192 DISC

Veterinary Dermatology 2000, 11, 241246

Progressive Langerhans' cell histiocytosis in a puppy

MASAHIKO NAGATA,* MASAHIKO HIRATA,{ TAKUO ISHIDA,{ SHINICHI
HIRATA{ and HIROKO NANKO}
*Animal Dermatology Centre, ASC, Jindaijihigashi Chofu, Tokyo, Japan
*Animal Medical Centre, Nihon University, Kanagawa, Japan
{Department of Clinical Pathology, Nippon Veterinary and Animal Science University School of Veterinary
Medicine, Tokyo, Japan
{Hirata Animal Hospital, Yamaguchi, Japan
}Department of Dermatology Tokyo Koseinenkin Hospital, Tokyo, Japan
(Received 5 January 1999; accepted 30 July 1999)

Abstract A 8-month-old, female miniature Dachshund dog was presented for the complaint of pruritic,
generalized, multiple nodules and plaques. Two months previously, a nodule on the left pinna was excised and
diagnosed as a cutaneous histiocytoma. One month post-excision, a nodule reappeared at the same site and,
shortly thereafter, additional nodules developed. Histopathological examination revealed a diuse
proliferation of histiocytic cells, which reacted strongly to antibodies for vimentin and lysozyme.
Immunophenotypic analysis showed that most of the cells expressed CD1a, CD1c, CD11c, CD18, CD45
and MHC class II markers. Electron microscopic examination revealed cytoplasmic lopodia and
paracrystalline structures. These ndings indicated that the cells originated from Langerhans' cell. The
disease progressed despite glucocorticoid therapy and griseofulvin was administered as an immunomodulating
drug. All lesions resolved completely after 7 weeks of griseofulvin treatment. The dog, however, died three
months later after discontinuation of griseofulvin therapy and a necropsy was not performed. It is considered
that the present canine dermatosis corresponds with a severe form of Langerhans' cell histiocytosis in humans
rather than canine cutaneous histiocytoma.
Keywords: cutaneous histiocytoma, dog, griseofulvin, immunophenotype, nodules.

INTRODUCTION
Primary histiocytic dermatoses are a group of skin
disorders in which histiocytic cells predominate
histologically in the absence of any known proliferative stimuli such as infectious agents, foreign
bodies and metabolic products. In dogs, there are
four well-dened histiocytic proliferative disorders.112
Canine cutaneous histiocytoma (CCH) usually occurs
as a single skin lesion in younger dogs.46 Lesions
tend to be localized on the ears, muzzle and the
extremities. Clinically they appear as rapidly growing, alopecic, erythematous, frequently ulcerated
dome-shaped nodules. Spontaneous regression is the
usual behaviour of CCH. Cutaneous histiocytosis
(CH) primarily involves the skin and subcutis.7,8 The
lesions are characterized by multiple plaques or
nodules that tend to wax and wane and eventually
may spontaneously regress. Systemic histiocytosis
(SH) has occurred most commonly in closely related
Bernese Mountain dogs and less commonly in other
breeds.9,10 The lesions are generalized with a marked
tendency to involve skin and peripheral lymph nodes.
Although the clinical course is variable, SH tends to

Correspondence: M. Nagata DVM, Animal Dermatology Centre,

ASC, 13-2 Jindaijihigashi Chofu, Tokyo 1820012, Japan. Tel.:
(81) 424809342/Fax: (81) 424416095.
# 2000 Blackwell Science Ltd

be a slowly progressive disease that often requires

continuous immunosuppressive therapy. Malignant
histiocytosis (MH), the fourth type, is a rapidly
progressive, multicentric disorder, which rarely involves the skin and is invariably fatal.11,12 However,
it is not always easy to dierentiate these disorders
from granulomatous, reactive inammatory disorders
or lymphoproliferative diseases, or to distinguish
between the syndromes in regular paran sections.
Recent studies have shown immunophenotypic analysis is useful to identify the cell lineages.46 It has
been demonstrated that the histiocytic diseases are
largely derived from a proliferation of Langerhans'
cells (LCs) rather than macrophages.46 This report
describes a miniature Dachshund puppy, with progressive, multiple CCH which was investigated by
immunohistochemical staining, immunophenotypic
analysis and electron microscopic examination.
CASE REPORT
A 8-month-old, female miniature Dachshund dog
was presented for the complaint of moderately
pruritic, generalized, multiple nodules and plaques
(Fig. 1). Two months previously, a nodule had been
observed on the left pinna. The nodule was excised at
a local veterinary clinic and diagnosed as CCH by
routine histopathology. One month after surgical
241

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M. Nagata et al.

