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The ciliated human keratinocyte
JOURNAL OF ULTRASTRUCTURERESEARCH 87, 212--220
(1984)
The Ciliated Human Keratinocyte R O L F ELOFSSON,* ANDERS ANDERSSON,t BENGT FALCK, t AND STEINAR SJOBORG~
Departments of*Zoology, tHistology, and ~.Dermatology, University of Lund, Lund, Sweden Received May 18, 1984 H u m a n keratinocytes were investigated for the presence of single cilia. Almost all basal keratinocytes were found to carry a single cilium in normal, occluded, and psoriatic skin. The ciliary structure was progressively reduced in keratinocytes approaching the surface. No remnants of the ciliary apparatus were found in the gramdar layer. In one case of nickel-allergic dermatitis (patch test), the keratinocytes had lost their cilia; the significance of this surprising finding remains to be elucidated.
© 1984 Academic Press, Inc.
Recently melanocytes in the human epi- acetate. One specimen obtained from normal skin was dermis were found to carry one Cilium each subjected to fixation according to Karnovsky (1965) (Elofsson et al., 1981). Single cilia occur with a buffer osmolality of 100 mosmole/kg and a total osmolality of 1810 mosmole/kg. After prefixation the scattered in many cell populations of the specimen was treated as above, Sections were cut on body (Afzelius, 1979; Flood and Totland, a diamond knife and were examined in a Zeiss EM 10 1977; Scherft and Daems, 1967; Wilsman, and a JEOL 100 CX. Consecutive sections were placed on one-hole grids 1978). Their presence on every cell of a coated with Formvar and strengthened with carbon whole-cell system, such as the melanocytes coating. Ten or twenty grids were produced from each of the epidermis, has so far been claimed biopsy. Each grid carried 20 sections of less than 100only for a few cell types (Flood and Totland, rim thickness each. They measured 150-250 #m in 1977; Wilsman and Fletcher, 1978). This length and comprised the whole epidermis. The analwork reports the regular appearance of sin- yses were made on 10 to 20 keratinocytes at a time and the cells were mapped and followed throughout gle cilia in the keratinocyte population. their whole soma. One or two such analyses were done on each series of sections.
MATERIALS AND METHODS Punch biopsies (3 mm) from adult h u m a n skin were obtained using local anesthesia (Xylocain 1%): clinicaUy normal skin from four persons, one specimen from a positive patch test reaction to nickel and one specimen from psoriatic skin. Two specimens were obtained after occluding normal skin by aluminum foil for 48 hr. The biopsies were trimmed from excess dermis immediately upon immersion in the fixative and were divided into two or three portions. With one exception, all specimens were processed in the same way. The prefixative used was 2.5% glutaraldehyde in 0.15 M cacodylate buffer (pH 7.3). The buffer osmolality was 300 and the total osmolality of the fixative was 555 mosmole/kg. The reason for using this fixative has been discussed elsewhere (Falck et aL, 1981). Prefixation lasted 2 hr and the postfixation in 2% OsO4 in the above buffer was performed for 1-2 hr. The specimens were dehydrated in ethanol starting at 50% and stained en bloc by 1% phosphotungstic acid and 0.5% uranyl acetate. Additional staining was done on the sections with 0.5% lead citrate and 1% uranyl
RESULTS
Normal skin. The keratinocytes of the basal layer were found, with rare exceptions (see below), to possess one cilium each (Fig. 1). The cilium was usually situated anywhere on the upper half of the keratinocyte. It could also be inserted deeper down on the cell surface but rarely below the nucleus. The ciliary shaft was always located in a channel continuous with the intercellular space, which invaginated deep into the ceil. The channel rarely followed a straight line to the nearest cell surface but rather ran a longer distance, sometimes with a curvature (Figs. 2-4), inside the cell before emerging (Fig. 4). The cilium could point in any direction in the epidermis.
212 0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All fights of reproduction in any form reserved.
