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Correspondence to: Raija Tammi, Dept. of Anatomy, U. of Kuopio, PO Box 1627, FIN-70210 Kuopio, Finland.
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Summary |
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We used immunogold staining and stereology to examine the ultrastructural localization and to estimate the relative content of CD44 in different strata and cell types of normal human epidermis. We found that CD44 existed almost exclusively on the plasma membranes; only rare labeling occurred on vesicular structures within the cytoplasm. Quantitation of the immunogold particles indicated that the labeling density of melanocytes corresponded to that of basal keratinocytes, and Langerhans cells displayed a labeling density of 10% that of the surrounding spinous cells. Among keratinocyte strata, the highest labeling density occurred on spinous cells, suggesting upregulation of CD44 after detachment from the basement membrane. The plasma membrane distribution of CD44 was compartmentalized, with little signal on cellcell and cellsubstratum contact sites such as desmosomes, the plasma membrane domain facing the basement membrane, and the close apposition of terminally differentiating granular cells. In contrast, CD44 was abundant on plasma membrane domains facing an open intercellular space, rich in hyaluronan. This distribution is in line with a role of CD44 as a hyaluronan receptor, important in the maintenance of the intercellular space for nutritional and cell motility functions in stratified epithelia. (J Histochem Cytochem 46:241248, 1998)
Key Words: CD44, electron microscopy, quantitation, human epidermis
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Introduction |
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CD44 (H-CAM) REPRESENTS a family of closely related plasma membrane glycoproteins involved in cellmatrix and cellcell adhesion and cell movements, and is therefore important in morphogenesis, wound healing, tumor invasion, and metastasis, as reviewed by
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Materials and Methods |
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Skin Samples and CD44 Immunostaining
Approval for obtaining skin samples for the study was obtained from the Ethical Committee of Kuopio University Hospital. Skin samples were obtained either from mastectomy surgery or from the volar surface of the arm in volunteers. The specimens were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 4 hr at 4C and were washed with the buffer. The samples were immersed in 2.3 M sucrose for 30 min and then frozen in liquid propane using a Reichert Jung KF 80 freezing instrument (Vienna, Austria). Thin sections with pale gold interference were cut using a Reichert Jung Ultracut E microtome equipped with a cryoset (FC4E) and were collected on Formvar-coated 200-mesh copper grids. Thick sections (1 µm) for light microscopy were cut and mounted on Organosilane-coated slides.
Immunostaining for CD44 of the thick sections, using the monoclonal antibody (Mab) Hermes-3 (a generous gift from Dr. Sirpa Jalkanen; University of Turku, Turku, Finland) was done as previously described (
Hyaluronan Staining
Skin samples were fixed in 2% paraformaldehyde and 2% glutaraldehyde in phosphate buffer and were embedded in paraffin (
For electron microscopic demonstration of hyaluronan, a pre-embedding staining protocol was used. The epidermis was separated from the dermal connective tissue using a 30-min incubation in 0.02 % EDTA in PBS (Sigma) at 37C (
Morphometry and CD44 Distribution
The sections were viewed and photographed with a JEOL 1200 EX electron microscope. Three photographs (original magnification x12,000) were systematically taken from each of the following strata of the epidermis: basal cells (basal surface and lateral surface), lower spinous cell layer, upper spinous cell layer, and granular cell layer (
The boundary length of the plasma membrane (Bpl) was estimated as described by White and co-workers (1982). The Bpl can be estimated from intersections through feature profiles (I) made by the test lines of spacing (h). The appropriate relation with the parallel line set is:
In addition to estimating the total boundary length of the plasma membrane, we estimated the boundary length of the plasma membrane belonging to villous processes and the planar cell surface and that in the desmosomes. Villous processes were defined as projections of plasma membrane clearly extending from the cell body (at least 40 nm).
The distribution of CD44 was determined by counting the number of gold particles (n) associated with the plasma membrane and distributing the counts to the different categories. The numbers of gold particles were expressed per boundary length of the plasma membrane (Bpl).
Statistics
The values given are means ± SE. The statistical significances of the differences between epidermal strata were tested using two-way analysis of variance and those between n/desmosomes and n/plasma membrane using three-way analysis of variance of the random effects of patients and fixed effects of epidermal strata and plasma membrane compartments. The means were compared using the simultaneous confidence limits test procedure of NewmanKeuls (
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Results |
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Light Microscopy
Epidermal cell surfaces were intensely stained with MAb Hermes 3 against an epitope close to the N-terminus of CD44 and common to all splice variants (Figure 1A). Immunoreactivity for CD44 was present on the lateral and apical surfaces of the basal cells, whereas the basal surface was negative (Figure 1A). The spinous cells displayed CD44 on all surfaces. The staining pattern typically had a somewhat punctate appearance (Figure 1A). The positive reaction continued up to the lower granular cell layer (Figure 1A), whereas the upper granular cells and cornified cells were always negative (Figure 1A).