Figure 1. Photograph of the dog. Note

generalized, multiple nodules and
plaques, especially on the face and
extremities.

excision, a nodule reappeared at the same site, and

the dog subsequently developed generalized nodules
that were most numerous on the face and the
extremities. The lesions consisted of erythematous
papules, plaques, nodules, ulcers, crusts and alopecia.
Other than for the skin, the dog appeared healthy.
The three littermates did not have any skin lesions.
A complete blood count, serum biochemical
analysis and urinalysis revealed no abnormalities.
Abdominal radiographs also were normal. Because of
the progressive nature of the skin masses, skin
biopsies from papules were obtained. Tissues were
xed in 10% neutral buered formalin and then
processed routinely. The sections were stained with
haematoxylin and eosin (H & E), and examined by
light microscopy. Histopathological examination of
the sections revealed a dense and diuse homogeneous accumulation of pleomorphic cells in the
dermis and the subcutis (Fig. 2). Intraepithelial

inltrations of proliferative cells were observed.

These cells were ovoid and had abundant pale
eosinophilic cytoplasm and oval, round, folded and
pleomorphic nuclei with one or sometimes two small,
well-stained nucleoli. Mitosis and atypia were not
easily recognized (Fig. 3).
Immunohistochemical staining of the biopsy specimens was performed for the following: vimentin
(M0725; DAKO, Denmark), lysozyme (A0099;
DAKO, Denmark), cytokeratin (M821; DAKO,
Denmark), S-100 (Z0311; DAKO, Denmark), a-1antichymotrypsin (A022; DAKO, Denmark), and a1-antitrypsin (A012; DAKO, Denmark) using the
avidin-biotin complex (ABC) peroxidase detection
system (2 V-1000010; Vector Laboratories,
USA).5,13 The cells reacted strongly to antibodies
for vimentin and lysozyme but were unreactive to
antibodies for cytokeratin, protein S-100, a-1-antichymotrypsin and a-1-antitrypsin.

Figure 2. Histopathology of the

nodule. Note a poorly demarcated
intracutaneous nodule with diuse
proliferations of histiocytic cells.
H&E6158.
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Progressive histiocytosis in a puppy

243

Figure 3. High magnication of Figure 2.

Note abundant pale eosinophilic cytoplasm
and oval, round, folded, and pleomorphic
nuclei. H&E6 2086.

In addition, immunophenotypic analysis of the

cells using ow cytometry was performed.14 A nodule
was aspirated using a 25 gauge needle and the cells
were resuspended in RPMI 1640 with 10% (v/v)
foetal bovine serum. The cell suspension was layered
over Ficoll (Histopaque 1.083, Sigma) and centrifuged at 1500 r.p.m., 400 g for 30 min. The tissue cell
band, located at the serumFicoll interface, was
removed and the cells were incubated twice with
phosphate-buered saline. The cells were reacted with
monoclonal antibodies specic for the following
leucocyte antigens: CD1a, CD1c, CD3, CD4, CD8,
CD11c, CD18, CD21, CD45, MHC class II and Thy1. Positive cells were counted using a ow cytometer
(Cyto ACE-150; Nihon Kohden, Japan). The results
were as follows: CD1a (97.1%), CD1c (96.8%), CD3
(0.7%), CD4 (0.7%), CD8 (4.3%), CD11a (27.2%),
CD11b (46.8%), CD11c (76.9%), CD18 (96.7%),
CD21 (0.7%), CD45 (99.5%), CD49d (84.4%), MHC
class II (99.7%) and Thy1 (3.2%).
Electron microscopy also was performed. The
formalin-xed tissues were rst rexed in 2.5%
glutaraldehyde after routine deparanation and then
embedded in epoxy resin (EPON812). Ultrathin
sections were cut and stained with uranyl acetate
and lead citrate for examination by transmission
electron microscopy (Hitachi H-7000). Electron
microscopic examination revealed convoluted nuclei,
abundant lysosomes, cytoplasmic lopodia and
paracrystalline structures (Fig. 4). These ndings
indicated that the cells originated from LC.46
Despite two months of oral prednisolone (maximum dosage 2 mg kg71 BID), the skin lesions
progressed and the dog's condition worsened, as
evidenced by signs of depression and anorexia.
Griseofulvin (Grisovin-FP, Sankyo, Tokyo, Japan)
30 mg kg71 BID was then administered as an
immunomodulating drug15,16 and the general condition of the dog improved one week later. In addition,

