J. Anat. (1975), 120, 2, pp. 337-348
337
With 15 figures Printed in Great Britain
The histochemistry of armadillo skin I. CAMPO-AASEN AND J. CONVIT
Histochemistry Department, Instituto Nacional de Dermatologia, Universidad Central de Venezuela, Apartado 4043, Caracas 101, Venezuela (Accepted 11 February 1975) INTRODUCTION
The identification by Storrs (1971) and by Kirchheimer & Storrs (1971), of Mycobacterium leprae in the skin of armadillos (Dasypus novemcinctus) 26 and 31 months after previous experimental infection has been satisfactorily confirmed by Convit & Pinardi (1974). These investigators, utilizing the staining reactions for M. leprae (Campo-Aasen & Convit, 1968; Convit & Pinardi, 1972; Fisher & Barksdale, 1973), the competence-in-clearing-bacilli (CCB) test (Convit, Avila, Goihman & Pinardi, 1972) and the Mitsuda test, have demonstrated that antigens prepared from armadillo skin and from infected human skin are similar in their reactions to those present in leprous and tuberculoid human patients (Convit et al. 1974). Owing to variations in host response to leprous infections, the disease as observed in the armadillo has thrown light upon the granulomatous infections generally. Transmission of leprosy to the local armadillo (D. sabanicola) has been successfully effected in the Laboratorio de Leprologia of the International Reference Center for Histological Identification by Convit et al. (personal communication). Thus the animal is established as an experimental model for epidemiological, immunological and biochemical investigation of leprous infections (Storrs, 1971; Kirchheimer & Storrs, 1971; Convit & Pinardi, 1974). Therefore, a histochemical study of normal armadillo skin should be of interest as a basis for a later comparative study of enzyme alterations in the macrophage and lymphocyte cellular populations, with a view to elucidating macrophage-lymphocyte interactions in the immunity phenomena of leprosy. The present work reports such a preliminary study. MATERIAL AND METHODS
Biopsies of the ventral skin were taken from healthy armadillo near the rear limbs. Pieces of skin approximately 1 cm2 were each divided in two parts, one being fixed immediately in cold neutral formalin, while the other was fixed in formol-calcium. Another piece (0 5 cm) was fixed in 2-5 % buffered glutaraldehyde for electron microscopy. In all, 13 staining methods were employed, including the following where it is desirable to give some technical details: Alkaline phosphatase (Burstone, 1958). A simultaneous coupling azo-dye method, using naphthol-AS-B1 phosphoric acid as substrate, Fast Red T.R. as salt, pH 9-2, 30 minutes' incubation at 22 'C.
338
I. CAMPO-AASEN AND J. CONVIT
Acid phosphatase (Gomori, 1950); 15 minutes' incubation at 37 °C, methyl green
counterstain, pH 5 0. Non-specific esterases. (1) Alpha-naphthyl acetate (Pearse, 1972); At pH 6 5, pararosanilin HCL as coupler (Barka & Anderson, 1962), 5 minutes' incubation. (2) Napthol-As acetate (Gomori method as modified by Pearse, 1955). Using Fast Red Violet LB salt as coupler, pH 6-8, 30 minutes' incubation at 22 'C. (3) Indoxyl-acetate esterase (Holt, 1956). Using 4-chloro-5-bromo-indoxyl as substrate, 2 hours' incubation at 37 'C, pH 6-2, carmalum counterstain. Beta-glucuronidase (Pugh & Walker, 1961; Hayashi, 1964). Post-coupling azo dye principle using the naphthol-AS-B-D glucuronide (Chugai Pharmaceutical, Tokyo) as substrate, pararosanilin HCL as coupler, incubation 2 hours at pH 5 0, methyl green counterstain. Incubations were also carried out at pH 4 0 and 4-5. Acetyl-cholinesterases (Karnovsky & Roots, 1964). For nerve endings and fibres. Dopa-oxidase (Pearse's 1960 adaptation of the method of Becker, Praven & Thatcher). Fresh sections were fixed in cold neutral formalin for only 15 minutes; 2 hours' incubation. Bodian (Armed Forces Institute of Pathology, 1969, pp. 162-163). For demonstration of fibres and nerve endings: 8 ,um sections in paraffin. Fontana (Culling, 1963). Silver impregnation technique for melanin, toluidine blue (pH 4 5) for metachromasia, methyl green pyronin for A.R.N., using a modification of the method of Trevan & Sharrock. Oil Red 0 (Lillie & Ashburn, 1943). For neutral fatty acids. RESULTS
