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Expression of Serotonin and its 5-HT1A Receptor in Canine Cutaneous Mast Cell Tumours
J. Comp. Path. 2009, Vol. 141, 89e97
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
Expression of Serotonin and its 5-HT1A Receptor in Canine Cutaneous Mast Cell Tumours G. Kastengren Fro¨berg*, R. Lindberg†, M. Ritter‡ and K. Nordlind‡ Albano Animal Hospital, Danderyd, † Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Uppsala and ‡ Unit of Dermatology and Venereology, Department of Medicine, Karolinska University Hospital, Solna, Sweden *
Summary Mast cells of a number of different animal species have been reported to contain serotonin (5-hydroxytryptamine; 5-HT), a monoamine capable of numerous and complex actions, which may include an impact on tumour growth. Limited previous studies have suggested that normal or neoplastic canine mast cells do not express 5-HT. In the present study, canine cutaneous mast cell tumours (MCTs) of Patnaik histological grades IeIII were investigated immunohistochemically for expression of 5-HT and its receptor (R) 5-HT1A. The proportion of positively labelled cells and the intensity of labelling of individual cells were determined. Both 5-HT and the 5-HT1A receptor were expressed by non-neoplastic dermal mast cells and neoplastic mast cells. More neoplastic mast cells expressed 5-HT than the 5-HT1AR. Poorly differentiated tumours expressed fewer of both molecules, but the better differentiated mast cells at the periphery of such lesions had more consistent 5-HT expression. 5-HT and the 5-HT1A receptor may be involved in the differentiation of canine MCTs and in the microvascular complications associated with these neoplasms. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: dog; mast cell tumour; receptor; serotonin
Introduction Mast cell tumours (MCTs) are one of the most common neoplasms of the canine skin (Dobson et al., 2002) and are most often graded histologically by the scheme described by Patnaik et al. (1984). Tumours of grade I are circumscribed, mainly dermal in location, and consist of well-differentiated mast cells with prominent metachromatic cytoplasmic granules. Poor cellular differentiation, aggressive growth pattern, moderate to high mitotic activity, mitotic atypia and sparse to absent cytoplasmic granulation are the major features of grade III tumours. Grade II MCTs constitute an intermediary form between the two other grades, but they tend to infiltrate more deeply than grade I neoplasms (Patnaik et al., 1984; Gross et al., 2005). The treatment of canine cutaneous MCTs remains challenging and available options include surgery,
Correspondence to: G. Kastengren Fro¨berg (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2008.08.002
chemotherapy and radiation therapy (Lemarie et al., 1995; Murphy, 2003). Recurrence of MCTs at the site of surgical excision is common and grade III tumours are prone to metastasize. Recent immunohistochemical studies have evaluated a series of markers as potential prognostic indicators including expression of c-KIT, AgNOR (argyrophilic nucleolar organizer regions), Ki-67 and PCNA (proliferating cell nuclear antigen) (Scase et al., 2006; Seguin et al., 2006; Webster et al., 2007). The biogenic amine serotonin (5-hydroxytryptamine; 5-HT) is implicated as a mediator in an array of physiological processes and is of importance for basic cell functions such as proliferation, differentiation/ maturation and migration (Azmitia, 2001; Mohammad-Zadeh et al., 2008). 5-HT is found in the mast cells of rodents (Edvinsson et al., 1977), guinea-pigs (Lehtosalo et al., 1984) and humans (Zochodne et al., 1997; Kushnir-Sukhov et al., 2007), and the enzyme that regulates the synthesis of 5-HT, tryptophane hydroxylase, has been demonstrated in both normal and neoplastic human mast cells (Slominski et al., 2003; Ó 2008 Elsevier Ltd. All rights reserved.
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Kushnir-Sukhov et al., 2007). By contrast, with one exception (Rice and Mitchener, 1961), the published studies of canine mast cells have failed to demonstrate the presence of 5-HT (Parratt and West, 1957; Meier, 1959; Beaven et al., 1982; Fernandez et al., 2005). Fifteen different receptor molecules have been described in mammals that are able to bind 5-HT (Bockaert et al., 2006). These are expressed in different tissues and coupled to diverse signalling systems. Of those, one of the best characterized receptors is the 5-HT1AR (Raymond et al., 1999) that has been shown to mediate the effects of 5-HT in murine and human mast cells (Lundeberg et al., 2002; KushnirSukhov et al., 2006). 5-HT is reported to act as a growth factor for several types of human cancer (Vicaut et al., 2000; Siddiqui et al., 2005). For example, in prostatic carcinoma an increased number of 5-HT releasing tumour cells with neuroendocrine differentiation correlates with tumour progression and poor prognosis (Abrahamsson, 1999; Bollito et al., 2001; Ito et al., 2001), and the cancer cells in the most poorly differentiated tumours express the most 5-HT1AR (Dizeyi et al., 2004). The purpose of this study was initially to re-examine whether canine mast cells express 5-HT and to determine if there may be expression of 5-HT1AR by these cells. As such expression was proven, the second aim of the investigation was to determine whether the degree of differentiation of canine cutaneous MCTs was correlated with this serotonergic activity.