CILIATED HUMAN KERATINOCYTE
F~G. 1. A schematic drawing of the human epidermis comprising the basal, spinous, and granular layers. The drawing illustrates the well-developed cilium of the keratinocytes in the basal layer and the gradual disappearance of the cilium in the spinous layer. The cilia varied in length within one specimen. The longest cilium measured ca. 3 # m and poked into a neighboring keratinocyte for m o r e than 1 /zm, ending close to the nucleus (Figs. 5-6). A cilium could be confined within the basal keratinocyte, but usually the cilia extended into the intercellular
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space o f the epidermis without coming into contact with other cells. The whole ciliary apparatus consisted o f a basal b o d y similar to a centriole pair and a finger-like extension from the cell surface, also called the ciliary shaft, containing the axoneme. The a x o n e m e o f the cilia had nine tubular duplets and no central tubuli but lost this regular arrangement toward the tip o f the cilium (Fig. 13). N o dynein arms were observed. The basal b o d y was usually situated close to the nucleus and in the n e i g h b o r h o o d o f a Golgi apparatus. It consisted o f one pair o f cylinders, with each cylinder f o r m e d by nine triplets o f tubuli approximately 230 n m in diameter and 350 n m in length. The two cylinders could have any direction within the cell and no preferred direction was found. The cylinders were sometimes oriented perpendicular to one another but other directions were c o m m o n . A n o t h e r variation occurred in the distance between them. The cylinders were usually very close (0.1 #m) but could be several micrometers apart. The basal b o d y carrying the ciliary shaft normally had basal feet. These appeared as five cones encircling the middle o f the cylinder, with their bases toward the tubuli. Only on two occasions did thin, feebly developed rootlets occur, very similar to those observed by W i l s o n and McW h o r t e r (1963) and D a r 6 c z y and F e l d m a n n (1974). Alar sheets were present which connected the distal tip o f the basal body, carrying the cilium, with the cell m e m b r a n e . Two basal keratinocytes deviated in some
FIGS. 2-4. Normal skin. Three sections from a series through a cilium in a basal keratinocyte. The ciliar shaft points toward the nucleus (Fig. 2), bends inside the cell (Fig. 3), and emerges in the intercellular space (Fig. 4). Scale bars = 0.5 fzm. FIG. 5. Normal skin. Survey of a cilium in a basal keratinocyte traversing the intercellular space and pointing into an adjacent keratinocyte (arrows). Epidermal surface to the left. Scale bar = 1 #m. FIG. 6. Detail of Fig. 5 from another section. Scale bar = 0.5/~m. FIG. 7. Normal skin. Two parallel basal bodies situated apically in a basal keratinocyte are shown. Scale bar = 0.5/zm. FIG. 8. Two sections away from that of Fig. 7, the two cilia appear in the extracellular channel formed inside the cell. Scale bar = 0.5 ~zm.
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FIG. 14. Psoriatic skin. T h e figure s h o w s two adjacent basal keratinocytes carrying one cilium each (arrows). Scale b a r = 1 /.tin.
respects. One was found carrying only the basal body and no ciliary shaft. The other carried two ciliary shafts lying contiguous to one another and having the two cylinders of the basal body parallel to one another (Figs. 7-8). The keratinocytes in the stratum spinosum also possessed cilia, however, reduced compared to those found in the basal layer. The reduction was gradual, increasing toward the surface. Short ciliary shafts usually confined to the small extracellular channel invaginating into the cell were found in the keratinocytes immediately above the basal
layer (Fig. 9). In the keratinocytes situated in the middle layers of the stratum spinosum and below the stratum granulosum, the ciliary shafts had disappeared. Remnants of them were found in the small vesicle connected to one of the cylinders of the basal body (Figs. 10-11). This vesicle is probably a remnant of the invaginated intercellular space housing the ciliary shaft in the more basally located keratinocytes. The ciliary apparatus and/or the remnants of it were found in the upper part of all keratinocytes above the basal layer. The basal bodies were always situated close to the nucleus. Their
FIG. 9. N o r m a l skin. T h e figure s h o w s a reduced cilium confined inside the keratinocyte, w h i c h is situated in the suprabasal layer. Scale b a r = 0.5 urn. FIG. 10. N o r m a l skin. Detail f r o m a kerafinocyte in the u p p e r part o f the s t r a t u m s p i n o s u m . T h e basal b o d y a n d the small r e m n a n t o f the extraceUular space are seen as a small vesicle attached to the basal b o d y (arrow). Scale b a r = 0.5 # m . FIG. 11. F o u r sections away f r o m that o f Fig. 10, the vesicle a n d the tiny rest o f the cilium (arrow) are shown. E p i d e r m a l surface to the left. Scale b a r = 0.5 ~zm. FIG. 12. N o r m a l skin fixed in a low-osmolality solution (Karnovsky). Arrows p o i n t at a cilium in a basal keratinocyte. Note the obliterated extracellular space. Scale b a r = 0.5 urn. FIG. 13. Skin as in Fig. 12 showing a cross section o f a cilium (arrow) inside a keratinocyte. T h e extracellular i n v a g i n a t e d c h a n n e l s u r r o u n d i n g t h e ciliary shaft is obliterated. Scale b a r = 0.5 urn.