Alkaline phosphatase. This was positively indicated by brick-red precipitate in longitudinal and transverse sections of the hairs, in the capillaries, in the central region of the crystals (arrow) which are common in the dermis of this animal, and in the outer layer of the external root sheath of the hairs. Dendritic cells or positive melanocytes were visible in the epidermis (Figs. 1, 2). Acidphosphatase. As in other animals, including man, the granular layer is intensely positive, as shown by the blackish-brown precipitate (Fig. 3). Alpha-naphthyl acetate esterase. Positive cells are marked by brownish-red precipitate. They are very large cells, extensively ramified into dendrites, some extending throughout the epidermis, others confined to the dermo-epidermal junction, while others were in the upper dermis. The brownish precipitate is coarse and granular, extending to the ends of the dendrites (Figs. 4-6) rendering these cells very conspicuous. Naphthol-AS acetatase. This enzyme does not show any marked peculiarity in armadillo skin. Indoxyl acetatase. The esterase activity is shown by a diffuse indigo-blue colour in the basement layer of the epidermis, and in the hair (Fig. 7). The blue deposit is also seen in cells surrounding the capillaries considered by us to be mastocytes (Fig. 8). Beta-glucuronidase. A brilliant red deposit demonstrates activity in the cornified
.~ ~ ~ ~ ~ . , . . ; . . Armadillo skin
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.......
339
......... ..
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.1" ,j, """t I Fig. 1. Alkaline phosphatase, Burstone technique. Brick-red precipitate in dermal capillaries, transverse hair sections. x 125. Fig. 2. Positivity of alkaline phosphatase in longitudinal section of hair. Birefringent crystals (arrows) are visible, with red alkaIine phosphatase deposit in the centres. x 125.
340
I. CAMPO-AASEN AND J. CONVIT
AV
3
...Kt&t
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Fig. 3. Acid phosphatase, Gomori's technique. Blackish-brown diffuse band in the granular layer. x 175. Fig. 4. Alpha-naphthyl-acetate esterase (technique of Nachlas & Seligman). Intensely positive dendritic cell, showing coarse, granular, brownish deposit around the nucleus and in the
dendrites. x 125.
Armadillo skin
341
5
Fig. 5. Alpha-naphthyl-acetate esterase. Activity demonstrated by granular precipitate. The dendrites extend through much of the epidermal layers. x 175. Fig. 6. The dendrites are extremely long and show many ramifications. x 175.
342
I. CAMPO-AASEN AND J. CONVIT
VY
8 Fig. 7. Indoxyl-acetate esterase, Holt's technique. Diffuse activity in epidermis, in transverse sections of hairs. x 125. Fig. 8. Indoxyl-acetate esterase, Holt's technique. Activity demonstrated by indigo blue precipitate in cells of the perivascular region, considered to be mastocytes. x 425.
Armadillo skin
343
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Fig. 9. Beta-glucuronidase. Positive cornified layers. x 125. Fig. 10. Beta-glucuronidase, post coupling azo dye technique. Giant cell stained brilliant red. Birefringent crystal visible in upper extremity. x 125.
344
*.
I. CAMPO-AASEN AND J. CONVIT
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11
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Fig. 11. Beta-glucuronidase. Positive giant cell, with crystal in its middle part. Methyl green stained nuclei of granuloma cells. x 175. Fig. 12. Acetyl-cholinesterase (Karnovsky-Roots). Nerve endings near basal layer of the epidermis. x 175.
Armadillo skin
345
Fig. 13. Dopa-oxidase. Becker technique. Mastocytes in the perivascular region and in upper part of the dermis intensely stained black. No reactivity of dendritic cells is visible. x 175. Fig. 14. Dopa-oxidase at higher magnification. Mastocytes show strong positivity. Dendritic cells are doubtfully positive. x 175.