Materials and Methods Samples
The study was based on surgical specimens of canine cutaneous MCTs archived in the Division of Pathology, Pharmacology and Toxicology of the Swedish University of Agricultural Sciences. The specimens had been fixed at the time of collection in 4% neutral buffered formalin, processed using routine methods and embedded in paraffin wax. Sections prepared from these blocks were stained by haematoxylin and eosin (HE) and toluidine blue. Twenty-four cases were selected from the archive, including 8 examples of each of the three histological grades defined above (Fig. 1). Biopsy specimens of non-lesional skin from the trunk of 4 dogs were also examined in order to evaluate non-neoplastic mast cells. Immunohistochemical Labelling Antibodies. Rabbit polyclonal antibodies to 5-HT (1 in 20,000; Diasorin, Stillwater, USA) and 5-HT1AR (1 in 5,000; S1A-170) were used as primary antisera. The 5-HT1AR antibody is directed against amino acids
170e186 in the second extracellular loop of the molecule (Azmitia et al., 1992), this amino acid sequence showing complete homology between humans and rats. The antiserum labels a major band of 64 kD on immunoblots of solubilized protein derived from the hippocampus and brainstem of neonatal rats and, in different recombinant systems, specifically labels cells transfected with constructs containing the 5-HT1AR coding sequence (DeFelipe et al., 2001). A mouse monoclonal antibody to tryptase (1 in 1,000; Chemicon, Temecula, USA) was used to confirm the identity of mast cells examined for 5-HT and 5-HT1AR (see below). Labelling Technique. Sections (4 mm) were dewaxed with Bio-ClearÔ (Bio-Optica, Milan, Italy) for 15 min, rehydrated in graded concentrations of ethanol and boiled in Tris-EDTA buffer (25 min at 98 C) for antigen retrieval. The avidinebiotin complex horseradish peroxidase (ABC-HRP) technique was used for detection of bound primary antibody. Endogenous peroxidase was quenched with H2O2 0.3% in PBS (pH 7.4). After applying 10% goat serum for 40 min to minimize non-specific protein binding, the sections were incubated with the primary antibodies overnight at 4 C in a humid atmosphere. Biotinylated goat anti-rabbit antibody (BA 1000, 1 in 200; Vector Laboratories, Burlingame, USA) was applied for 40 min and avidinebiotin complex (PK6100, Vector Laboratories) for 30 min, both at room temperature. Amino-ethylcarbazole (FK-4200, Vector Laboratories) was used as chromogen. Sections were counterstained with haematoxylin and mounted under glycerol gelatin. As negative controls, the primary antibodies were omitted or replaced by normal rabbit immunoglobulins (X0936, Dako, Glostrup, Denmark). Immunofluorescence Labelling
In order to further demonstrate the presence of 5-HT and 5-HT1AR in mast cells, a double immunofluorescence (IF) technique was applied. Sections of normal skin were incubated in sequence with 10% goat serum, polyclonal antibodies to 5-HT or 5-HT1AR, biotinylated goat anti-rabbit secondary antibody and streptavidin-conjugated Texas Red (1 in 2,000; Vector Laboratories). After this procedure the mouse anti-tryptase antibody, followed by rabbit anti-mouse fluorescein isothiocyanate (FITC)-conjugated antibody (dilution 1 in 40; Dako), was applied. Slides were mounted under glycerol gelatin. Microscopy
One examiner (GKF), blinded to the grading of the individual tumours, evaluated all slides for
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Fig. 1. Representative photomicrographs of mast cell tumours of different Patnaik grades. (a) Grade I, (b) grade II and (c) grade III. HE. Bar, 50 mm.
immunohistochemical expression of 5-HT and 5HT1AR. Each slide was scored for the intensity of labelling of individual cells as: () no labelling, (+) weak, (++) moderate or (+++) strong labelling. The number of positively labelled cells in different areas of the tumour was evaluated semi-quantitatively and expressed as 0e25%, 25e50%, 50e75% and 75e100% of tumour cells. For the evaluation of the two-colour immunofluorescence, slides were examined with a fluorescence microscope at the excitation wavelengths of 465e495 (FITC) and 540e580 nm (Texas Red).
