Immunohistochemical study of carbonic anhydrase isozymes in human skin

ARTICLE

Auteur(s) : Antonio MASTROLORENZO¹, Giuliano ZUCCATI¹, Daniela MASSI², Maria G GABRIELLI³, Angela CASINI⁴, Andrea SCOZZAFAVA⁴, Claudiu T SUPURAN⁴

¹ Dipartimento di Scienze Dermatologiche, Università degli Studi di Firenze, via Degli Alfani, 37, 50121 Firenze, Italy. ² Dipartimento Patologia Umana e Oncologia, Università degli Studi di Firenze, Viale Morgagni 85, 50134 Firenze, Italy. ³ Dipartimento di Scienze Biochimiche e Morfologiche Comparate, Università degli Studi di Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy. ⁴ Università degli Studi, Polo Scientifico, Lab. Chimica Bioinorganica, Rm 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy

Article accepted on 26\05\2003

Carbonic anhydrases (CAs) are ubiquitous zinc proteins, present in archaeobacteria, prokaryotes and eukaryotes. In these organisms, they are encoded by three distinct, evolutionarily unrelated gene families: the α‐CA (mainly in vertebrates, eubacteria, algae and cytoplasm of green plants), the β‐CA (predominantly in eubacteria, algae and chloroplasts of both mono‐ as well as dicotyledons) and γ‐CA (in archaeobacteria and some eubacteria) families, respectively [1‐5]. To date, fourteen different carbonic anhydrase isoforms (CAs) have been described in higher vertebrates including humans (Table I) [2‐18]. CAs have been found in all tissues and are involved in crucial physiological processes because they are able to catalyse the hydration of CO2 to bicarbonate at the physiological pH [7, 8]. Bicarbonate is the substrate for several carboxylation steps in a number of fundamental metabolic pathways such as gluconeogenesis, biosynthesis of several amino acids, lipogenesis, ureagenesis and pyrimidine synthesis [19]. In addition to these biosynthetic reactions, some of the CAs are involved in other physiological processes connected with respiration and transportation of CO2\bicarbonate between metabolising tissues and the lungs, pH homeostasis and electrolyte secretion in a variety of tissues [7, 8].Table 1. Higher vertebrate α‐CA isozymes. A summary of expression in normal skin and oro‐genital tissues

Materials and Methods

The skin specimens which were used for immunohistochemical analyses were collected from either biopsied or excised materials from 10 patients. Informed consent was obtained from each patient. Skin samples from patients older than 60 and younger than 20 were excluded.

Antibodies

Sheep polyclonal antibodies to human CA I and CA II, purified from human erythrocytes, were purchased from The Binding Site (Birmingham, UK). The anti‐CA I and anti‐CA II were used diluted 1:300 and 1:500, respectively. Monoclonal antibody (M75) recognizing the N‐terminal domain of MN\CA IX protein was obtained by Dr. Silvia Pastoreková (Slovak Academy of Sciences; Bratislava, Slovak Republic) [27]. It was used diluted 1:100. Biotinylated rabbit anti‐sheep IgG or biotinylated goat anti‐mouse IgG (Vector Laboratories, Burlingame, CA, USA) were used as secondary antibodies.

Tissue preparation

The fresh human specimens were fixed in Carnoy‘s fluid for 6 h at 4°C. The samples were then dehydrated and embedded in paraffin wax in a vacuum oven at 68°C. For immunohistochemistry, 5 microscope slides were prepared from each specimen, each one containing 6 sections of 5 µm each.

Immunohistochemistry

CA isozymes CA I, CA II, and CA IX were visualized using the avidin‐biotin peroxidase complex (ABC) method [28]. The ABC method was performed with reagents purchased in kit form (Vectastain ABC kit; Vector Lab.). Paraffin sections were rehydrated and immersed in 0.3% H2O2 in methanol for 30 min to inactivate endogenous peroxidase activity. Blocking of endogenous avidin‐binding activity was performed using an avidin‐biotin blocking kit (Vector Lab.). The slides were then washed three times (5 min each) in 0.05 M phosphate‐buffered saline (PBS), pH 7.6 and pre‐incubated for 20 min in appropriate non‐immune serum diluted 1:5 with 1% bovine serum albumin (BSA; Sigma Chemical Co., St.Louis, MO, USA) in PBS. Thereafter, the sections were treated with primary antibody overnight at room temperature. After three rinses in PBS, biotinylated secondary antibody (1:200) was applied for 45 min, followed by further washing in PBS and by incubation with the ABC reagent for 30 min. The localization of peroxidase was detected using substrate 3‐3‘diaminobenzidine (DAB\substrate kit; Vector Lab.). The sections were counterstained with haematoxylin or methylene blue. The negative control consisted of substituting the primary antibody with PBS.

