Immunohistochemical Analysis of Cytokeratin Expression in Dog Skin

FULL PAPER Internal Medicine

Mihoko KOZAKI1), Yuka NAKAMURA2,3)*, Midori IGUCHI1), Rui KANO1), Shinichi WATANABE3), Kosaku FUJIWARA1) and Atsuhiko HASEGAWA1)

1)Departments of Pathobiology,2)Veterinary Internal Medicine, Nihon University, School of Veterinary Medicine, 1866, Kameino, Fujisawa, Kanagawa 252–8510 and 3)Department of Dermatology, Teikyo University School of Medicine, 11–1 Kaga-2, Itabashi, Tokyo 173–8605, Japan

(Received 23 February 2000/Accepted 5 September 2000)

ABSTRACT. The expression of cytokeratins and involucrin was analyzed to identify the skin cells which compose the epidermis of dogs. The distribution of cytokeratins and involucrin in normal dog skin was immunohistochemically examined with 27 commercial monoclonal antibodies for human use. Antibodies, No.4, OV-TL12/13, 35βH11, 4.1.18, CAM5.2, NCL5D3, Ks.13.1, Ks.18.04, Ks.19.1, 170.2.14 and Ks.20.8 stained hair follicles and/or the sweat gland duct, but not the epidermis. Antibodies, 34βB4, AE3, 34βE12, LP34, RCK102, MNF116, AE1, KL1, DE-K10 and DE-K13 reacted with every layer of the epidermis, hair follicles and the sweat gland duct. These results were similar to those reported in the human skin. No positive staining, however, could be detected in the epidermis, hair follicles and the sweat gland duct with commercial antibodies, 6B10, Ks.7.18, Mu146-uc, E3, RCK108 and involucrin. Therefore, immunohistochemical investigation with these commercial antibodies developed for human skin examination might be available for investigating the origin of skin tumors in dogs. KEY WORDS: canine skin, immunohistochemistry, monoclonal antikeratin antibody J. Vet. Med. Sci. 63(1): 1–4, 2001

Keratinocytes in normal human and dog epidermis 10% buffered formaldehyde, and paraffin-embedded. undergo an orderly pattern of maturation during their migra- The tissue blocks were cut into 4 µm sections, deparaf- tion from the basal layer to the cornified layer through the finized, rehydrated, rinsed in phosphate-buffered saline spinous and granular layers. This progression is accompa- (PBS), and then stained by the avidin-biotin complex method nied by changes in the synthesis of cytokeratins (CKs) [3, 8] [5]. and involucrin [9]. Distribution of these proteins has also Before staining with monoclonal antikeratin antibody been found in different layers of the epidermis [1, 2] and dif- (Ab), the sections were digested with a 0.25% (w/v) solution ferent structures of the cutaneous appendages [6]. This dis- of trypsin (Gibco, Grand Island, NY, U.S.A.) in Tris-HCl tribution pattern of CKs and involucrin, characteristic to the buffer at 37°C for 20–40 min, and washed in cold PBS. normal epidermis and cutaneous appendages, has been Then, all sections were incubated for 20 min in 0.3% (v/v) shown to be different in malignant transformation and abnor- hydrogen peroxide solution, to block endogenous peroxidase mal proliferation of human skin, although it is usually activity. The sections were washed with PBS, and incubated retained in benign tumors from epidermis or cutaneous with 2% (v/v) normal horse serum to block non-specific anti- appendages. Recently, immunohistochemical staining with body bindings. After absorption of excess serum, all sections antibodies against these proteins has become available for were incubated with the primary monoclonal Ab against the histopathology of human skin tumors [4, 7, 10]. cytokeratins, followed by incubation with biotin-conjugated To investigate dog skin tumors, antibodies against dog horse anti-mouse IgG or IgM serum, and then finally with cytokeratins are required, however the antibodies for animal avidin-biotin-conjugated peroxidase complex (Vector Labo- use are not yet available. Therefore, commercial antibodies ratories, Inc., Burlingame, CA, U.S.A.). developed for the analysis of human keratin expression were For staining against involucrin, rabbit anti-involucrin examined for their reactivity in normal dog skin, and to deter- serum was used as the primary Ab, and biotinylated goat mine whether it would be possible to use them for the identi- anti-rabbit immunoglobulin serum as the second Ab. Before fication of the cells of dogs. staining, endogenous peroxidase activity was blocked as described above, and non-specific Ab binding was also MATERIALS AND METHODS blocked with normal goat serum. All incubations were for 30 min at 37°C, and the slides Biopsy specimens of normal skin were collected from were rinsed in PBS extensively between incubations. The nose, cheek, neck, abdomen and thigh of seven healthy bea- sections were then incubated with a 3,3’-diaminobenzidine gle dogs kept at our laboratory. Each specimen was fixed in (DAB) (Sigma Chemical Co., St Louis, MO, U.S.A.) solution for 5 min and counterstained with Mayer’s hematoxylin, *CORRESPONDENCE TO AND PRESENT ADDRESS: NAKAMURA, Y., Department of Veterinary Internal Medicine, Nihon University and finally mounted. The DAB preparation used was 3 mg School of Veterinary Medicine, 1866 Kameino, Fujisawa, DAB/ml of Tris-HCl buffer containing 0.03% hydrogen per- Kanagawa 252–8510, Japan oxide. The trypsin digestion period was adjusted for each 2 M. KOZAKI ET AL.