Results Normal Skin
Scattered mast cells were found in the superficial dermis of the normal skin, generally in association with small blood vessels. These cells had strong cytoplas-
mic expression of 5-HT and 5-HT1AR by immunohistochemistry (IHC) (Fig. 2). The identity of these mast cells was further confirmed by the double immunofluorescence technique, demonstrating co-localization of tryptase with 5-HT and 5-HT1AR (Fig. 3). Mast Cell Tumours
5-HT expression was found in 8/8 grade I, 7/8 grade II and 6/8 grade III MCTs. Grade I tumours had the greatest number of positively labelled cells and the majority of these had strong expression (+++) of 5-HT. In 4/8 tumours, 5-HT expression was greater in neoplastic cells located beneath the epidermis than in deeper infiltrates (Fig. 4). In 6/8 tumours, small clusters of positively labelled cells were found in the peritumoural connective tissue. In grade II tumours, immunolabelling was more variable. In 4/8 tumours almost all mast cells were positively labelled, although generally much less
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Fig. 2. Sections of normal skin, showing immunoreactivity for (a) 5-HT and (b) the 5-HT1A receptor in dermal mast cells. IHC. Bar, 50 mm.
Fig. 3. Co-localization of tryptase with 5-HT (aec) or 5-HT1AR (def) in normal cutaneous mast cells. (a) Tryptase (FITC). (b) 5-HT (Texas Red). (c) Tryptase + 5-HT. (d) Tryptase (FITC). (e) 5-HT1AR (Texas Red). (f) Tryptase + 5-HT1AR. IF. Bar, 50 mm.
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Fig. 4. Expression of 5-HT within a grade I mast cell tumour. (a) Strongly immunolabelled mast cells in superficial tumour area. (b) Predominance of weakly stained cells in deeper area. IHC. Bar, 50 mm.
intensely than those of grade I (75e100% of cells were of labelling intensity +). One tumour had 75e100% of mast cells strongly labelled (+++), while the remainder (3/8) had areas with 25e50% of mast cells moderately positive (++). Positively labelled cells were more often observed close to the epidermis or in more deeply located border areas of the tumours. Grade III MCTs had the smallest complement of positively labelled cells. In 4/8 tumours, 75e100% of cells were very weakly labelled or negative (Fig. 5a). The positive mast cells in these tumours were scattered or appeared in small clusters, often at the periphery of the tumour or in the adjacent connective tissue (Fig. 5b). These mast cells appeared well differentiated and were often intensely labelled (+++). In 2/8 tumours, positively labelled mast
cells tended to occur in the subepidermal area. In 2/8 tumours, mast cells were universally negative for 5-HT expression. 5-HT1AR expression was identified in 7/8 grade I, 5/8 grade II and 3/8 grade III tumours. In grade I tumours there was a gradient of labelling, with most positive cells situated near the epidermis (Fig. 6a). In these tumours, approximately 50e75% of the cells in the upper dermis were positively labelled and the intensity of labelling varying between + and +++ (most often ++). Grade II tumours had fewer immunolabelled cells than grade I, and in two of these tumours the largest component of positively labelled mast cells was in the subepidermal area. 5-HT1AR expressing mast cells were scattered or present in small clusters. The
Fig. 5. Expression of 5-HT within a grade III mast cell tumour. (a) Weakly labelled mast cells. (b) Strongly labelled mast cells at the edge of the tumour contrast with weakly labelled tumour cell mass at left. IHC. Bar, 50 mm.
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Discussion
Fig. 6. Expression of the 5-HT1A receptor. (a) Grade I mast cell tumour, superficial tumour area, with immunolabelled mast cells. (b) Grade III mast cell tumour showing essentially negative immunolabelling. IHC. Bar, 50 mm.
intensity of labelling varied, but weakly stained cells predominated. Tumours of grade III had the fewest immunolabelled cells and the majority of these tumours were essentially negative for 5-HT1AR expression (Fig. 6b). The immunoreactive mast cells were consistently only weakly labelled, and were found either subepidermally or clustered in the loose connective tissue surrounding the tumour.