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cylinders were several micrometers apart, with the distance increasing even more just beneath the granular layer. No traces of the ciliary apparatus were found in the granular layer. Occluded and psoriatic skin. In psoriatic skin both rete ridges and suprapapillary epidermal areas were scrutinized for the presence of cilia. As in normal skin, cilia displaying different degrees of differentiation were found within the keratinocytes (Fig. 14). However, a few of the basal keratinocytes lacked a ciliary shaft but had the basal body. Skin fixed in low-osmolality buffer (Karnovsky). It has recently been demonstrated that, in a fixative consisting of 2.5% glutaraldehyde dissolved in cacodylate buffer having an osmolality above 230 mosmole/ kg, the epidermal intercellular spaces are prominent (Falck et al., 1981). This was also the case with all biopsies in this study except in the one fixed in Karnovsky solution. This fixative produced closely packed keratinocytes with obliterated intercellular space as do glutaraldehyde-based fixatives having a cacodylate buffer osmolality well below 230 mosmole/kg (Falck et aL, 1981). After Karnovsky fixation the cilia were difficult to find among the contiguous cells of the epidermis, and the number of basal keratinocytes observed to carry a cilium was much lower than that reported above. The cilia displayed the same features as described above and were even seen to protrude into the extremely narrow intercellular space (Figs. 12-13). Nickel contact allergy. In the epidermis of a positive patch test reaction to nickel, no ciliary apparatuses or remnants of them were observed in the keratinocytes. A structure similar to a basal body or a centriole was situated in the basal half of the keratinocytes. DISCUSSION
Scattered cells having one cilium are found in most tissues of the body (see Afzelius, 1979; Flood and Totland, 1977; Scherft and
Daems, 1967; Wilsman, 1978), whereas all chondrocytes from articular cartilage of neonatal dog and epithelial cells along the nephron and collecting ducts of the adult mouse have been reported to carry a cilium (Wilsman and Fletcher, 1978; Flood and Totland, 1977). Such cilia are referred to in different terms, e.g., single, solitary, primary, isolated, residual, vestigial, or immature cilia. The first ultrastructural study of single cilia in human keratinocytes was made by Wilson and McWhorter (1963). They found cilia (flagella) in some basal keratinocytes in normal epidermis and in basal cell carcinoma. Anton-Lamprecht (1972) described cilia as intracellular structures in some basal keratinocytes of normal epidermis as well as in three patients with KleinWaardenburg Syndrome. Dar6czy and Feldmann (1974) reported the presence of cilia on some keratinocytes in a number of pathological situations. More keratinocytes carried single cilia in spinocellular carcinoma and bullous skin diseases after UV treatment. Single cilia have also been observed in some cells of fetal skin and human gingiva (Breathnach, 1971; Nikai et aL, 1970). In contrast to these observations on human keratinocytes the present study has revealed a regular occurrence of single cilia on practically all basal keratinocytes. In addition it has been disclosed that the ciliary apparatus gradually disappears with the progressive differentiation of the keratinocytes with no remnant of it being found in the granular layer. This finding could be obtained by taking advantage of some technical precautions which greatly facilitate the study of this organelle. First, the ~hoice of fixating solution is important. By varying the buffer osmolality the dimensions of the epidermal intercellular space can be changed within a broad range (Falck et aL, 1981). A buffer osmolality of 300 mosmole/kg produces wide intercellular spaces and makes the mapping of the individual keratinocyte in serial sections and tracing of ciliary shafts easy.
CILIATEDHUMAN KERATINOCYTE Moreover, the invaginating channels harboring these shafts are well preserved. With an almost obliterated intercellular space, as produced by the Karnovsky fixative, the keratinocyte mapping is very difficult. As a consequence of the cellular swelling an important landmark, i.e., the invaginated channel containing the ciliary shaft, is eradicated. Also the ciliary shafts protruding in between the ceils tend to be masked. Thus, the low-osmolality buffers will reduce the number of ciliated keratinocytes observed. This cellular swelling probably contributed to the misinterpretation that keratinocytic cilia are intracellular, especially in those cases where the ciliary shaft is short. In the biopsy from contact allergic dermatitis caused by nickel, the intercellular spaces were at least as prominent as in normal skin and there was no sign of artifactual swellings of epidermal cells; thus the absence of ciliary shafts cannot be attributed to technical failure. Second, serial sectioning is a prerequisite for the mapping of keratinocytes and the revealing of cilia. Since there is no preferred location of the cilium on the cell, the whole keratinocyte has