Table 1. Antibodies used in this study Antibody Specificity Antibody source 34βB4 Cytokeratin 1 Enzo AE3 Cytokeratins1 to 8 Boeheringer KL1 Cytokeratins1, 2, 5 to 8, 11, Immunotech 14, 16 to 18 34βE12 Cytokeratins1, 5, 10, 11 Enzo 6B10 Cytokeratin 4 Sigma No.4 Cytokeratin 4 Boeheringer RCK102 Cytokeratins 5, 8 Sanbio LP34 Cytokeratins 5, 6, 18 DAKO MNF116 Cytokeratins 5, 6, 8, 17, 19 DAKO Ks.7.18 Cytokeratin 7 Progen OV-TL12/30 Cytokeratin 7 DAKO 35βH11 Cytokeratin 8 Enzo 4.1.18 Cytokeratin 8 Boeheringer CAM5.2 Cytokeratins 8, 18 Becton Dickinson Fig. 1. Immunohistochemical staining of normal dog skin with NCL5D3 Cytokeratins 8, 18, 19 Sanbio RCK102 (Ab against CKs 5 and 8). The basal layer of the epider- DE-K10 Cytokeratin 10 DAKO mis is stained strongly, but the suprabasal layer is not. AE1 Cytokeratins 10,14 to 16, 19 Boeheringer Ks13.1 Cytokeratin 13 Progen DE-K13 Cytokeratins 10, 13 DAKO MU146-uc Cytokeratin 14 Biogenex E3 Cytokeratin 17 DAKO Ks18.04 Cytokeratin 18 Progen RCK108 Cytokeratin 19 DAKO Ks19.1 Cytokeratin 19 Sanbio 170.2.14 Cytokeratin 19 Boeheringer IT-Ks20.8 Cytokeratin 20 Progen Involucrin Involucrin Biomedical Tech specimen, and the preparation giving optimum staining in each individual case was used. In controls, DAB was applied and the primary and secondary antibodies were not used in the incubation. The antibodies used in this study are shown in Table 1. Fig. 2. Immunohistochemical staining of normal dog skin with RCK102 (Ab against CKs 5 and 8). The outer root sheath is RESULTS stained strongly, but the inner root sheath is not.