The present study has demonstrated that normal and neoplastic canine mast cells express 5-HT and 5-HT1AR. Neoplastic mast cells expressed more 5HT than 5-HT1AR and more differentiated tumours had greater expression of both molecules. Few previous studies have examined expression of 5-HT by canine mast cells or MCTs. An early biochemical and histochemical study (Meier, 1959) showed only negligible quantities of 5-HT in canine MCTs. Another early investigation suggested the presence of 5-HT in a single canine MCT using a variety of histochemical techniques (Rice and Mitchener, 1961), but no 5-HT was found by enzymatic isotopic assay in canine fundic mucosal mast cells (Beaven et al., 1982). Fernandez et al. (2005) failed to demonstrate 5-HT by immunohistochemically labelling 21 canine cutaneous MCTs of various Patnaik grades. The discrepancies between the results of that study and ours may be explained by the different techniques used for antigen retrieval and the use of different anti-serotonin antibodies. Our observation that normal canine mast cells express 5-HT1AR is consistent with findings on human and murine mast cells (Lundeberg et al., 2002; Kushnir-Sukhov et al., 2006), and indicates that 5-HT1AR functions as an autoreceptor on mast cells, analogous to 5-HT producing rodent neural cells (Azmitia, 2001). 5-HT is known to act as a growth factor for several types of benign and malignant human tumour cell lines (Seuwen and Pouyssegur, 1990; Vicaut et al., 2000; Siddiqui et al., 2005), but 5-HT agonists may also inhibit tumour growth, possibly via effects on blood vessels (Baguley et al., 1993; Stu¨cker et al., 1997). We have shown a gradual loss of both 5-HT and 5-HT1AR expression with decreasing levels of differentiation of canine MCTs. The fact that canine MCTs have less serotonergic activity with increasing anaplasia is in contrast to studies of human prostatic carcinoma, where the most poorly differentiated cancer cells have the strongest 5-HT1AR signal (Dizeyi et al., 2004). The 5-HT1A receptor may have a differentiating effect, as reported in studies of neural cells (Azmitia, 2001), and our results may give indirect support for this mechanism acting in canine MCTs. This suggests that pharmacological activation of the 5-HT1A receptor may be one way to alter the level of differentiation of the tumour. However, the simultaneous loss of the ligand and its receptor with decreasing differentiation may also be interpreted as an effect, rather than a cause, of poor differentiation of the MCT.
Serotonin and 5-HT1A Receptor in Mast Cell Tumours
In some tumours, strongly 5-HT positive and welldifferentiated mast cells were noted at the periphery of the lesion and in the adjacent connective tissue. This was most obvious in grade III tumours, where these cells contrasted with the largely 5-HT negative poorly differentiated tumour cells. Mast cells may have different tumour-promoting actions, but they may also be part of an immune response aimed at eliminating tumour cells (Ch’ng et al., 2006). The well-differentiated appearance of the peripheral mast cells, irrespective of tumour grade, may suggest that these are a reactive, rather than neoplastic, population. This possibility has been highlighted by retrospective studies of canine cutaneous MCTs showing a low rate of local recurrence despite non-tumour-free surgical margins (Michels et al., 2002; Seguin et al., 2006). Erythema, oedema and itch (Darier’s sign), traditionally attributed to the release of histamine, heparin and other inflammatory mediators, can be detected upon manipulation of canine MCTs (Fox, 1998; Murphy, 2003). Systemic antihistamines are often used in an attempt to decrease such effects, especially when dealing with multiple tumours. It has been shown that 5-HT can produce itching, flare and wheals when applied to the human skin (Fjellner and Ha¨germark, 1979; Weissaar et al., 1997). 5-HT released from neoplastic mast cells may therefore contribute to the microvascular and pruritic phenomena associated with palpation of the tumour. Using a 5-HT antagonist in addition to antihistamines may thus be warranted to reduce these symptoms in dogs. In this respect, various symptoms of cutaneous mastocytosis in humans have been reported to be reduced after treatment with cyproheptadine, a combined histamine and 5-HT antagonist (Haneke, 1970; Fenske et al., 1985; Gasior-Chrzan and Falk, 1992; Enomoto et al., 1999). Mechanisms responsible for the infiltration of canine MCTs by eosinophils (Gross et al., 2005), have not yet been explored. Among mediators of eosinophil accumulation in inflammatory conditions of various species, eotaxin and other chemokines have been ascribed an important role, and activated mast cells may release eotaxin or generate its production by other cells by histamine release (Das et al., 1998; Williams and Galli, 2000; Menzies-Gow et al., 2004). However, a recent study in humans and mice indicates that 5-HT also acts as a chemoattractant for eosinophils and that this chemotaxis can be blocked by cyproheptadine treatment (Boehme et al., 2004). It is therefore possible that serotonergic activity of the neoplastic cells may be one mechanism underlying infiltration of eosinophils into canine MCTs.
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In summary, we have demonstrated the expression of 5-HT and its 5-HT1A receptor in canine cutaneous MCTs. This expression diminishes with decreasing differentiation of the neoplasm. Our findings imply that the impact of various biological actions of 5HT in the pathogenesis and therapeutic management of canine MCTs should be considered.
Acknowledgments We thank Professor E.C. Azmitia, New York University, for kindly supplying the 5-HT1A antibody and Mrs. K. Curra Holmberg and Mrs. A.-L. Kastman for their technical assistance.