to be analyzed. A keratinocyte may be found on 60 to 180 consecutive sections, and a cilium can in extreme cases appear on only 5 consecutive sections. Randomly chosen sections thus rarely contain any cilia. Third, the use of one-hole grids is another prerequisite. A comparative study of consecutive sections on net grids showed that the number of cilia discovered was low compared to that on one-hole grids, the reason being that grid bars totally or partly mask the cilium. Generally single cilia seem to have the same construction. The ciliary shaft usually has nine peripheral double tubules, lacking the central ones (the 9 + 0 pattern). It attaches deeply into the cell and protrudes from an extracellular channel surrounding the proximal part of the ciliary shaft. The axoneme loses its regular pattern close to the tip of the shaft. The basal body is sit-
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uated near the nucleus and in the neighborhood of a Golgi zone. The size and additional structures (basal feet, alar sheet, and rootlets) often conform with the results presented here. The basal body appears as a pair of cylinders similar to a centriole. The function of these cilia has been much discussed. It has been suggested that they are functionless evolutionary remnants, motile organelles, regulators of mitosis, and stimuli receptors. Considering the fact that they appear on every basal keratinocyte, which has to develop and maintain them with an expenditure of energy, it is difficult to believe that they are functionless vestiges. The 9 + 0 pattern of the tubules and the lack of dynein arms strongly suggest that they are nonmotile (Afzelius, 1979). The remaining two hypotheses seem to be more acceptable. Single cilia have been found to be less common in mitotic than in nonmitotic tissue (Fonte et al., 1971; Rash et al., 1969; Rieder et al., 1979; Wheatley, 1971). This observation has led to the suggestion that they may be involved in the regulation of mitosis. The keratinocytic single cilia should belong to nonmotile modified cilia. Cilia of this type have been found to function as photo-, chemo-, or mechanoreceptors (Afzelius, 1969; Altner and Prillinger, 1977; Barber, 1974). It is thus tempting to emphasize the hypothesis that the keratinocytic cilia can also have a sensory function. In this context it is of great interest that human epidermal melanocytes also possess single modified cilia (Elofsson et al., 1981). Thus, the possibility can be considered that the two epidermal ciliary systems are involved in the reception of UV light and regulation of pigmentation. This investigation has disclosed the presence in the basal keratinocytes of a ciliary apparatus which dedifferentiates during the progressive differentiation of the keratinocytes. The function of these single cilia remains to be elucidated. We are verygratefulto ProfessorBj~rnAfzelius,who generouslyshared his great knowledgeof cilia with us.
ELOFSSON ET AL.
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The work has been supported by grants from the Swedish Work Environment Fund and the Swedish Medical Science Research Council (00712). Note added in proof Recently (B. B. Boycott and J. M. Hopkins, 1984, J. Cell Sci. 66, 95-118) single cilia were reported to occur on all cells of five different neuron types in the cat and rabbit retina. This finding conforms with that of the keratinocytes of the present investigation as well as the age-dependent disappearance of cilia in a subpopulation of ganglion cells.
REFERENCES AFZELIUS, B. A. (1969) in LIMA-DE-FARIA,A. (Ed.), Handbook of Molecular Cytology: Ultrastructure of Cilia and Flagella, pp. 1219-1242, North-Holland, Amsterdam. AFZELIUS, B. A. (1979) Int. Rev. Exp. Pathol. 19, 143. ALTNER, H., AND PRILLINGER, L. (1977) Int. Rev. Cytol 67, 69-139. ANTON-LAMPRECI-IT,I. (1972) Arch. Dermatol. Res. 243, 199-208. BARBER, V. C. (1974) in SLEIGH, M. A. (Ed.), Cilia and Flagella: Cilia in Sense Organs, pp. 403-433, Academic Press, New York. BREATHNACH,A. S. (1971) An Arias of the Ultrastructure of Human Skin, Churchill, London.
DAROCZY,J., ANDFELDMANN,J. ( 1974) Zentralbl. Allg. PathoL Pathol. Anat. 118, 314-320. ELOFSSON, R., ANDERSSON, A., FALCK, B., AND SJOBORG,S. (1981) Acta Derm. Venereol. Suppl. 99, 4952. FALCK, B., ANDERSSON, A., ELOFSSON, R., AND SJOBORG, S. (1981)Acta Derm. Venereol. SuppL 99, 327. FLOOD, P. R., AND TOTLAND,G. K. (1977) Cell Tissue Res. 183, 281-290. FONTE, V. (~., SEARLS,R. L., AND HILFER, S. R. (1971) J. Cell Biol. 49, 226-229. KARNOVSKY,M. J. (1965)J, Cell Biol. 27, 270A. NIKAI, H., ROSE, G. G., AND CATTONI, M. (1970) J. Dent. Res. 49, 1141-1153. RASH, J. E., SHAY, J. W., AND BIESELE,J. J. (1969) J. Ultrastruct. Res. 29, 470-484. RIEDER, C. L., JENSEN, C. (]., AND JENSEN, L. C. W. (1979) J. Ultrastruct. Res. 68, 173-185. SCHERF'F,J. P., AND DAEMS, W. Z. (1967) J. Ultrastruct. Res. 19, 546-555. WHEATLEY, D. N. (1971) J. Anat. 110, 367-382. WILSMAN, N. J. (1978) J. Ultrastruct. Res. 64, 270282. WILSMAN, N. J., AND FLETCHER, T. F. (1978) Anat. Rec. 190, 871-890. WILSON, R. B., AND McWHORTER, C. A. (1963) Lab. Invest. 12, 242-249.