No positive immunoreactivity could be detected in the epi- although all parts of the inner root sheath showed a negative dermis, inner and outer root sheath, sweat gland, sebaceous reaction. With AE3, moderate reactivities were detected in gland and hair matrix with the antibodies 6B10 (Ab against all parts of the inner root sheath and the infundibular outer CK 4), Ks 7.18 (Ab against CK 7), OV-TL12/13 (Ab against root sheath. The isthmus and inferior portion in the outer CK 7), Ks 13.1 (Ab against CK 13), Mu146-uc (Ab against root sheath also showed a strong reaction. With 170.2.14 CK 14), E3 (Ab against CK 17), RCK108 (Ab against CK (Ab against CK 19), a moderate reaction was detected in all 19), Ks 19.1 (Ab against CK 19), and Involucrin (Ab against parts of the outer root sheath. Moderate reactivity in the involucrin). Keratinocytes in the basal epidermis were infundibulum inner root sheath was noted with 34βB4, No.4 stained strongly with AE3 (Ab against CKs 1-8), 34βE12 (Ab against CK4), DE-K10 and DE-K13. With AE3, (Ab against CKs 1, 5, 10, and 14), RCK102 (Ab against CKs RCK102, LP34, MNF116, 4.1.18 (Ab against CK 8), 5 and 8; Figs. 1 and 2), LP34 (Ab against CKs 5, 6, and 18) CAM5.2 (Ab against CKs 8 and 18), NCL5D3 (Ab against and MNF116 (Ab against CKs 5, 6, 8, 17, and 19), and mod- CKs 8, 18, and 19), AE1, Ks18.04 (Ab against CK 18) and erate reactivity was detected with IT-Ks20.8 (Ab against 170.2.14 (Fig. 3) strong or moderate reactivities were CK20). Also, keratinocytes in the suprabasal epidermis were observed in the sweat gland ducts and secretory portion. stained strongly with 34βB4 (Ab against CK 1), AE3, KL1 With 35βH11 (Ab against CKs 5 and 8), moderate reactivity (Ab against CKs 1, 2, 5–8, 11, 14, and 16–18), LP34, was detected only in the secretory portion of the sweat gland. MNF116, DE-K10, Ks18.04 (Ab against CK 18), AE1 (Ab Moderate reactivity only in the myoepithelial cells of the against CKs 10, 14-16, and 19) and DE-K13 (Ab against CKs sweat gland secretory portion was noted with 34βE12. With 10 and 13). With RCK102, LP34, MNF116 and AE1, strong 34βE12, RCK102, MNF116 and AE1, moderate reactivities reactivities were observed in all pars of the outer root sheath, were detected in the sebaceous gland. The only positive CYTOKERATIN EXPRESSION IN DOG SKIN 3 reaction in the hair matrix was observed with AE1 partially. against CKs and involucrin developed for human dermatol- These immunohistochemical observations are summa- ogy might be available for the detection of cytoskeletal inter- rized in Table 2. mediate filament proteins in canine normal skin using routine avidin-biotin-peroxidase immunohistochemical methods. DISCUSSION The reactions of 21 antibodies to canine skin were almost similar to those of human, although six antibodies were not The results of this study revealed that 27 antibodies able to react with canine epidermis, hair follicles, sweat gland, sebaceous gland and hair matrix cells. The canine inner root sheath indicated moderate reactivities with only 6 antibodies, suggesting minor differences of the epitope between canine and human inner root sheaths. However, these Abs might be useful for the analysis of the inner root sheath, since the reactivity of each Ab indicated a high specificity to a part of the inner root sheath. RCK102, LP34, MNF116 and AE1 might be useful for diagnozing neoplasms of the outer root sheath such as trichilemmoma. Further- more, 34βB4, KL1, 34βE12, RCK102, DE-K10 and DE-K13 might be useful for investigating the origin of basal cell tumors since they specifically react with basal cells or suprabasal cells. Also, 35βH11 might be available for confirming tumors in the sweat gland secretory portion. On the other hand, Ks18.04 might be useful for analyzing tumors in the Fig. 3. Immunohistochemical staining of normal dog skin with sweat gland, since it was reactive in the sweat gland. More- 170.2.14 (Ab against CK 19). The outer root sheath and sweat over, AE3 was useful as a positive control for checking each gland duct is stained strongly, but the inner root sheath is not. procedure, since it showed positive reactions in all epithelial