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Serotonin and 5-HT1A Receptor in Mast Cell Tumours
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May 26th, 2008 ½ Received, Accepted, August 18th, 2008
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www.elsevier.com/locate/jcpa
Expression of Serotonin and its 5-HT1A Receptor in Canine Cutaneous Mast Cell Tumours G. Kastengren Fro¨berg*, R. Lindberg†, M. Ritter‡ and K. Nordlind‡ Albano Animal Hospital, Danderyd, † Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Uppsala and ‡ Unit of Dermatology and Venereology, Department of Medicine, Karolinska University Hospital, Solna, Sweden *
Summary Mast cells of a number of different animal species have been reported to contain serotonin (5-hydroxytryptamine; 5-HT), a monoamine capable of numerous and complex actions, which may include an impact on tumour growth. Limited previous studies have suggested that normal or neoplastic canine mast cells do not express 5-HT. In the present study, canine cutaneous mast cell tumours (MCTs) of Patnaik histological grades IeIII were investigated immunohistochemically for expression of 5-HT and its receptor (R) 5-HT1A. The proportion of positively labelled cells and the intensity of labelling of individual cells were determined. Both 5-HT and the 5-HT1A receptor were expressed by non-neoplastic dermal mast cells and neoplastic mast cells. More neoplastic mast cells expressed 5-HT than the 5-HT1AR. Poorly differentiated tumours expressed fewer of both molecules, but the better differentiated mast cells at the periphery of such lesions had more consistent 5-HT expression. 5-HT and the 5-HT1A receptor may be involved in the differentiation of canine MCTs and in the microvascular complications associated with these neoplasms. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: dog; mast cell tumour; receptor; serotonin
Introduction Mast cell tumours (MCTs) are one of the most common neoplasms of the canine skin (Dobson et al., 2002) and are most often graded histologically by the scheme described by Patnaik et al. (1984). Tumours of grade I are circumscribed, mainly dermal in location, and consist of well-differentiated mast cells with prominent metachromatic cytoplasmic granules. Poor cellular differentiation, aggressive growth pattern, moderate to high mitotic activity, mitotic atypia and sparse to absent cytoplasmic granulation are the major features of grade III tumours. Grade II MCTs constitute an intermediary form between the two other grades, but they tend to infiltrate more deeply than grade I neoplasms (Patnaik et al., 1984; Gross et al., 2005). The treatment of canine cutaneous MCTs remains challenging and available options include surgery,
Correspondence to: G. Kastengren Fro¨berg (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2008.08.002
chemotherapy and radiation therapy (Lemarie et al., 1995; Murphy, 2003). Recurrence of MCTs at the site of surgical excision is common and grade III tumours are prone to metastasize. Recent immunohistochemical studies have evaluated a series of markers as potential prognostic indicators including expression of c-KIT, AgNOR (argyrophilic nucleolar organizer regions), Ki-67 and PCNA (proliferating cell nuclear antigen) (Scase et al., 2006; Seguin et al., 2006; Webster et al., 2007). The biogenic amine serotonin (5-hydroxytryptamine; 5-HT) is implicated as a mediator in an array of physiological processes and is of importance for basic cell functions such as proliferation, differentiation/ maturation and migration (Azmitia, 2001; Mohammad-Zadeh et al., 2008). 5-HT is found in the mast cells of rodents (Edvinsson et al., 1977), guinea-pigs (Lehtosalo et al., 1984) and humans (Zochodne et al., 1997; Kushnir-Sukhov et al., 2007), and the enzyme that regulates the synthesis of 5-HT, tryptophane hydroxylase, has been demonstrated in both normal and neoplastic human mast cells (Slominski et al., 2003; Ó 2008 Elsevier Ltd. All rights reserved.
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Kushnir-Sukhov et al., 2007). By contrast, with one exception (Rice and Mitchener, 1961), the published studies of canine mast cells have failed to demonstrate the presence of 5-HT (Parratt and West, 1957; Meier, 1959; Beaven et al., 1982; Fernandez et al., 2005). Fifteen different receptor molecules have been described in mammals that are able to bind 5-HT (Bockaert et al., 2006). These are expressed in different tissues and coupled to diverse signalling systems. Of those, one of the best characterized receptors is the 5-HT1AR (Raymond et al., 1999) that has been shown to mediate the effects of 5-HT in murine and human mast cells (Lundeberg et al., 2002; KushnirSukhov et al., 2006). 5-HT is reported to act as a growth factor for several types of human cancer (Vicaut et al., 2000; Siddiqui et al., 2005). For example, in prostatic carcinoma an increased number of 5-HT releasing tumour cells with neuroendocrine differentiation correlates with tumour progression and poor prognosis (Abrahamsson, 1999; Bollito et al., 2001; Ito et al., 2001), and the cancer cells in the most poorly differentiated tumours express the most 5-HT1AR (Dizeyi et al., 2004). The purpose of this study was initially to re-examine whether canine mast cells express 5-HT and to determine if there may be expression of 5-HT1AR by these cells. As such expression was proven, the second aim of the investigation was to determine whether the degree of differentiation of canine cutaneous MCTs was correlated with this serotonergic activity.