(1984)
The Ciliated Human Keratinocyte R O L F ELOFSSON,* ANDERS ANDERSSON,t BENGT FALCK, t AND STEINAR SJOBORG~
Departments of*Zoology, tHistology, and ~.Dermatology, University of Lund, Lund, Sweden Received May 18, 1984 H u m a n keratinocytes were investigated for the presence of single cilia. Almost all basal keratinocytes were found to carry a single cilium in normal, occluded, and psoriatic skin. The ciliary structure was progressively reduced in keratinocytes approaching the surface. No remnants of the ciliary apparatus were found in the gramdar layer. In one case of nickel-allergic dermatitis (patch test), the keratinocytes had lost their cilia; the significance of this surprising finding remains to be elucidated.
© 1984 Academic Press, Inc.
Recently melanocytes in the human epi- acetate. One specimen obtained from normal skin was dermis were found to carry one Cilium each subjected to fixation according to Karnovsky (1965) (Elofsson et al., 1981). Single cilia occur with a buffer osmolality of 100 mosmole/kg and a total osmolality of 1810 mosmole/kg. After prefixation the scattered in many cell populations of the specimen was treated as above, Sections were cut on body (Afzelius, 1979; Flood and Totland, a diamond knife and were examined in a Zeiss EM 10 1977; Scherft and Daems, 1967; Wilsman, and a JEOL 100 CX. Consecutive sections were placed on one-hole grids 1978). Their presence on every cell of a coated with Formvar and strengthened with carbon whole-cell system, such as the melanocytes coating. Ten or twenty grids were produced from each of the epidermis, has so far been claimed biopsy. Each grid carried 20 sections of less than 100only for a few cell types (Flood and Totland, rim thickness each. They measured 150-250 #m in 1977; Wilsman and Fletcher, 1978). This length and comprised the whole epidermis. The analwork reports the regular appearance of sin- yses were made on 10 to 20 keratinocytes at a time and the cells were mapped and followed throughout gle cilia in the keratinocyte population. their whole soma. One or two such analyses were done on each series of sections.
MATERIALS AND METHODS Punch biopsies (3 mm) from adult h u m a n skin were obtained using local anesthesia (Xylocain 1%): clinicaUy normal skin from four persons, one specimen from a positive patch test reaction to nickel and one specimen from psoriatic skin. Two specimens were obtained after occluding normal skin by aluminum foil for 48 hr. The biopsies were trimmed from excess dermis immediately upon immersion in the fixative and were divided into two or three portions. With one exception, all specimens were processed in the same way. The prefixative used was 2.5% glutaraldehyde in 0.15 M cacodylate buffer (pH 7.3). The buffer osmolality was 300 and the total osmolality of the fixative was 555 mosmole/kg. The reason for using this fixative has been discussed elsewhere (Falck et aL, 1981). Prefixation lasted 2 hr and the postfixation in 2% OsO4 in the above buffer was performed for 1-2 hr. The specimens were dehydrated in ethanol starting at 50% and stained en bloc by 1% phosphotungstic acid and 0.5% uranyl acetate. Additional staining was done on the sections with 0.5% lead citrate and 1% uranyl
RESULTS
Normal skin. The keratinocytes of the basal layer were found, with rare exceptions (see below), to possess one cilium each (Fig. 1). The cilium was usually situated anywhere on the upper half of the keratinocyte. It could also be inserted deeper down on the cell surface but rarely below the nucleus. The ciliary shaft was always located in a channel continuous with the intercellular space, which invaginated deep into the ceil. The channel rarely followed a straight line to the nearest cell surface but rather ran a longer distance, sometimes with a curvature (Figs. 2-4), inside the cell before emerging (Fig. 4). The cilium could point in any direction in the epidermis.
212 0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All fights of reproduction in any form reserved.
