Canine melanocytes: Immunohistochemical expression of melanocytic markers in different somatic areas

Case selection

Samples were harvested from five dogs, with no clinically reported dermatological or mucosal lesions, submitted for necropsy after owner's consent. Samples (two to three) were collected from different cutaneous and mucosal sites, less than 3 h after the death of the dog. Sampling was performed harvesting an 8 mm punch biopsy (measured parallel to the epithelial surface) to guarantee a proper evaluation. In the present study samples were collected from: oral mucosa (internal aspect of the cheek), oral mucocutaneous junction (lateral upper lip the canine tooth level), eyelid (lower right eyelid), nose, pinna (tip), ventral abdomen (cranial to the umbilical scar, on the median line), back (lumbar area, on the median line) and head (between the pinnae, on the median line).

Pigmentation evaluation

The presence of pigmentation was evaluated macroscopically on epithelium and hair both before and after inclusion in paraffin and sectioning. Subgrossly, the degree of pigmentation was semiquantitatively evaluated on the epidermis/mucosal surface and assigned one of the following values: 0, pink/whitish skin with no spots; 1, light brown skin with no spots; 2, dark brown skin with no spots; 3, black skin with no spots; P, patchy, because of the presence of spots or irregular distribution of pigment. The same grades were assigned for the evaluation of hair coat pigmentation: 0, white hair; 1, light brown hair; 2, dark brown hair; 3, black hair; P, patchy, because of the presence of spots or different coloration of hair.

Histological and immunohistochemical evaluation

From all samples a routinely stained haematoxylin & eosin slide was prepared. Five micrometre sections were cut from formalin-fixed and paraffin-embedded samples and mounted on poly-l-lysine-coated slides, which then were dewaxed and dehydrated. Immunohistochemical evaluation was performed on serial sections with antibodies reported to be cross-reactive in canine species, raised against Melan A,¹⁸ PNL2,¹⁸ TRP1, TRP2, SOX-10 and MITF. Positive controls were obtained from canine melanomas for Melan A, PNL2, TRP1, TRP2 and MITF, while mammary gland was used as control for SOX-10.¹⁹ AEC was used as a chromogen for its red colour, to differentiate from melanin. Bleaching was not performed. Negative controls were run omitting the primary antibody and incubating control sections with TBS. IBA1 immunolabelling was performed, following protocols reported previously, in selected cases to differentiate melanocytes from melanin-laden macrophages.²⁰ Immunohistochemical protocols and antibodies used in this study are summarised in Table 1.

For each antibody, the presence of positive cells was assessed within the cutaneous/mucosal epithelium, in the hair bulb and follicular wall and in the other cutaneous/mucosal structures (e.g. glands, nerves, dermis).

Immunofluorescence

Double immunofluorescence was performed on selected samples from nose, oral mucosal and haired skin. The co-expression of melanocytic markers was assessed pairing antibodies with similar antigen retrieval methods. For this purpose, primary antibodies previously described for immunohistochemical evaluation (TRP1, SOX-10 and MITF) were co-localised with anti-Melan A conjugated with Alexa Fluor 594, while TRP2 was co-localised with anti-PNL2 conjugated with Alexa Fluor 488 antibodies (Santa Cruz). SOX10 and TRP1 also could be co-localised, yet co-localisation of TRP1 and TRP2 was not possible, both being primary antibodies raised in rabbit. After dewaxing and dehydrating, the slides were incubated with a 3% hydrogen peroxide methanol solution. Antigen retrieval was performed as reported for immunohistochemical evaluation and primary antibodies were incubated overnight at 4°C with the appropriate concentration. After washing with tris-buffered saline, a secondary goat anti-mouse conjugated with a red fluorochrome (Goat Anti-Mouse IgG H&L-Alexa Fluor 647) and with a 1:200 dilution, was applied for 2 h at room temperature (RT). After a careful wash, a protein block step was performed before incubation with Melan A and PNL2 fluorochrome-conjugated antibodies. To co-localise SOX-10 and TRP1, after incubation of TRP1, a secondary anti-rabbit antibody conjugated with a fluorochrome (Goat Anti-Rabbit IgG H&L-Alexa Fluor 594; Abcam) was used. Finally, a drop of aqueous mounting medium containing DAPI (Abcam) was added and slides were incubated for 5 min at RT, before coverslip mounting. Negative controls were run omitting the primary antibodies and incubating the sections with phosphate-buffered saline.