Materials and Methods Samples
The study was based on surgical specimens of canine cutaneous MCTs archived in the Division of Pathology, Pharmacology and Toxicology of the Swedish University of Agricultural Sciences. The specimens had been fixed at the time of collection in 4% neutral buffered formalin, processed using routine methods and embedded in paraffin wax. Sections prepared from these blocks were stained by haematoxylin and eosin (HE) and toluidine blue. Twenty-four cases were selected from the archive, including 8 examples of each of the three histological grades defined above (Fig. 1). Biopsy specimens of non-lesional skin from the trunk of 4 dogs were also examined in order to evaluate non-neoplastic mast cells. Immunohistochemical Labelling Antibodies. Rabbit polyclonal antibodies to 5-HT (1 in 20,000; Diasorin, Stillwater, USA) and 5-HT1AR (1 in 5,000; S1A-170) were used as primary antisera. The 5-HT1AR antibody is directed against amino acids
170e186 in the second extracellular loop of the molecule (Azmitia et al., 1992), this amino acid sequence showing complete homology between humans and rats. The antiserum labels a major band of 64 kD on immunoblots of solubilized protein derived from the hippocampus and brainstem of neonatal rats and, in different recombinant systems, specifically labels cells transfected with constructs containing the 5-HT1AR coding sequence (DeFelipe et al., 2001). A mouse monoclonal antibody to tryptase (1 in 1,000; Chemicon, Temecula, USA) was used to confirm the identity of mast cells examined for 5-HT and 5-HT1AR (see below). Labelling Technique. Sections (4 mm) were dewaxed with Bio-ClearÔ (Bio-Optica, Milan, Italy) for 15 min, rehydrated in graded concentrations of ethanol and boiled in Tris-EDTA buffer (25 min at 98 C) for antigen retrieval. The avidinebiotin complex horseradish peroxidase (ABC-HRP) technique was used for detection of bound primary antibody. Endogenous peroxidase was quenched with H2O2 0.3% in PBS (pH 7.4). After applying 10% goat serum for 40 min to minimize non-specific protein binding, the sections were incubated with the primary antibodies overnight at 4 C in a humid atmosphere. Biotinylated goat anti-rabbit antibody (BA 1000, 1 in 200; Vector Laboratories, Burlingame, USA) was applied for 40 min and avidinebiotin complex (PK6100, Vector Laboratories) for 30 min, both at room temperature. Amino-ethylcarbazole (FK-4200, Vector Laboratories) was used as chromogen. Sections were counterstained with haematoxylin and mounted under glycerol gelatin. As negative controls, the primary antibodies were omitted or replaced by normal rabbit immunoglobulins (X0936, Dako, Glostrup, Denmark). Immunofluorescence Labelling
In order to further demonstrate the presence of 5-HT and 5-HT1AR in mast cells, a double immunofluorescence (IF) technique was applied. Sections of normal skin were incubated in sequence with 10% goat serum, polyclonal antibodies to 5-HT or 5-HT1AR, biotinylated goat anti-rabbit secondary antibody and streptavidin-conjugated Texas Red (1 in 2,000; Vector Laboratories). After this procedure the mouse anti-tryptase antibody, followed by rabbit anti-mouse fluorescein isothiocyanate (FITC)-conjugated antibody (dilution 1 in 40; Dako), was applied. Slides were mounted under glycerol gelatin. Microscopy
One examiner (GKF), blinded to the grading of the individual tumours, evaluated all slides for
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Fig. 1. Representative photomicrographs of mast cell tumours of different Patnaik grades. (a) Grade I, (b) grade II and (c) grade III. HE. Bar, 50 mm.
immunohistochemical expression of 5-HT and 5HT1AR. Each slide was scored for the intensity of labelling of individual cells as: () no labelling, (+) weak, (++) moderate or (+++) strong labelling. The number of positively labelled cells in different areas of the tumour was evaluated semi-quantitatively and expressed as 0e25%, 25e50%, 50e75% and 75e100% of tumour cells. For the evaluation of the two-colour immunofluorescence, slides were examined with a fluorescence microscope at the excitation wavelengths of 465e495 (FITC) and 540e580 nm (Texas Red).