CILIATED HUMAN KERATINOCYTE
F~G. 1. A schematic drawing of the human epidermis comprising the basal, spinous, and granular layers. The drawing illustrates the well-developed cilium of the keratinocytes in the basal layer and the gradual disappearance of the cilium in the spinous layer. The cilia varied in length within one specimen. The longest cilium measured ca. 3 # m and poked into a neighboring keratinocyte for m o r e than 1 /zm, ending close to the nucleus (Figs. 5-6). A cilium could be confined within the basal keratinocyte, but usually the cilia extended into the intercellular
213
space o f the epidermis without coming into contact with other cells. The whole ciliary apparatus consisted o f a basal b o d y similar to a centriole pair and a finger-like extension from the cell surface, also called the ciliary shaft, containing the axoneme. The a x o n e m e o f the cilia had nine tubular duplets and no central tubuli but lost this regular arrangement toward the tip o f the cilium (Fig. 13). N o dynein arms were observed. The basal b o d y was usually situated close to the nucleus and in the n e i g h b o r h o o d o f a Golgi apparatus. It consisted o f one pair o f cylinders, with each cylinder f o r m e d by nine triplets o f tubuli approximately 230 n m in diameter and 350 n m in length. The two cylinders could have any direction within the cell and no preferred direction was found. The cylinders were sometimes oriented perpendicular to one another but other directions were c o m m o n . A n o t h e r variation occurred in the distance between them. The cylinders were usually very close (0.1 #m) but could be several micrometers apart. The basal b o d y carrying the ciliary shaft normally had basal feet. These appeared as five cones encircling the middle o f the cylinder, with their bases toward the tubuli. Only on two occasions did thin, feebly developed rootlets occur, very similar to those observed by W i l s o n and McW h o r t e r (1963) and D a r 6 c z y and F e l d m a n n (1974). Alar sheets were present which connected the distal tip o f the basal body, carrying the cilium, with the cell m e m b r a n e . Two basal keratinocytes deviated in some
FIGS. 2-4. Normal skin. Three sections from a series through a cilium in a basal keratinocyte. The ciliar shaft points toward the nucleus (Fig. 2), bends inside the cell (Fig. 3), and emerges in the intercellular space (Fig. 4). Scale bars = 0.5 fzm. FIG. 5. Normal skin. Survey of a cilium in a basal keratinocyte traversing the intercellular space and pointing into an adjacent keratinocyte (arrows). Epidermal surface to the left. Scale bar = 1 #m. FIG. 6. Detail of Fig. 5 from another section. Scale bar = 0.5/~m. FIG. 7. Normal skin. Two parallel basal bodies situated apically in a basal keratinocyte are shown. Scale bar = 0.5/zm. FIG. 8. Two sections away from that of Fig. 7, the two cilia appear in the extracellular channel formed inside the cell. Scale bar = 0.5 ~zm.
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FIG. 14. Psoriatic skin. T h e figure s h o w s two adjacent basal keratinocytes carrying one cilium each (arrows). Scale b a r = 1 /.tin.
respects. One was found carrying only the basal body and no ciliary shaft. The other carried two ciliary shafts lying contiguous to one another and having the two cylinders of the basal body parallel to one another (Figs. 7-8). The keratinocytes in the stratum spinosum also possessed cilia, however, reduced compared to those found in the basal layer. The reduction was gradual, increasing toward the surface. Short ciliary shafts usually confined to the small extracellular channel invaginating into the cell were found in the keratinocytes immediately above the basal
layer (Fig. 9). In the keratinocytes situated in the middle layers of the stratum spinosum and below the stratum granulosum, the ciliary shafts had disappeared. Remnants of them were found in the small vesicle connected to one of the cylinders of the basal body (Figs. 10-11). This vesicle is probably a remnant of the invaginated intercellular space housing the ciliary shaft in the more basally located keratinocytes. The ciliary apparatus and/or the remnants of it were found in the upper part of all keratinocytes above the basal layer. The basal bodies were always situated close to the nucleus. Their
FIG. 9. N o r m a l skin. T h e figure s h o w s a reduced cilium confined inside the keratinocyte, w h i c h is situated in the suprabasal layer. Scale b a r = 0.5 urn. FIG. 10. N o r m a l skin. Detail f r o m a kerafinocyte in the u p p e r part o f the s t r a t u m s p i n o s u m . T h e basal b o d y a n d the small r e m n a n t o f the extraceUular space are seen as a small vesicle attached to the basal b o d y (arrow). Scale b a r = 0.5 # m . FIG. 11. F o u r sections away f r o m that o f Fig. 10, the vesicle a n d the tiny rest o f the cilium (arrow) are shown. E p i d e r m a l surface to the left. Scale b a r = 0.5 ~zm. FIG. 12. N o r m a l skin fixed in a low-osmolality solution (Karnovsky). Arrows p o i n t at a cilium in a basal keratinocyte. Note the obliterated extracellular space. Scale b a r = 0.5 urn. FIG. 13. Skin as in Fig. 12 showing a cross section o f a cilium (arrow) inside a keratinocyte. T h e extracellular i n v a g i n a t e d c h a n n e l s u r r o u n d i n g t h e ciliary shaft is obliterated. Scale b a r = 0.5 urn.