I. CAMPO-AASEN AND J. CONVIT
346
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.1... _g......
iia;
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layer (Fig 9)Inthedermisaspartof scantygranlomas A,
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are observed very large elongated cells having several nuclei stained with methyl green and the cytoplasm completely covered with scarlet red deposit. These cells are always seen around the crystals (arrows) which are frequent in the skin of this animal (Figs. 10, 1 1). The results were similar at pH 40 and 45. These crystals are negative to cholesterol and fat stains. Acetyl-cholinesterase. This enzyme was present in afferent nerve fibres with endings at the basal layer of the epidermis (Fig. 12). Dopa-oxidase. Cells with the characteristics of mastocytes and showing a black precipitate are seen in the perivascular region and in the upper dermis (Figs. 13, 14) near the dermo-epidermal junction. This observation was made with the electron microscope. There were no dopa-positive melanocytes in the sections studied. Metachromasia was not very evident with toluidine blue in mastocytes at 4e5, pH nor were mastocytes very evident with methyl green pyronin. The Bodian method failed to demonstrate nerve fibres in the skin of the armadillo despite the evidence of their presence in the acetyl cholinesterase preparations. DISCUSSION
The skin of the armadillo (D. sabanicola) shows certain enzymic peculiarities. In the epidermis, the dendritic cells (melanocytes?) were dopa-negative, but were strongly positive for such enzymes as alkaline phosphatase and alpha-naphthyl acetate esterase. These cells were very large and often staining for alkaline phospha-
Armadillo skin
347
tase and alpha naphthyl acetatase it was clear that their dendrites extended throughout the layers of the epidermis. They were found in all layers of the epidermis, in the dermo-epidermal junction, and even in the upper dermis. The ability of these cells in the armadillo to synthetize the enzymatic complex of tyrosinase is problematical. In the upper dermis and in the perivascular region, certain intensely dopa-positive cells are considered to be mastocytes from their morphology. However, little metachromasia was revealed by toluidine blue, and affinity for methyl green pyronine was slight. Three cells did not fluoresce nor did they show acid phosphatase or betaglucuronidase activity. The non-specific positivity of mastocytes for dopa has been observed in other species (Okun, Niebaner & Hamada, 1969) and Campo-Aasen & Convit (1973): it is similar to that observed in eosinophils and erythrocytes by van Duijn (1953), and it has been identified as due to peroxidase rather than tyrosinase (Okun et al. 1970). It is possible that the crystals in the dermis of the armadillo may have been observed by others; they are easily demonstrated with hematoxylin and eosin (Fig. 15). Curiously enough, their centres are alkaline phosphatase-positive, suggesting that they may be formed about a capillary (as shown in Fig. 2). They are negative for cholesterol and neutral fat. Possibly these crystals are associated with giant cells which are positive for beta-glucuronidase. This seems to be the case in other granulomas (e.g. human granulomas produced by P. brasiliensis, personal observation). There is no ready explanation for the presence of these cells, but they may be a by-product of the intense metabolism of the cells which defend the organism against alien invaders of the dermis. The ultrastructure of these cells in the skin of the armadillo is being studied by us in the hope that their morphology and function will become clearer. SUMMARY
The histochemistry of armadillo skin has been studied. The dendritic cells are extremely large, very sharply outlined by methods for alkaline phosphatase and alpha-naphthyl-acetate esterase, and they are dopa-negative. The mastocytes, however, are dopa-oxidase-positive, probably due to peroxidase rather than tyrosinase activity. The giant cells of the granulomas normally seen in the dermis of the armadillo are strongly beta-glucuronidase-positive. These giant cells are evidently foreign body cells reacting to the crystals always present in the dermis of the armadillo. The centre of these crystals, which are cholesterol and fat-negative, is alkaline phosphatase-positive. Further study of the mastocytes and dendritic cells is necessary to elucidate their nature.
This work was supported by Grant no. DF-S1-153 from Consejo de Investigaciones Cientificas y Tecnologicas (C.O.N.I.C.I.T.) of Caracas.