Results Normal Skin
Scattered mast cells were found in the superficial dermis of the normal skin, generally in association with small blood vessels. These cells had strong cytoplas-
mic expression of 5-HT and 5-HT1AR by immunohistochemistry (IHC) (Fig. 2). The identity of these mast cells was further confirmed by the double immunofluorescence technique, demonstrating co-localization of tryptase with 5-HT and 5-HT1AR (Fig. 3). Mast Cell Tumours
5-HT expression was found in 8/8 grade I, 7/8 grade II and 6/8 grade III MCTs. Grade I tumours had the greatest number of positively labelled cells and the majority of these had strong expression (+++) of 5-HT. In 4/8 tumours, 5-HT expression was greater in neoplastic cells located beneath the epidermis than in deeper infiltrates (Fig. 4). In 6/8 tumours, small clusters of positively labelled cells were found in the peritumoural connective tissue. In grade II tumours, immunolabelling was more variable. In 4/8 tumours almost all mast cells were positively labelled, although generally much less
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Fig. 2. Sections of normal skin, showing immunoreactivity for (a) 5-HT and (b) the 5-HT1A receptor in dermal mast cells. IHC. Bar, 50 mm.
Fig. 3. Co-localization of tryptase with 5-HT (aec) or 5-HT1AR (def) in normal cutaneous mast cells. (a) Tryptase (FITC). (b) 5-HT (Texas Red). (c) Tryptase + 5-HT. (d) Tryptase (FITC). (e) 5-HT1AR (Texas Red). (f) Tryptase + 5-HT1AR. IF. Bar, 50 mm.
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Fig. 4. Expression of 5-HT within a grade I mast cell tumour. (a) Strongly immunolabelled mast cells in superficial tumour area. (b) Predominance of weakly stained cells in deeper area. IHC. Bar, 50 mm.
intensely than those of grade I (75e100% of cells were of labelling intensity +). One tumour had 75e100% of mast cells strongly labelled (+++), while the remainder (3/8) had areas with 25e50% of mast cells moderately positive (++). Positively labelled cells were more often observed close to the epidermis or in more deeply located border areas of the tumours. Grade III MCTs had the smallest complement of positively labelled cells. In 4/8 tumours, 75e100% of cells were very weakly labelled or negative (Fig. 5a). The positive mast cells in these tumours were scattered or appeared in small clusters, often at the periphery of the tumour or in the adjacent connective tissue (Fig. 5b). These mast cells appeared well differentiated and were often intensely labelled (+++). In 2/8 tumours, positively labelled mast
cells tended to occur in the subepidermal area. In 2/8 tumours, mast cells were universally negative for 5-HT expression. 5-HT1AR expression was identified in 7/8 grade I, 5/8 grade II and 3/8 grade III tumours. In grade I tumours there was a gradient of labelling, with most positive cells situated near the epidermis (Fig. 6a). In these tumours, approximately 50e75% of the cells in the upper dermis were positively labelled and the intensity of labelling varying between + and +++ (most often ++). Grade II tumours had fewer immunolabelled cells than grade I, and in two of these tumours the largest component of positively labelled mast cells was in the subepidermal area. 5-HT1AR expressing mast cells were scattered or present in small clusters. The
Fig. 5. Expression of 5-HT within a grade III mast cell tumour. (a) Weakly labelled mast cells. (b) Strongly labelled mast cells at the edge of the tumour contrast with weakly labelled tumour cell mass at left. IHC. Bar, 50 mm.
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Discussion
Fig. 6. Expression of the 5-HT1A receptor. (a) Grade I mast cell tumour, superficial tumour area, with immunolabelled mast cells. (b) Grade III mast cell tumour showing essentially negative immunolabelling. IHC. Bar, 50 mm.
intensity of labelling varied, but weakly stained cells predominated. Tumours of grade III had the fewest immunolabelled cells and the majority of these tumours were essentially negative for 5-HT1AR expression (Fig. 6b). The immunoreactive mast cells were consistently only weakly labelled, and were found either subepidermally or clustered in the loose connective tissue surrounding the tumour.