Figure 4. Electron microscopic examination revealed convoluted

nuclei, abundant lysosomes, and cytoplasmic lopodia.
(bar = 1mm)

the size of the skin lesions gradually diminished and

complete resolution of all lesions was noted after 7
weeks of the treatment (Fig. 5). Griseofulvin was
discontinued at the owner's request. Within one
month lymphadenopathy appeared and within two
months hepatosplenomegaly was palpable. The dog
died three months after discontinuing the therapy.
Unfortunately, necropsy was not allowed.
DISCUSSION
We conrmed that the histiocytic cells in this dog's
skin disease originated from LC based on the results
of immunohistochemical staining, immunophenotypic analysis and electron microscopic examination.46
However, it was not easy to dierentiate this
dermatosis from the canine histiocytic syndromes
observed previously. In the early stages, clinical and
histopathological ndings in this dog were characterized as CCH.16 With time, multiple lesions developed, but the proliferative cells lacked expression of
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M. Nagata et al.

Figure 5. (a) Clinical ndings of the dog before griseofulvin

therapy. (b) Dramatic improvement was obtained after 7 weeks of
the griseofulvin treatment.

CD4 (a marker of LC activation) and Thy-1 (a

marker of dermal dendritic antigen presenting cells).
Moore et al. indicated that histiocytes in CCH lack
expression of CD4 and Thy-1, which are consistently
expressed by histiocytes in CH and SH.6 All initial
ndings were consistent with a case of multiple
CCH,12,4,6 but the dog became depressed, anorexic
and died. The clinical course of disease did not reveal
features common to multiple CCH, in which the
regression of lesions can occur spontaneously.4,6
Moreover, the histopathologic examination did not
show any cytologically malignant features indicative
of MH.12
In humans, Langerhans' cell histiocytosis (LCH)
refers to a group of several histiocytic cell syndromes
previously known as histiocytosis X, Letterer-Siwe
disease, Hand-Schuller-Christian disease, eosinophilic granuloma and self-healing reticulohistiocytoma.
LCH is a condition characterized by an accumulation
and/or proliferation of cells expressing phenotypic
markers S-100, CD1a, CD11c, CD16, CD33, HLADR and the Fc receptor.17,18 Almost all organs and
systems can be aected by the LCH; the most
common sites of involvement include the skin, bone,
lymph nodes, liver, spleen, lung and posterior
pituitary gland.17 The clinical spectrum of LCH can
# 2000 Blackwell Science Ltd, Veterinary Dermatology, 11, 241246

be extremely variable, ranging from asymptomatic

single-system involvement to fulminant fatal multisystem disease.17 Mortality is highest in infants under
2 years of age with multisystem disease.19 In this dog,
the biological behaviour of the proliferative histiocytic cells closely resembled that of a severe form of
LCH in humans rather than multiple CCH in dogs.
Although the aetiology and pathogenesis of LCH
remain uncertain, it is likely caused by an immunological dysregulation.17 Human LCH cells showed
strong expression of cellular adhesion molecules such
as CD54, CD58 and b-integrin a4 that are related
with up-regulation during activation of normal
LCs.20 This would suggest that a `down-regulatory'
signal is lacking in human LCH, resulting in
accumulation and/or proliferation of abnormal
LCs.20 Furthermore, human LCH cells have IFN-g
receptor, which is valuable for the dierentiation of
normal LCs from LCH cells.21 Moreover, cytokines
were abundant within human LCH lesions, including
interleukin (IL)-1a, IL-1b, IL-4, and IFN-g.22 These
ndings suggest that some cytokines, especially IFN,
may play an important role in inducing `downregulatory' signals. In fact, intralesional interferon
(IFN)-b and systemic IFN-a have been reported to be
eective for cutaneous LCH in humans.23,24
In this case, griseofulvin appeared to be eective in
controlling symptoms. Griseofulvin is a fungistatic
antibiotic that also has immunomodulating properties.15 It has been reported that griseofulvin decreases
IFN-g-induced HLA-DR molecules on human epidermal keratinocytes.16 In addition, ultraviolet (UV)
radiation is acknowledged to impair cutaneous
immune functions25 and following treatment the
number of HLA-DR on LCs are immediately
reduced.26 Thus, it is postulated that griseofulvin
could induce `down-regulatory' signals within LCH
lesions. Further investigations will be needed.
ACKNOWLEDGEMENTS
We thank Dr Peter F. Moore (University of California, Davis, USA) for providing monoclonal antibodies
against CD1a, CD1c, CD3, CD4, CD8, CD11c,
CD18, CD21, CD45, MHC class II, and Thy-1.
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1. Scott, D.W., Miller, W.H., Grin, C.E. Muller and
Kirk's Small Animal Dermatology. 5th edn.
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2. Gross, T.L., Ihrke, P.J., Walder, E.J. Histiocytic and
mast cell tumors. Veterinary Dermatopathology: A
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feline skin disease. St. Louis: Mosby Year Book Co.,
1992, 46573.
3. Goldschmidt, M.H., Shofer, F.S. Canine cutaneous
histiocytoma. Skin Tumors of the Dog and Cat. Oxford:
Pergamon Press., 1992: 22230.