23
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I. CAMPO-AASEN AND J. CONVIT
REFERENCES BARKA, T. & ANDERSON, P. J. (1962). Histochemical methods for acid phosphatase using hexazonium pararosanilin as a coupler. Journal of Histochemistry and Cytochemistry 10, 741-763. BURSTONE, M. S. (1958). Histochemical comparison of Naphthol-AS substrates using frozen-dried tissues. Journal of Histochemistry and Cytochemistry 6, 87-89. CAMPO-AASEN, I. & CONVIT, J. (1968). Identification of the noncultivable pathogenic mycobacteria (M. leprae and M. lepramurium). International Journal ofLeprosy 36, 166-170. CAMPo-AASEN, I. & CONVIT, J. (1973). Histochemical identification of non-culturable myobacteria (M. leprae and M. lepremurium). II. Dopa-oxidase. Histochimie 35, 63-66. CAMPo-AASEN, I. & ALBORNOZ, M. C. (1975). The carboxylic activity of giant cells in human granuloma produced by P. brasiliensis. Sabourandia. (In the Press.) CONVIT, J., AVILA, J. L., GOIHMAN, M. & PINARDI, M. E. (1972). A test for the determination of competency in clearing bacilli in leprosy patients. Bulletin of the World Health Organization 46, 821-826. CONVIT, J. & PINARDI, M. E. (1972). A simple method for the differentiation of M. leprae from other mycobacteria through routine staining technics. International Journal of Leprosy 40, 130-132. CONVIT, J. & PINHARDI, M. E. (1974). Confirmation in the armadillo. Science 184, 1191-1192. CONVIT, J., PINARDI, M. E., RODRIGUEZ-OCHOA, G., ULRICH, M., AVILA, J. L. & GOIMAN, M. (1974). Elimination of Mycobacterium leprae subsequent to local in vivo activation of macrophages in lepromatous leprosy by other mycobacteria. Clinical and Experimental Immunology 17, 261-265. CULLING, C. F. A. (1963). Handbook of Histopathological Techniques, 2nd ed., pp. 254, 232, 219. London: Butterworth. DUIJN, P. VAN (1953). Inactivation experiments on the dopa-factor. Journal of Histochemistry and Cytochemistry 1, 143-150. FISHER, C. A. & BARKSDALE, L. (1973). Cytochemical reactions of human leprosy bacilli and mycobacteria: ultrastructural implications. Journal of Bacteriology 113, 1389-1399. GOMORI, G. (1950). An improved histochemical technic for acid phosphatase. Stain Technology 25, 81-85. HAYASHI, M. (1964). Distribution of fl-glucuronidase activity in rat tissues employing the naphthol AS-BI glucuronide hexazonium pararosanilin method. Journal of Histochemistry and Cytochemistry 12, 659-669. HOLT, S. J. (1956). The value of fundamental studies of staining reactions in enzyme histochemistry with reference to indoxyl methods for esterases. JournalofHistochemistry and Cytochemistry 4,541-554. KARNOVSKY, M. & RO3TS, L. (1964). A 'direct coloring' thiocholine method for cholinesterases. Journal of Histochemistry and Cytochemistry 12, 219-221. KIRCHHEIMER, W. F. & STORRS, E. E. (1971). Attempts to establish the armadillo (Dasypus ntovemcinctus Linn.) as a model for the study of leprosy. International Journal of Leprosy 39, 693-702. LEWIS, P. R. & LOBBAN, M. C. (1961). The chemical specificity of the Schultz test for steroids. Journal of Histochemistry and Cytochemistry 9, 2-10. LILLIE, R. D. & ASHBURN, L. L. (1943). Supersaturated solutions of fat stains in dilute isopropanol for demonstration of acute fatty degeneration not shown by Herzheimer technique. Archives of Pathology 36, 432-435. OKUN, M., NIEBAUER, G. & HAMADA, G. (1969). The histochemical tyrosine-dopa and its use in localizing activity in mast cells. Journal of Investigative Dermatology 53, 39-45. OKUN, M. R., EDELSTEIN, L. M., OR, N., HAMADA, G. & DONNELAN, B. (1970). The role of peroxidase vs. the role of tyrosinase enzymatic conversion of tyrosine to malanin in melanocytes, mast cells and eosinophils. Journal of Investigative Dermatology 55, 1-12. PEARSE, A.G. E. (1955). Azo dye methods in enzyme histochemistry. International Review of Cytology 111, 329-358. PEARSE, A. G. E. (1960). Histochemistry Theoretical and Applied, p. 905. London: Churchill. PEARSE, A. G. E. (1972). Histochemistry, Theoretical and Applied, 3rd ed., p. 1303. London: Churchill. PUGH, D. & WALKER, P. G. (1961). Histochemical localization of 8-glucuronidase and N-acetyl-flglucosaminidase. Journal of Histochemistry and Cytochemistry 9, 105-106. STORRS, E. E. (1971). The nine-banded armadillo: A model for leprosy and other bio-medical research. International Journal of Leprosy 39, 703-714.