Samples were evaluated using an Olympus BX51 fluorescent microscope equipped with a Nikon DS-Qi2Mc camera. NIS-ELEMENTS D software was used for image acquisition and analysis.

Case selection and macroscopical evaluation

The five dogs selected for this preliminary investigation were a chow chow, a Labrador retriever, a Siberian husky, a mixed English setter and a mixed breed dog. The five animals showed a variable degree of pigmentation of both skin/mucosa and of hair coat in the sampled areas. The results of macroscopical evaluation are reported in Tables 2 and 3, respectively.

Melan A

Melan A immunolabelling was observed as a granular cytoplasmic reaction. The reaction was strong and specific, and no signs of background stain were present. Melan A positive cells were observed in the basal and suprabasal layers of the epithelium in mucosal, junctional, nasal and cutaneous samples (Table 2; Figure 1a–e). One of the dogs (Siberian husky, Case 5) showed mucosa with a macroscopical patchy pigmentation. In this case, corresponding to the macroscopically evident patcahy pigmentation (P), there were areas with numerous Melan A⁺ melanocytes alternating with areas where no positive cells could be observed. A strong expression of this marker also was seen in the hair bulbs and in the infundibula of hair follicles (Table 3; Figure 2a). In hair bulbs, where pigmentation was prominent with H&E visualisation, the expression of Melan A was more intense and located in a higher number of cells. Nevertheless, the expression of Melan A also could be observed in scattered cells in those samples where hair pigmentation was not histologically evident. In one of the samples from the abdominal skin (Case 4), a mild hyperplasia of the epidermis was seen, associated with multifocal, mild lymphocytic and plasmacellular infiltrates in the superficial dermis; in this case, the number of melanocytes appeared to be increased. Generally, no melanocyctes were observed in sebaceous glands, except for Meibomian glands and periocular sebaceous glands, where occasional Melan A⁺ cells were observed among the basal cells (Figure 2b). Only in one case (Case 1) were Melan A⁺ cells present near apocrine glands in the dermis and these were interpreted as dermal melanocytes (Figure 2c).

PNL2

The expression of PNL2 was seen as a finely granular, cytoplasmic immunolabelling. The expression of this marker was variable and sometimes very faint, with an occasional uneven distribution of the immunoreactivity in the cytoplasm. Positive cells were seen in the basal and suprabasal layers of the epithelium (Table 2; Figure 1f–j). In a similar way to Melan A, patchy-pigmented oral mucosa showed areas with the presence of PNL2⁺ melanocytes alternating with areas with no detectable cells. Occasionally, in homogeneously pigmented epithelia such as the nose and the mucosal junctions, PNL2⁺ melanocytes were present with a multifocal distribution, unlike the regular presence of immunoreactive melanocytes seen for other markers, particularly Melan A and SOX-10. The immunolabelling was stronger in follicular melanocytes, in both hair bulb (Table 3; Figure 2d) and follicular wall. Scattered PNL2⁺ cells were present in the basal compartment of Meibomian glands (Figure 2 e), yet, in a similar way to Melan A, not observed in sebaceous glands of the other cutaneous areas sampled for this study. Dermal melanocytes were negative in all samples examined, including for Case 1 (Figure 2f).

TRP1

The immunolabelling for TRP1 was cytoplasmic and coarsely granular; the intensity of the labelling was variable, from moderate to strong. In eyelid and oral mucocutaneous junctions (Figure 1k–o), the marker's expression was strong and similar to what observed with Melan A, while no TRP1⁺ intraepithelial melanocytes were present in most of the samples harvested from the cutaneous sites (Table 2). As for hair bulbs (Figure 2g) and follicular walls (Table 3), the immunolabelling was strong and specific, and apparently increased with the quantity of melanin-containing cells. In all of the samples from haired skin, sparse TRP1⁺ positive round cells, characterised by moderate amounts of cytoplasm and rounded nucleus, were observed in particular in the superficial dermis. Rare immunolabelled cells were observed in the basal compartment of Meibomian and periocular sebaceous glands (Figure 2h). Dermal melanocytes did not label with TRP1, yet some positive cells were seen among negative melanocytes (Figure 2i) and were interpreted as mast cells.