Results

Both CA I and CA II were focally expressed in the basal and spinous layers of the skin. In particular, a moderate staining was predominantly observed in the cytoplasm, basolateral and apical plasma membrane of the prickle cells (Fig. 1A, B). The basal cells displayed moderate CA positivity in their cytoplasm and in the apical and lateral membranes. However, the immunoreaction within the basal cell layer facing the basal lamina was difficult to evaluate due to the presence of melanin pigment. No staining was found in the granular and horny layer. Keratinocytes forming the intraepidermal spiraled portion of the eccrine duct (i.e., the acrosyringium) showed a moderate positivity (Fig. 1C). CA I and II positivity was the same in eccrine and apocrine sweat units that showed a similar pattern of staining, although in some sections staining for CA II was stronger and more homogeneous as compared with CA I. In particular, the secretory portion composed of coiled secretory glands was homogeneously and strongly stained while the ductal epithelium appeared more weakly stained (Fig. 2). In most capillaries the endothelial cells were strongly stained especially towards their luminal surface (Fig. 3). Cells of the outer root sheath surrounding the hair shaft were stained (not shown), while no staining was found in the sebaceous glands. As regards to CA IX in the normal skin, all epithelial cells, capillaries, sweat and sebaceous glands were negative. A weak staining was found on the plasma membrane of the outer root sheath cells in hair follicles (not shown).

Discussion

CAs have been shown to be present in the skin of various mammals, humans included, but the isozymes have not been identified [15, 18, 20‐26]. To achieve a more comprehensive view of CA isozymes represented in human skin we examined the expression of three isozymes CA I, CA II and CA IX in specimens of normal human skin. The data presented here clearly show that in the epidermis the CA I and II positivity is present in the stratum spinosum where the cytoplasm and all membranes of the cells were stained and in the basal layer with a staining of the cytoplasm, apical and lateral membranes. To our knowledge this is the first comprehensive analysis of the cutaneous expression of these isozymes. Our data also provide additional evidence to the previously reported demonstration of a CA positivity in normal skin by Hansson [25] and Eichhorn et al. [26] although in those studies the CA was evaluated by histochemical techniques and not by means of immunohistochemistry. We found a detectable staining of the endothelial cells of the capillaries in the papillary dermis in contrast with their results, and a similar pattern of immunoreactivity within sweat glands. In general, the immunohistochemical distribution of CA I and II was similar in all skin specimens studied, with CA II being more intense than CA I in the eccrine and apocrine sweat glands. These findings are in agreement with previous immunohistochemical investigation concerning CA localization in sweat glands [16, 17, 20, 21]. In evident disaccordance are our negative results regarding the sebaceous glands [16, 17, 20]. This discrepancy should benefit from our attention in future investigations.

Recent immunohistochemical studies [24] have demonstrated the presence of CA IX in basal cells in and nearby the infudibulum and medulla of hair follicles whereas the basal cells of the epidermis and the sweat glands were negative. In line with those observations, in our investigation all epithelial cells of the normal skin and all endothelial cells of capillaries and sweat glands remained totally unstained to CA IX. A faint but always present staining was identified in the cells surrounding the hair shaft.

Even though the biological function of these cytoplasmic and cell ‐ surface CA isozymes in the skin is not completely known at present, it is possible to speculate the biological significance of these findings by analysing the specific location of CAs I and II in the active sites. Both isozymes are expressed in the cytoplasm and the basolateral membranes of the epithelial cells of the spinosum and basal layers (where CA IX is negative) suggesting a potential involvement in the physiological cell proliferation and adhesion. Earlier studies focusing on CAs I and II revealed no clear relationship with tumorigenesis [29]. The immunohistochemical observation of those isozymes on the luminal aspect of endothelial cells of the capillaries and sweat glands may be related to the complex phenomena of macromolecular secretion into luminal structures or bicarbonate and other ion exchange, as well documented by other investigators in eccrine sweat and large salivary glands [16, 18, 20‐23].

In striking contrast to CAs I and II expression and in accordance with Ivanov et al. [24] CA IX was not expressed in normal human skin. Recent studies [30] have shown distinct expression of CA IX protein in cervical displasia and carcinoma. This could suggest that CA IX expression is more restricted to abnormal cell growth thus strengthening the hypothesis of its role in neoplastic cell proliferation and, possibly, in malignant transformation [31, 32, 34, 35, 37]. However, its function in reactive, hyperplastic processes as well as intercellular interactions and cell adhesion in different organs other than the skin, cannot be completely ruled out [9, 33, 36].

On the basis of the above considerations and current data we suggest that comparative analysis for the expression and interplay of these CA isozymes on normal human skin, skin diseases and skin malignancies will be required to better understand their role on epithelial cell regulation, proliferation and neoplastic transformation. Acknowledgements. 

We are grateful to Dr. Silvia Pastoreková (Slovak Academy of Sciences; Bratislava, Slovak Republic) for providing the Monoclonal antibody (M75) recognizing the N‐terminal domain of MN\CA IX protein.

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