dimensional gel electrophoresis were all performed as described previously [411.
Results
Table 2 summarizes the immunohistochemical results obtained using frozen sections. All of the monoclonal anti-
bodies directed against a broad range of cytokeratins stained epithelial cells throughout the human fetal enamel organ. However, different patterns were obtained when antibodies specific for individual cytokeratin polypeptides (Table 1) were applied. The antibody AE14 against cytokeratin 5 stained most epithelial cells including the dental lamina (Figs. 1a, 2a), whereas cytokeratin 13 as detected by antibody 207 was found to be restricted to a few cells of the enamel organ (Fig. l a , inset). Incubation with the antibodies against cytokeratin 10 (RKSE 60, Kk 8.60) produced a positive reaction in the suprabasal cells of fetal oral stratified squamous epithelium but not in the enamel organ (not shown). Whereas cytokeratin 8 was detected in most cells of the enamel organ (Fig. 1 b), the exception being the internal enamel epithelium at later stages, cytokeratin 18 was only weakly expressed or appeared to be absent at this site (Table 2; for the antibodies, see Table 1). On the other hand, cytokeratin 19 was detectable in most epithelial cells of the enamel organ (Fig. 2b) and dental lamina. Cytokeratin 7 was consistently detected in the stellate reticulum, in certain cells of the dental lamina, and also, in 16- and 18-week-old fetuses, in the external epithelium of the enamel organ (Figs. l c , 2c). This cytokeratin was not detectable in the oral stratified squamous epithelium but was observed in the inner cells of the peg-like epithelial outgrowths, located immediately beneath the oral surface epithelium, which might have been salivary-gland germs (not shown). Cytokeratin 4 was not found in the enamel organ (Fig. 2d), except for a few cells of the external enamel epithelium of week 26 that were positive, but was detected in the upper layers of the oral squamous epithelium (not shown).
Fig. 2s-e. Distribution of cytokeratins and vimentin in fetal enamel-organ anlage at a more advanced stage (1 6-week-old-human fetus; immunoperoxidase staining) during which enamel production begins (not illustrated). a, b Antibodies AE14 against cytokeratin 5 (a) and BA 16 against cytokeratin 19 (b) stained most cells of the enamel organ (EEE, external enamel epithelium; SR,stellate reticulum; IEE, internal enamel epithelium). c Antibody RCK 105 against cytokeratin 7 decorated the externat enamel epithelium and the stellate reticulum. d Negative reaction obtained with antibody 6B10 against cytokeratin 4. e Antibody V9 against vimentin stained the basal portions of cells of the internal enamel epithelium as well as stellate-reticulum cells. Bars, 50 pm
21 1
’
TNEPHG
S
SD
Discussion
._ **
J1
.*: ”*
Fig. 3. Cytokeratin-polypeptidepattern of human fetal enamel-organ anlage (10-week-old fetus) as revealed by two-dimensional gel electrophoresis of cytoskeletal material obtained by microdissection (NEPHG, direction of the first dimension using nonequilibrium pH gradient electrophoresis; SDS, direction of the seconddimension electrophoresis in the presence of sodium dodecyl sulfate; silver staining). Cytokeratin polypeptides present are indicated by their numbers, according to the previously published catalog of human cytokeratins [41]. V , vimentin; A , actin; P, 3-phosphoglycerokinase from yeast, which was added as an internal marker protein
In formalin-fixed tissues of older tooth germs (newborn children), antibody lu-5, which recognizes a cytokeratin epitope resistant to treatment with formaldehyde, stained the resting “atrophic” epithelial cells, including the secretory ameloblasts (not shown). The antibodies against vimentin stained not only the stromal elements but also the stellate reticulum of the enamel organ (Figs. Id, 2e). The coexpression of cytokeratins and vimentin was not only apparent from serial sections but could also be confirmed by double-labeling immunofluorescence microscopy (not shown). Vimentin immunostaining was also observed in some cells of the external enamel epithelium and in the internal epithelium; in the latter, the positive reaction was restricted to the basal portions of the cells (Fig. 2e, Table 2). The dental lamina was always negative for vimentin (not shown). During later stages of tooth development (26th and 27th gestational week and in neonates), the epithelial cells still expressed cytokeratins, but no vimentin expression was detectable (not shown). The cells of the enamel organ were always negative with antibodies against desmin and glial fibrillary acidic protein (GFAP; not shown). Negative results were also obtained when the primary antibody was replaced by PBS. Gel electrophoresis was performed on a cytoskeletal preparation obtained by microdissecting the enamel organ from frozen sections through the upper jaw of a 10-weekold fetus. Silver staining of the two-dimensional gel revealed cytokeratins 5, 14, and 17 (stratified epithelial type) to be the major components, while only minor amounts of the simple-epithelium-typecytokeratins 7, 8 and 19 were present (Fig. 3). In addition, a sizeable quantity of vimentin was detected in this preparation.