TRP2

Immunolabelling for TRP2 was strong and coarsely granular, with a cytoplasmic distribution. The intensity of the staining was unpredictably variable, being strong and neat in some cases, in particular on the nose, oral mucocutaneous junction and eyelid junction (Table 2; Figure 1q,rt, respectively), while in haired skin and oral mucosa (Figure 1p,s), the immunolabelling often was faint. Generally, immunolabelling was stronger in hair bulbs (Table 3; Figure 2j) than in intraepithelial melanocytes. In a similar way to PNL2, the immunolabelling of this marker showed a multifocal distribution in all cases in some macroscopically heavily pigmented samples, such as the nose (Table 2). Rare positive cells were observed in the basal compartment of Meibomian glands (Figure 2k). Dermal melanocytes did not label with TRP2, yet in some samples, TRP2⁺ cells were seen in the dermis (Figure 2l). Morphologically these cells were characterised by a moderate quantity of cytoplasm and interpreted as histiocytes.

SOX-10

SOX-10 expression appeared as a strong and diffuse nuclear immunolabelling, regularly expressed in both haired skin, mucocutaneous junctions and oral mucosa (Figure 1u–y). The immunolabelling of the nucleus was sharp and strong and allowed the rapid and solid identification of positive cells. Besides, this neat immunolabelling granted a better identification of melanocytes also in highly pigmented sites, such as the hair bulbs (Table 3; Figure 2m). All positive cells within the epithelia and in hair follicles were morphologically compatible with melanocytes, being characterised by scant clear cytoplasm or filled with melanin pigment and showing dendritic prolongations of the cytoplasm. Positive cells among the basal component of Meibomian glands (Figure 2n) were frequently observed; on other samples from haired skin, no positivity was assessed yet, differently from the other markers, rare positive nuclei were observed in the cutaneous samples from the head of Case 1. Dermal melanocytes were positive (Figure 2o). The same cells were negative for the macrophagic marker IBA1 (inset in Figure 2o). (Positivity also was observed in dermal nerves and in myoepithelial cells of apocrine glands and of mammary gland, that were sampled together with the abdominal skin.)

MITF

Immunolabelling of MITF was seen as a finely granular nuclear reactivity. Often, the immunoreactivity was faint when compared to the other markers, yet nuclear expression was helpful particularly in highly pigmented samples. In a similar way to PNL2, TRP1 and TRP2, melanocyte immunolabelling with MITF was more rarely observed within haired epithelia (Table 2; Figure 1z), while it was more prominent in samples harvested from mucocutaneous junctions (Figure 1bb, dd), oral mucosa (Figure 1cc) and, even if weaker, from the nose (Figure 1aa).

The labelling of melanocytes in hair bulbs was more intense and clearer when compared to the intraepithelial samples (Table 3; Figure 2p). Rare positive cells were observed in the basal compartment of Meibomian glands (Figure 2q). Moreover, the inner root sheet showed a moderate-to-intense immunolabelling in hair follicles (Figure 2r). Dermal melanocytes showed a variable positivity. MITF⁺ cells were scattered in the superficial to mid-dermis; these cells were characterised by rounded shape, a moderate amount of cytoplasm and a central rounded nucleus, morphologically similar to mast cells.

Melanocytic marker co-expression

Melan A showed a clear and moderate-to-strong expression and an almost total co-localisation with TRP1 on oral mucosa and nose (Figure 3a). PNL2 confirmed a lower sensitivity in detecting melanocytes and the signal often was weaker. The co-localisation was possible only with TRP2, because both antibodies required antigen retrieval with citrate buffer. TRP2 was more sensitive than PNL2 in detecting melanocytes (Figure 3b). Melan A and SOX-10 showed an almost complete co-localisation (Figure 3c), with SOX-10 being more sensitive and, as a consequence of the nuclear staining, easier for operators to recognise. On the nose and mucosal sites, SOX-10 showed a sensitivity, which was similar to TRP1 (Figure 3d). Co-localisation of Melan A and MITF confirmed that the latter was less sensitive; in a similar way to immunohistochemical findings, the signal was weak (Figure 3e).