337
With 15 figures Printed in Great Britain
The histochemistry of armadillo skin I. CAMPO-AASEN AND J. CONVIT
Histochemistry Department, Instituto Nacional de Dermatologia, Universidad Central de Venezuela, Apartado 4043, Caracas 101, Venezuela (Accepted 11 February 1975) INTRODUCTION
The identification by Storrs (1971) and by Kirchheimer & Storrs (1971), of Mycobacterium leprae in the skin of armadillos (Dasypus novemcinctus) 26 and 31 months after previous experimental infection has been satisfactorily confirmed by Convit & Pinardi (1974). These investigators, utilizing the staining reactions for M. leprae (Campo-Aasen & Convit, 1968; Convit & Pinardi, 1972; Fisher & Barksdale, 1973), the competence-in-clearing-bacilli (CCB) test (Convit, Avila, Goihman & Pinardi, 1972) and the Mitsuda test, have demonstrated that antigens prepared from armadillo skin and from infected human skin are similar in their reactions to those present in leprous and tuberculoid human patients (Convit et al. 1974). Owing to variations in host response to leprous infections, the disease as observed in the armadillo has thrown light upon the granulomatous infections generally. Transmission of leprosy to the local armadillo (D. sabanicola) has been successfully effected in the Laboratorio de Leprologia of the International Reference Center for Histological Identification by Convit et al. (personal communication). Thus the animal is established as an experimental model for epidemiological, immunological and biochemical investigation of leprous infections (Storrs, 1971; Kirchheimer & Storrs, 1971; Convit & Pinardi, 1974). Therefore, a histochemical study of normal armadillo skin should be of interest as a basis for a later comparative study of enzyme alterations in the macrophage and lymphocyte cellular populations, with a view to elucidating macrophage-lymphocyte interactions in the immunity phenomena of leprosy. The present work reports such a preliminary study. MATERIAL AND METHODS
Biopsies of the ventral skin were taken from healthy armadillo near the rear limbs. Pieces of skin approximately 1 cm2 were each divided in two parts, one being fixed immediately in cold neutral formalin, while the other was fixed in formol-calcium. Another piece (0 5 cm) was fixed in 2-5 % buffered glutaraldehyde for electron microscopy. In all, 13 staining methods were employed, including the following where it is desirable to give some technical details: Alkaline phosphatase (Burstone, 1958). A simultaneous coupling azo-dye method, using naphthol-AS-B1 phosphoric acid as substrate, Fast Red T.R. as salt, pH 9-2, 30 minutes' incubation at 22 'C.
338
I. CAMPO-AASEN AND J. CONVIT
Acid phosphatase (Gomori, 1950); 15 minutes' incubation at 37 °C, methyl green
counterstain, pH 5 0. Non-specific esterases. (1) Alpha-naphthyl acetate (Pearse, 1972); At pH 6 5, pararosanilin HCL as coupler (Barka & Anderson, 1962), 5 minutes' incubation. (2) Napthol-As acetate (Gomori method as modified by Pearse, 1955). Using Fast Red Violet LB salt as coupler, pH 6-8, 30 minutes' incubation at 22 'C. (3) Indoxyl-acetate esterase (Holt, 1956). Using 4-chloro-5-bromo-indoxyl as substrate, 2 hours' incubation at 37 'C, pH 6-2, carmalum counterstain. Beta-glucuronidase (Pugh & Walker, 1961; Hayashi, 1964). Post-coupling azo dye principle using the naphthol-AS-B-D glucuronide (Chugai Pharmaceutical, Tokyo) as substrate, pararosanilin HCL as coupler, incubation 2 hours at pH 5 0, methyl green counterstain. Incubations were also carried out at pH 4 0 and 4-5. Acetyl-cholinesterases (Karnovsky & Roots, 1964). For nerve endings and fibres. Dopa-oxidase (Pearse's 1960 adaptation of the method of Becker, Praven & Thatcher). Fresh sections were fixed in cold neutral formalin for only 15 minutes; 2 hours' incubation. Bodian (Armed Forces Institute of Pathology, 1969, pp. 162-163). For demonstration of fibres and nerve endings: 8 ,um sections in paraffin. Fontana (Culling, 1963). Silver impregnation technique for melanin, toluidine blue (pH 4 5) for metachromasia, methyl green pyronin for A.R.N., using a modification of the method of Trevan & Sharrock. Oil Red 0 (Lillie & Ashburn, 1943). For neutral fatty acids. RESULTS