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Electron Microscopy
Ultracryosections of human skin were immunogold-stained using the same antibody as for light microscopy, resulting in a general tissue distribution corresponding to that observed in light microscopy (Figure 1 and Figure 2). As suggested by light microscopy, most of the gold particles were detected in close vicinity to the plasma membranes (Figure 1 and Figure 2). Very few particles were present on structures such as mitochondria or nuclei, confirming the specificity of the staining (Figure 1 and Figure 2). Rarely, positive labeling was found in association with vesicular structures within the cells (Figure 2B). Although most gold particles were associated with the microvilli, some label was also found on the actual keratinocyte cell body (Figure 1C, Figure 1E, Figure 1F, Figure 2A, and Figure 2B). Desmosomes were almost devoid of CD44 (Figure 1E, Figure 1F, Figure 2A, and Figure 2B), probably explaining the punctate staining pattern seen in light microscopy (Figure 1A). In many cases the opposing villi were closely apposed to each other on one surface while the other side faced an open space (Figure 1E, Figure 1F, and Figure 2B). The signal for CD44 was then preferentially found on the side facing the open space (Figure 1E, Figure 1F, and Figure 2B). The basal surface of the basal cells and the hemidesmosomes were almost negative (Figure 1C).
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Epidermal melanocytes showed intense staining with MAb Hermes 3 (Figure 2A). In particular, the processes of the melanocytes were heavily labeled (Figure 1C and Figure 2A). The melanosomes inside the epidermal cells were negative (Figure 2A). The basal surface of the melanocytes (towards the basal lamina) was negative, like that of the keratinocytes (not shown). The plasma membrane of Langerhans cells was generally less intensely stained than that of keratinocytes (Figure 2B).
The control specimens, stained either without a primary antibody (not shown) or using a nonrelated MAb (Figure 1B), exhibited very low labeling.
Morphometric Analysis of the Immunogold Labeling
Table 1 summarizes the numbers of gold particles per plasma membrane boundary length (Ng/pl) in nonkeratinocytes and keratinocytes in different epidermal strata. The Ng/pl was highest in the lower spinous cell layer. The values in the upper spinous cell layer and lateral surfaces of the basal layer were slightly lower than that of the lower spinous cells, whereas the Ng/pl on granular cells and basal surface of the basal cells was low. ANOVA showed that there was a statistically significant difference in the number of gold particles among the different strata (p<0.05). The differences between the lateral surfaces of the basal layer, the lower spinous cell layer, and the upper spinous cell layer were not statistically significant, whereas they all differed significantly from those of the basal surface of the basal cells and the granular cell layer.
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Approximately 72% of all gold particles were located on the villous processes. However, when the numbers of gold particles were calculated per plasma membrane boundary length, there was no difference in the labeling density between the villi and cell body (data not shown). The labeling density of the desmosomes was, however, significantly lower than the rest of the plasma membrane in all epidermal layers.
Melanocytes showed about the same Ng/pl as the lateral side of basal keratinocytes, whereas Langerhans cells had an Ng/pl level about 10% of that in the surrounding spinous cells (Table 1).
Hyaluronan Affinity Staining
The biotinylated high-affinity probe for hyaluronan produced a strong extracellular signal thoughout the vital layers of the epidermis in paraffin-embedded sections (Figure 3A). Hyaluronan was present up to the granular layer, like CD44, whereas the stratum corneum was negative. A similar pattern was obtained with the pre-embedding protocol for EM. Figure 3B, from the spinous cell layer, shows the coalescence of multiple 1-nm gold particles by silver enhancement, demonstrating the presence of hyaluronan in the pouches lined with CD44-rich membrane areas. Control samples stained with a probe pretreated with hyaluronan oligosaccharides showed no signal (data not shown).
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Discussion |
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Ultrastructural localization of CD44 has previously been performed on synovial (
The volume of a keratinocyte increases by 50% when it moves from the basal to the spinous layer (Tammi and Santti 1989), and plasma membrane surface area per cell volume increases by about 40% (
The heavy labeling of melanocyte cell processes, also found in melanocyte cultures (
The enrichment of CD44 on the plasma membranes of microvilli lining the hyaluronan-stuffed pouches conforms well with the concept that CD44 acts as an anchor for hyaluronan (
CD44 acts as an endocytosis receptor for lysosomal degradation of hyaluronan in macrophages and chondrocytes (
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Acknowledgments |
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The skillful technical assistance of Ms Eija Voutilainen, Mr Kari Kotikumpu, Ms Raija Pitkänen, and Ms Eija Antikainen is gratefully acknowledged.
Received for publication February 14, 1997; accepted August 5, 1997.
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