While studies on molecular aspects of the differentiation of tooth-forming tissue have mainly been concerned with molecules functionally related to biomineralization (e.g. [59]), relatively little work has been done on the expression of intermediate filaments proteins, which is related to differentiation processes in a more-general way [ 5 , 34, 36, 441. We selected the developing human enamel organ for investigation, because, according to our working hypothesis, it seemed a likely candidate for a tissue expressing both cytokeratins and vimentin. Although the results justified our expectation, all of the available data concerning the coexpression of cytokeratins and vimentin could be explained within the framework of several hypotheses: 1. This coexpression may occur as a transient event during ontogenetic development. Many fetal tissues such as the collecting tubules of the kidneys as well as rat and human Sertoli cells and some inner ear epithelia coexpress cytokeratins and vimentin [15, 481, our own unpublished data); in some other cases, desmin is also present [31, 661. The data obtained in the present study show that, in the enamel organ, the coexpression of cytokeratins and vimentin is restricted to a brief ontogenetic phase, the so-called bell stage. 2. The expression of vimentin in epithelial cells is a typical feature of proliferative (including reactive-proliferative) situations, e.g., in proliferative mesothelial cells [54], in proliferative endometrial glands [6, 371, and in regenerating kidney-tubule epithelia [13]. Our findings for the proliferative stage of stellate reticulum development could also be interpreted along these lines. 3. Vimentin expression in epithelial cells may be connected with the loss of cell-to-cell contact [3] or with reduced epithelial-cell density in vivo [49]. This may be applicable to stellate-reticulum cells. 4. The coexpression of cytokeratins and vimentin appears to be associated with the secretory and/or resorptive function(s) of nonglandular epithelia bordering cavities filled with low-protein body fluids as well as with cells submerged in such fluids [19, 24281. The stellate reticulum is an atypical epithelium submerged in a proteoglycan-rich matrix [57] and, in this respect, is comparable to, for example, the stellate cells of Wharton’s jelly in the umbilical cord, which also simultaneously express cytokeratins, vimentin, and also desmin [26]. Furthermore, complex secretion processes are involved in enamel formation [55]. Which of these possible explanations is applicable to the coexpression of cytokeratins and vimentin in the human enamel organ must remain an open question. It is also noteworthy that several of the cytokeratins coexpressed with vimentin in the enamel organ belong to the “ stratified-epithelial type” (see below). This unusual combination appears to also occur occasionally in the corneal (271 and the fetal tongue epithelium [66]. Species differences are another factor that has to be considered. Lesot et al. [34] have analyzed the localization of prekeratin and vimentin in the mouse enamel organ, but were unable to find any vimentin reactivity in the enamel epithelia. Other investigators have described heterogeneity of cytokeratins during rat odontogenesis [5, 441. It is interesting to note that ameloblastomas express various cytokeratins [41] as well as focal concentrations of vimentin [A. Similarly,coexpression of cytokeratins and vimentin has recently been found in certain cells of adenomatoid odontogenetic tumors [61]. These findings
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confirm the general observation that tumor cells containing more than one IF protein often have normal precursor cells that exhibit similar coexpression (for reviews, see [19, 37, 401. Thus, it appears that such coexpressions are, in many cases, not random, but are, to some extent, intrinsic and cell-type-related. The findings of a slight staining for cytokeratins, in addition to strong staining for vimentin, in fibromatous cells of some ameloblastic fibromas [62] cannot be explained on the basis of the present results. The complex combinations of the individual cytokeratin polypeptides expressed in the epithelial cells of the