Alkaline phosphatase. This was positively indicated by brick-red precipitate in longitudinal and transverse sections of the hairs, in the capillaries, in the central region of the crystals (arrow) which are common in the dermis of this animal, and in the outer layer of the external root sheath of the hairs. Dendritic cells or positive melanocytes were visible in the epidermis (Figs. 1, 2). Acidphosphatase. As in other animals, including man, the granular layer is intensely positive, as shown by the blackish-brown precipitate (Fig. 3). Alpha-naphthyl acetate esterase. Positive cells are marked by brownish-red precipitate. They are very large cells, extensively ramified into dendrites, some extending throughout the epidermis, others confined to the dermo-epidermal junction, while others were in the upper dermis. The brownish precipitate is coarse and granular, extending to the ends of the dendrites (Figs. 4-6) rendering these cells very conspicuous. Naphthol-AS acetatase. This enzyme does not show any marked peculiarity in armadillo skin. Indoxyl acetatase. The esterase activity is shown by a diffuse indigo-blue colour in the basement layer of the epidermis, and in the hair (Fig. 7). The blue deposit is also seen in cells surrounding the capillaries considered by us to be mastocytes (Fig. 8). Beta-glucuronidase. A brilliant red deposit demonstrates activity in the cornified
.~ ~ ~ ~ ~ . , . . ; . . Armadillo skin
*~~~~~~~~~~~~~~~~~~
:.fstfs,
.......
339
......... ..
i : . , -i ! I-I |
...
''..... ......
#t:.
'U.
.1" ,j, """t I Fig. 1. Alkaline phosphatase, Burstone technique. Brick-red precipitate in dermal capillaries, transverse hair sections. x 125. Fig. 2. Positivity of alkaline phosphatase in longitudinal section of hair. Birefringent crystals (arrows) are visible, with red alkaIine phosphatase deposit in the centres. x 125.
340
I. CAMPO-AASEN AND J. CONVIT
AV
3
...Kt&t
__o:
Fig. 3. Acid phosphatase, Gomori's technique. Blackish-brown diffuse band in the granular layer. x 175. Fig. 4. Alpha-naphthyl-acetate esterase (technique of Nachlas & Seligman). Intensely positive dendritic cell, showing coarse, granular, brownish deposit around the nucleus and in the
dendrites. x 125.
Armadillo skin
341
5
Fig. 5. Alpha-naphthyl-acetate esterase. Activity demonstrated by granular precipitate. The dendrites extend through much of the epidermal layers. x 175. Fig. 6. The dendrites are extremely long and show many ramifications. x 175.
342
I. CAMPO-AASEN AND J. CONVIT
VY
8 Fig. 7. Indoxyl-acetate esterase, Holt's technique. Diffuse activity in epidermis, in transverse sections of hairs. x 125. Fig. 8. Indoxyl-acetate esterase, Holt's technique. Activity demonstrated by indigo blue precipitate in cells of the perivascular region, considered to be mastocytes. x 425.
Armadillo skin
343
,.Ae'
e-
VM
3WI
....9
Fig. 9. Beta-glucuronidase. Positive cornified layers. x 125. Fig. 10. Beta-glucuronidase, post coupling azo dye technique. Giant cell stained brilliant red. Birefringent crystal visible in upper extremity. x 125.
344
*.
I. CAMPO-AASEN AND J. CONVIT
BiSjS _
11
lk
*-
1 2
Fig. 11. Beta-glucuronidase. Positive giant cell, with crystal in its middle part. Methyl green stained nuclei of granuloma cells. x 175. Fig. 12. Acetyl-cholinesterase (Karnovsky-Roots). Nerve endings near basal layer of the epidermis. x 175.
Armadillo skin
345
Fig. 13. Dopa-oxidase. Becker technique. Mastocytes in the perivascular region and in upper part of the dermis intensely stained black. No reactivity of dendritic cells is visible. x 175. Fig. 14. Dopa-oxidase at higher magnification. Mastocytes show strong positivity. Dendritic cells are doubtfully positive. x 175.