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cylinders were several micrometers apart, with the distance increasing even more just beneath the granular layer. No traces of the ciliary apparatus were found in the granular layer. Occluded and psoriatic skin. In psoriatic skin both rete ridges and suprapapillary epidermal areas were scrutinized for the presence of cilia. As in normal skin, cilia displaying different degrees of differentiation were found within the keratinocytes (Fig. 14). However, a few of the basal keratinocytes lacked a ciliary shaft but had the basal body. Skin fixed in low-osmolality buffer (Karnovsky). It has recently been demonstrated that, in a fixative consisting of 2.5% glutaraldehyde dissolved in cacodylate buffer having an osmolality above 230 mosmole/ kg, the epidermal intercellular spaces are prominent (Falck et al., 1981). This was also the case with all biopsies in this study except in the one fixed in Karnovsky solution. This fixative produced closely packed keratinocytes with obliterated intercellular space as do glutaraldehyde-based fixatives having a cacodylate buffer osmolality well below 230 mosmole/kg (Falck et aL, 1981). After Karnovsky fixation the cilia were difficult to find among the contiguous cells of the epidermis, and the number of basal keratinocytes observed to carry a cilium was much lower than that reported above. The cilia displayed the same features as described above and were even seen to protrude into the extremely narrow intercellular space (Figs. 12-13). Nickel contact allergy. In the epidermis of a positive patch test reaction to nickel, no ciliary apparatuses or remnants of them were observed in the keratinocytes. A structure similar to a basal body or a centriole was situated in the basal half of the keratinocytes. DISCUSSION
Scattered cells having one cilium are found in most tissues of the body (see Afzelius, 1979; Flood and Totland, 1977; Scherft and
Daems, 1967; Wilsman, 1978), whereas all chondrocytes from articular cartilage of neonatal dog and epithelial cells along the nephron and collecting ducts of the adult mouse have been reported to carry a cilium (Wilsman and Fletcher, 1978; Flood and Totland, 1977). Such cilia are referred to in different terms, e.g., single, solitary, primary, isolated, residual, vestigial, or immature cilia. The first ultrastructural study of single cilia in human keratinocytes was made by Wilson and McWhorter (1963). They found cilia (flagella) in some basal keratinocytes in normal epidermis and in basal cell carcinoma. Anton-Lamprecht (1972) described cilia as intracellular structures in some basal keratinocytes of normal epidermis as well as in three patients with KleinWaardenburg Syndrome. Dar6czy and Feldmann (1974) reported the presence of cilia on some keratinocytes in a number of pathological situations. More keratinocytes carried single cilia in spinocellular carcinoma and bullous skin diseases after UV treatment. Single cilia have also been observed in some cells of fetal skin and human gingiva (Breathnach, 1971; Nikai et aL, 1970). In contrast to these observations on human keratinocytes the present study has revealed a regular occurrence of single cilia on practically all basal keratinocytes. In addition it has been disclosed that the ciliary apparatus gradually disappears with the progressive differentiation of the keratinocytes with no remnant of it being found in the granular layer. This finding could be obtained by taking advantage of some technical precautions which greatly facilitate the study of this organelle. First, the ~hoice of fixating solution is important. By varying the buffer osmolality the dimensions of the epidermal intercellular space can be changed within a broad range (Falck et aL, 1981). A buffer osmolality of 300 mosmole/kg produces wide intercellular spaces and makes the mapping of the individual keratinocyte in serial sections and tracing of ciliary shafts easy.