The present study has demonstrated that normal and neoplastic canine mast cells express 5-HT and 5-HT1AR. Neoplastic mast cells expressed more 5HT than 5-HT1AR and more differentiated tumours had greater expression of both molecules. Few previous studies have examined expression of 5-HT by canine mast cells or MCTs. An early biochemical and histochemical study (Meier, 1959) showed only negligible quantities of 5-HT in canine MCTs. Another early investigation suggested the presence of 5-HT in a single canine MCT using a variety of histochemical techniques (Rice and Mitchener, 1961), but no 5-HT was found by enzymatic isotopic assay in canine fundic mucosal mast cells (Beaven et al., 1982). Fernandez et al. (2005) failed to demonstrate 5-HT by immunohistochemically labelling 21 canine cutaneous MCTs of various Patnaik grades. The discrepancies between the results of that study and ours may be explained by the different techniques used for antigen retrieval and the use of different anti-serotonin antibodies. Our observation that normal canine mast cells express 5-HT1AR is consistent with findings on human and murine mast cells (Lundeberg et al., 2002; Kushnir-Sukhov et al., 2006), and indicates that 5-HT1AR functions as an autoreceptor on mast cells, analogous to 5-HT producing rodent neural cells (Azmitia, 2001). 5-HT is known to act as a growth factor for several types of benign and malignant human tumour cell lines (Seuwen and Pouyssegur, 1990; Vicaut et al., 2000; Siddiqui et al., 2005), but 5-HT agonists may also inhibit tumour growth, possibly via effects on blood vessels (Baguley et al., 1993; Stu¨cker et al., 1997). We have shown a gradual loss of both 5-HT and 5-HT1AR expression with decreasing levels of differentiation of canine MCTs. The fact that canine MCTs have less serotonergic activity with increasing anaplasia is in contrast to studies of human prostatic carcinoma, where the most poorly differentiated cancer cells have the strongest 5-HT1AR signal (Dizeyi et al., 2004). The 5-HT1A receptor may have a differentiating effect, as reported in studies of neural cells (Azmitia, 2001), and our results may give indirect support for this mechanism acting in canine MCTs. This suggests that pharmacological activation of the 5-HT1A receptor may be one way to alter the level of differentiation of the tumour. However, the simultaneous loss of the ligand and its receptor with decreasing differentiation may also be interpreted as an effect, rather than a cause, of poor differentiation of the MCT.
Serotonin and 5-HT1A Receptor in Mast Cell Tumours
In some tumours, strongly 5-HT positive and welldifferentiated mast cells were noted at the periphery of the lesion and in the adjacent connective tissue. This was most obvious in grade III tumours, where these cells contrasted with the largely 5-HT negative poorly differentiated tumour cells. Mast cells may have different tumour-promoting actions, but they may also be part of an immune response aimed at eliminating tumour cells (Ch’ng et al., 2006). The well-differentiated appearance of the peripheral mast cells, irrespective of tumour grade, may suggest that these are a reactive, rather than neoplastic, population. This possibility has been highlighted by retrospective studies of canine cutaneous MCTs showing a low rate of local recurrence despite non-tumour-free surgical margins (Michels et al., 2002; Seguin et al., 2006). Erythema, oedema and itch (Darier’s sign), traditionally attributed to the release of histamine, heparin and other inflammatory mediators, can be detected upon manipulation of canine MCTs (Fox, 1998; Murphy, 2003). Systemic antihistamines are often used in an attempt to decrease such effects, especially when dealing with multiple tumours. It has been shown that 5-HT can produce itching, flare and wheals when applied to the human skin (Fjellner and Ha¨germark, 1979; Weissaar et al., 1997). 5-HT released from neoplastic mast cells may therefore contribute to the microvascular and pruritic phenomena associated with palpation of the tumour. Using a 5-HT antagonist in addition to antihistamines may thus be warranted to reduce these symptoms in dogs. In this respect, various symptoms of cutaneous mastocytosis in humans have been reported to be reduced after treatment with cyproheptadine, a combined histamine and 5-HT antagonist (Haneke, 1970; Fenske et al., 1985; Gasior-Chrzan and Falk, 1992; Enomoto et al., 1999). Mechanisms responsible for the infiltration of canine MCTs by eosinophils (Gross et al., 2005), have not yet been explored. Among mediators of eosinophil accumulation in inflammatory conditions of various species, eotaxin and other chemokines have been ascribed an important role, and activated mast cells may release eotaxin or generate its production by other cells by histamine release (Das et al., 1998; Williams and Galli, 2000; Menzies-Gow et al., 2004). However, a recent study in humans and mice indicates that 5-HT also acts as a chemoattractant for eosinophils and that this chemotaxis can be blocked by cyproheptadine treatment (Boehme et al., 2004). It is therefore possible that serotonergic activity of the neoplastic cells may be one mechanism underlying infiltration of eosinophils into canine MCTs.
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In summary, we have demonstrated the expression of 5-HT and its 5-HT1A receptor in canine cutaneous MCTs. This expression diminishes with decreasing differentiation of the neoplasm. Our findings imply that the impact of various biological actions of 5HT in the pathogenesis and therapeutic management of canine MCTs should be considered.
Acknowledgments We thank Professor E.C. Azmitia, New York University, for kindly supplying the 5-HT1A antibody and Mrs. K. Curra Holmberg and Mrs. A.-L. Kastman for their technical assistance.
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May 26th, 2008 ½ Received, Accepted, August 18th, 2008