Table 2. Immunohistochemical staining in normal dog skin Epidermis Inner root sheath Outer root sheath Sweat gland Antibody BSa) SBb) IFc) ISd) IPe) IFa) ISb) IPc) Duct Gland SGf) HMg) 34βB4 – ++ + – – – – – – – – – AE3 ++ ++ + + + + ++ ++ ++ + – – KL1 – ++ – – – ++ + + – – – – 34βE12 ++ – – – – ++ ++ + – +h) +– 6B10 – – – – – – – – – – – – No.4 – – + – – – – – – – – – RCK102 ++ – – – – ++ ++ ++ + ++ + – LP34 ++ ++ – – – ++ ++ ++ + + – – MNF116 ++ ++ – – – ++ ++ ++ ++ ++ + – Ks.7.18 – – – – – – – – – – – – OV-TL12/30 – – – – – – – – – – – – 35βH11 – – – – – – – – – + – – 4.1.18 – – – – – – – – + ++ – – CAM5.2 – – – – + – – – + + – – NCL5D3 – – – – – – – – ++ + – – DE-K10 – ++ + + – – – – – – – – AE1 + ++ – – – ++ ++ ++ ++ + + +i) Ks13.1 – – – – – – – – – – – – DE-K13–++ + –– ––– – – – – MU146-uc – – – – – – – – – – – – E3 – – – – – – – – – – – – Ks18.04 – – – – – – – – ++ ++ – – RCK108 – – – – – – – – – – – – Ks19.1 – – – – – – – – – – – – 170.2.14 – – – – – + + + ++ ++ – – IT-Ks20.8 + – – – – – – – – – – – Involucrin – – – – – – – – – – – – a) BS:basal cell, b) SB:suprabasal cell, c) IF:infundibulum, d) IS:isthmus, e) IP:inferior portion, f) SG:sebaceous gland, g) HM:hair matrix, h) only myoepithelial cells showed positive reaction, i) positive reaction partially –, +, ++ staining intensity: negative, moderate, and strong, respectively. 4 M. KOZAKI ET AL. layers, the inner and outer root sheath, and the sweat gland. ment proteins in normal human tissues. Am. J. Pathol. 114: From these results, commercial monoclonal antibodies of 309–321. CKs for human use, especially 34βB4, AE3, KL1, 34βE12, 5. Hsu, S. M., Raine, L. and Fanger, H. 1981. Use of avidin-biotin- No.4, RCK102, LP34, MNF116, 35βH11, CAM5.2, DE- peroxidase complex (ABC) in immunoperoxidase techniques: a K10, AE1, DE-K13 and Ks18.04 might be available for the comparison between ABC and unlabeled antibody (PAP) proce- dures. J Histochem. Cytochem. 29: 577–580. immunohistochemical investigation of various dog epithelial 6. Lane, E. B., Bártek, J., Purkis, P. E. and Leigh, I. M. 1985. Ker- tumors. atin antigens in differentiating skin. Ann. New York Acad. Sci. U.S.A. 455: 241–258. REFERENCES 7. Osborn, M. 1983. Intermediate filaments as histologic markers: an overview. J. Invest. Dermatol. 81(Suppl.): 104–109S. 1. Banks-Schlegel, S. P., Schlegel, R. and Pinkus, G. S. 1981. Ker- 8. Skerrow, D. and Skerrow, C. J. 1983. Tonofilament differentia- atin protein domains within the human epidermis. Exp. Cell Res. tion in human epidermis. Isolation and polypeptide chain com- 136: 465–469. position of keratinocyte subpopulations. Exp. Cell Res. 043: 27– 2. Bowden, P. E. and Cunliffe, W. J. 1981. Modification of human 35. prekeratin during epidermal differentiation. Biochem. J. 199: 9. Watt, F. M. 1983. Involucrin and other markers of keratinocyte 145–154. terminal differentiation. J. Invest. Dermatol. 81(Suppl.): 100– 3. Fuchs, E. and Green, H. 1980. Changes in keratin gene expres- 103S. sion during terminal differentiation of the keratinocyte. Cell 19: 10. Watanabe, S., Mogi, S., Ichikawa, E. Takahashi, H., Minami, H. 1033–1042. and Harada, S. 1993. Immunohistochemical analysis of keratin 4. Gown, A. H. and Vogel, A. M. 1984. Monoclonal antibodies to distribution in eccrine poroma. Am. J. Pathol. 142: 231–239. human intermediate filament proteins. II. Distribution of fila-