CILIATEDHUMAN KERATINOCYTE Moreover, the invaginating channels harboring these shafts are well preserved. With an almost obliterated intercellular space, as produced by the Karnovsky fixative, the keratinocyte mapping is very difficult. As a consequence of the cellular swelling an important landmark, i.e., the invaginated channel containing the ciliary shaft, is eradicated. Also the ciliary shafts protruding in between the ceils tend to be masked. Thus, the low-osmolality buffers will reduce the number of ciliated keratinocytes observed. This cellular swelling probably contributed to the misinterpretation that keratinocytic cilia are intracellular, especially in those cases where the ciliary shaft is short. In the biopsy from contact allergic dermatitis caused by nickel, the intercellular spaces were at least as prominent as in normal skin and there was no sign of artifactual swellings of epidermal cells; thus the absence of ciliary shafts cannot be attributed to technical failure. Second, serial sectioning is a prerequisite for the mapping of keratinocytes and the revealing of cilia. Since there is no preferred location of the cilium on the cell, the whole keratinocyte has to be analyzed. A keratinocyte may be found on 60 to 180 consecutive sections, and a cilium can in extreme cases appear on only 5 consecutive sections. Randomly chosen sections thus rarely contain any cilia. Third, the use of one-hole grids is another prerequisite. A comparative study of consecutive sections on net grids showed that the number of cilia discovered was low compared to that on one-hole grids, the reason being that grid bars totally or partly mask the cilium. Generally single cilia seem to have the same construction. The ciliary shaft usually has nine peripheral double tubules, lacking the central ones (the 9 + 0 pattern). It attaches deeply into the cell and protrudes from an extracellular channel surrounding the proximal part of the ciliary shaft. The axoneme loses its regular pattern close to the tip of the shaft. The basal body is sit-
219
uated near the nucleus and in the neighborhood of a Golgi zone. The size and additional structures (basal feet, alar sheet, and rootlets) often conform with the results presented here. The basal body appears as a pair of cylinders similar to a centriole. The function of these cilia has been much discussed. It has been suggested that they are functionless evolutionary remnants, motile organelles, regulators of mitosis, and stimuli receptors. Considering the fact that they appear on every basal keratinocyte, which has to develop and maintain them with an expenditure of energy, it is difficult to believe that they are functionless vestiges. The 9 + 0 pattern of the tubules and the lack of dynein arms strongly suggest that they are nonmotile (Afzelius, 1979). The remaining two hypotheses seem to be more acceptable. Single cilia have been found to be less common in mitotic than in nonmitotic tissue (Fonte et al., 1971; Rash et al., 1969; Rieder et al., 1979; Wheatley, 1971). This observation has led to the suggestion that they may be involved in the regulation of mitosis. The keratinocytic single cilia should belong to nonmotile modified cilia. Cilia of this type have been found to function as photo-, chemo-, or mechanoreceptors (Afzelius, 1969; Altner and Prillinger, 1977; Barber, 1974). It is thus tempting to emphasize the hypothesis that the keratinocytic cilia can also have a sensory function. In this context it is of great interest that human epidermal melanocytes also possess single modified cilia (Elofsson et al., 1981). Thus, the possibility can be considered that the two epidermal ciliary systems are involved in the reception of UV light and regulation of pigmentation. This investigation has disclosed the presence in the basal keratinocytes of a ciliary apparatus which dedifferentiates during the progressive differentiation of the keratinocytes. The function of these single cilia remains to be elucidated. We are verygratefulto ProfessorBj~rnAfzelius,who generouslyshared his great knowledgeof cilia with us.
ELOFSSON ET AL.
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The work has been supported by grants from the Swedish Work Environment Fund and the Swedish Medical Science Research Council (00712). Note added in proof Recently (B. B. Boycott and J. M. Hopkins, 1984, J. Cell Sci. 66, 95-118) single cilia were reported to occur on all cells of five different neuron types in the cat and rabbit retina. This finding conforms with that of the keratinocytes of the present investigation as well as the age-dependent disappearance of cilia in a subpopulation of ganglion cells.
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DAROCZY,J., ANDFELDMANN,J. ( 1974) Zentralbl. Allg. PathoL Pathol. Anat. 118, 314-320. ELOFSSON, R., ANDERSSON, A., FALCK, B., AND SJOBORG,S. (1981) Acta Derm. Venereol. Suppl. 99, 4952. FALCK, B., ANDERSSON, A., ELOFSSON, R., AND SJOBORG, S. (1981)Acta Derm. Venereol. SuppL 99, 327. FLOOD, P. R., AND TOTLAND,G. K. (1977) Cell Tissue Res. 183, 281-290. FONTE, V. (~., SEARLS,R. L., AND HILFER, S. R. (1971) J. Cell Biol. 49, 226-229. KARNOVSKY,M. J. (1965)J, Cell Biol. 27, 270A. NIKAI, H., ROSE, G. G., AND CATTONI, M. (1970) J. Dent. Res. 49, 1141-1153. RASH, J. E., SHAY, J. W., AND BIESELE,J. J. (1969) J. Ultrastruct. Res. 29, 470-484. RIEDER, C. L., JENSEN, C. (]., AND JENSEN, L. C. W. (1979) J. Ultrastruct. Res. 68, 173-185. SCHERF'F,J. P., AND DAEMS, W. Z. (1967) J. Ultrastruct. Res. 19, 546-555. WHEATLEY, D. N. (1971) J. Anat. 110, 367-382. WILSMAN, N. J. (1978) J. Ultrastruct. Res. 64, 270282. WILSMAN, N. J., AND FLETCHER, T. F. (1978) Anat. Rec. 190, 871-890. WILSON, R. B., AND McWHORTER, C. A. (1963) Lab. Invest. 12, 242-249.