I. CAMPO-AASEN AND J. CONVIT
346
P~..
pwm.
t,.,t.lr10Xli;l>
.1... _g......
iia;
;f
t
'....
i- t d
s x
layer (Fig 9)Inthedermisaspartof scantygranlomas A,
:'-
g
are observed very large elongated cells having several nuclei stained with methyl green and the cytoplasm completely covered with scarlet red deposit. These cells are always seen around the crystals (arrows) which are frequent in the skin of this animal (Figs. 10, 1 1). The results were similar at pH 40 and 45. These crystals are negative to cholesterol and fat stains. Acetyl-cholinesterase. This enzyme was present in afferent nerve fibres with endings at the basal layer of the epidermis (Fig. 12). Dopa-oxidase. Cells with the characteristics of mastocytes and showing a black precipitate are seen in the perivascular region and in the upper dermis (Figs. 13, 14) near the dermo-epidermal junction. This observation was made with the electron microscope. There were no dopa-positive melanocytes in the sections studied. Metachromasia was not very evident with toluidine blue in mastocytes at 4e5, pH nor were mastocytes very evident with methyl green pyronin. The Bodian method failed to demonstrate nerve fibres in the skin of the armadillo despite the evidence of their presence in the acetyl cholinesterase preparations. DISCUSSION
The skin of the armadillo (D. sabanicola) shows certain enzymic peculiarities. In the epidermis, the dendritic cells (melanocytes?) were dopa-negative, but were strongly positive for such enzymes as alkaline phosphatase and alpha-naphthyl acetate esterase. These cells were very large and often staining for alkaline phospha-
Armadillo skin
347
tase and alpha naphthyl acetatase it was clear that their dendrites extended throughout the layers of the epidermis. They were found in all layers of the epidermis, in the dermo-epidermal junction, and even in the upper dermis. The ability of these cells in the armadillo to synthetize the enzymatic complex of tyrosinase is problematical. In the upper dermis and in the perivascular region, certain intensely dopa-positive cells are considered to be mastocytes from their morphology. However, little metachromasia was revealed by toluidine blue, and affinity for methyl green pyronine was slight. Three cells did not fluoresce nor did they show acid phosphatase or betaglucuronidase activity. The non-specific positivity of mastocytes for dopa has been observed in other species (Okun, Niebaner & Hamada, 1969) and Campo-Aasen & Convit (1973): it is similar to that observed in eosinophils and erythrocytes by van Duijn (1953), and it has been identified as due to peroxidase rather than tyrosinase (Okun et al. 1970). It is possible that the crystals in the dermis of the armadillo may have been observed by others; they are easily demonstrated with hematoxylin and eosin (Fig. 15). Curiously enough, their centres are alkaline phosphatase-positive, suggesting that they may be formed about a capillary (as shown in Fig. 2). They are negative for cholesterol and neutral fat. Possibly these crystals are associated with giant cells which are positive for beta-glucuronidase. This seems to be the case in other granulomas (e.g. human granulomas produced by P. brasiliensis, personal observation). There is no ready explanation for the presence of these cells, but they may be a by-product of the intense metabolism of the cells which defend the organism against alien invaders of the dermis. The ultrastructure of these cells in the skin of the armadillo is being studied by us in the hope that their morphology and function will become clearer. SUMMARY
The histochemistry of armadillo skin has been studied. The dendritic cells are extremely large, very sharply outlined by methods for alkaline phosphatase and alpha-naphthyl-acetate esterase, and they are dopa-negative. The mastocytes, however, are dopa-oxidase-positive, probably due to peroxidase rather than tyrosinase activity. The giant cells of the granulomas normally seen in the dermis of the armadillo are strongly beta-glucuronidase-positive. These giant cells are evidently foreign body cells reacting to the crystals always present in the dermis of the armadillo. The centre of these crystals, which are cholesterol and fat-negative, is alkaline phosphatase-positive. Further study of the mastocytes and dendritic cells is necessary to elucidate their nature.
This work was supported by Grant no. DF-S1-153 from Consejo de Investigaciones Cientificas y Tecnologicas (C.O.N.I.C.I.T.) of Caracas.
23
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