human enamel organ are unusual. Using two-dimensional gel electrophoresis, we found cytokeratin polypeptides 5, 14, and 17 [41] to be the major components. On the basis of their distribution in the various epithelial cell types [38, 641, cytokeratins 5, 14 and 17 are often regarded as markers of stratification or of keratinocytes [4,45]; in fact, cytokeratins 5 and 14, and 5, 14, and 17 are the main constituent polypeptides of epidermal basal keratinocytes (11, 42, 581 and cutaneous basal-cell carcinomas (undoubtedly keratinocyte-type tumors [30,41, 42]), respectively. Similarly, isolated basal cells of young cultures of rabbit corneal epithelial cells have been found to primarily contain keratins 5 and 14 [56]. The detection of this basal-keratinocyte-typical set of cytokeratins in cells of the enamel organ indicates the maintenance of a squamous epithelial differentiation component during development, even in stellate-reticulum cells that have lost a great deal of their squamous-epithelialtype coherence. This finding corresponds to ultrastructural demonstrations of the presence of bundles of tonofilaments and well-developed desmosomes connecting the epithelial cells to each other in the fetal enamel organ, including the stellate reticulum [38. 391. In the human enamel epithelia, cytokeratins 10 and 1 1 , and cytokeratins 4 and 13, which are markers of maturing layers of cornifying and noncornifying stratified squamous epithelia, respectively [4], are absent or very sparsely distributed. This indicates that, in the enamel organ, no terminal squamous epithelial differentiation occurs, this being in accordance with the available ultrastructural data [38, 391. Among the cytokeratins of the simple-epithelium type, cytokeratins 8 and 19, which are present in cells of the fetal oral epithelium, are apparently retained during the formation of the enamel organ. Perhaps our most interesting finding for the human enamel organ is our detection of the expression of cytokeratin 7, since during all of the ontogenetic stages so far studied, the squamous oral surface epithelia are negative for this cytokeratin, as are most other stratified squamous epithelia of the human body [52], with the squamous nests of the pituitary gland representing an exception [19]. If it is correct to assume that cytokeratin 7 is also absent in the oral epithelium at the very early stage during which oral epithelial cells enter the enamel organ lineage, this cytokeratin must be “switched on” in the sense of a “neoexpression”. Thus, we consider that this increase in simple-epithelium-type cytokeratins represents a characteristic differentiation step in the development of the enamel organ. Interestingly, a complex cytokeratin pattern similar to those observed by us in fetal odontogenic epithelia has recently been reported for the proliferating epithelium within periapical granulomas that is derived from remnants of those fetal epithelia [12], suggesting that this fetal phenotype may, under certain circumstances, be
reexpressed in adults. However, tests for vimentin have not been performed in that study. In conclusion, we were able to show that the patterns of IF expression in the enamel organ of human fetuses are unusually complex, particularly in the external enamel epithelium and in the stellate reticulum. The full functional significance of these patterns and their alterations remains to be elucidated. Acknowledgemenfs. We are grateful to Dr. J. Bartek (Research Institute of Clinical and Experimental Oncology, Brno, Czechoslovakia), Dr. W.W. Franke (German Cancer Research Center, Heidelberg, FRG), Dr. F.C.S. Ramaekers (University of Nijmegen, Nijmegen, The Netherlands), and Dr. T.-T. Sun (New York University, NY) for their generous gifts of antibodies. The excellent technical assistance of Mrs. M. Haase (Gorlitz) and Mrs. C. Zech, J. Jacobi, H. Breitbach, and K. Essling (Mainz) is gratefully acknowledged. We thank Mrs. C. Biirkner for her careful typing of the manuscript. This work was supported by grants from the Ministry of Health of the German Democratic Republic (to M.K.) and the Deutsche Forschungsgemeinschaft (to R.M.).
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Accepted in revised form April 29, 1989