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Progressive histiocytosis in a puppy
4. Moore, P.F., Schrenzel, M.D., Aolter, V.K., Olivry,
T., Naydan, D. Canine cutaneous histiocytoma is an
epidermotropic Langerhans cell histiocytosis that
expresses CD1 and specic b2-integrin molecules.
American Journal of Pathology 1996; 148: 1699708.
5. Marchal, T., Dezutter-Dambuyant, C., Fournel, C.,
Magnol, J.P., Schmitt, D. Immunophenotypic and
ultrastructural evidence of the Langerhans cell origin
of the canine cutaneous histiocytoma. Acta Anatomica
1995; 153: 189202.
6. Moore, P., Aolter, V.K., Olivry, T., Schrenzel, M.D.
The use of immunological reagents in dening the
pathogenesis of canine skin diseases involving
proliferation of leukocytes. In: Kwochka, K.W.,
Willemse, T., Von Tscharner, C. eds. Advances in
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7. Taylor, D.N., Dorn, C.R., Luis, O. Morphologic and
biologic characteristics of the canine cutaneous
histiocytoma. Cancer Research 1969; 29: 8392.
8. Calderwood Mays, M.B., Bergeron, J.A. Cutaneous
histiocytosis in dogs. Journal of the American
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9. Moore, P.F. Systemic histiocytosis of Bernese
mountain dogs. Veterinary Pathology 1984; 21: 55463.
10. Scott, D.W., Angarano, D.K., Suter, M.M. Systemic
histiocytosis in two dogs. Canine Practice 1987; 14: 712.
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12. Hayden, D.W., Waters, D.J., Burke, B.A., Manivel,
J.C. Disseminated malignant histiocytosis in a Golden
Retriever: Clinicopathologic, ultrastructural, and
immunohistochemical ndings. Veterinary Pathology
1993; 30: 25664.
13. Nagata, M., Nanko, H., Moriyama, A., Washizu, T.,
Ishida, T. Pigmented plaques associated with
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Epidermodysplasia
Verruciformis?
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14. Rottman, J.B., Tompkins, W.A.F., Tompkins, M.B. A
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16. Tamaki, K., Saitoh, A., Yasaka, N. Dierential eect
of griseofulvin on interferon-g-induced HLA-DR and
intercellular adhesion molecule-1 expression of human
keratinocytes. British Journal of Dermatology 1992;
127: 25861.
17. Gadner, H., Grois, N. The histiocytosis syndromes. In:
Fitzpatrick, T.B., Eisen, A.Z., Wol, K., Freedberg,
I.M., Austen, K.F. Dermatology in General Medicine
VI. New York: McGraw-Hill Book Co., 1993: 200317.
18. Lever, W.F., Schaumburg-Lever, G. Histiocytosis X.
Histopathology of the skin. 7th edn. Philadelphia: J.B.
Lippincott Co., 1990: 43640.
19. Yu, R.C., Chu, A.C. Langerhans cell histiocytosis
clinicopathological reappraisal and human leucocyte
antigen association. British Journal of Dermatology
1996; 135: 3641.
20. Graaf, J.H.D., Tamminga, R.Y.J., Kamps, W.A.,
Timens, W. Expression of cellular adhesion molecules
in Langerhans cell histiocytosis and normal
Langerhans cells. American Journal of Pathology
1995; 147: 116171.
21. Chu, T., Jae, R. The normal Langerhans cell and
LCH cell. British Journal of Cancer 1994: 70: S4S10.
22. Graaf, J.H.D., Tamminga, R.Y.J., Dam-Meiring, A.,
Kamps, W.A., Timens, W. The presence of cytokines in
Langerhans' cell histiocytosis. Journal of Pathology
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24. Kwong, Y.L., Chan, A.C.L., Chan, T.K. Widespread
skin-limited Langerhans cell histiocytosis: complete
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American Academy of Dermatology 1997; 36: 6289.
25. Kwon, O.S., Cho, K.H., Song, K.Y. Primary cutaneous
Langerhans cell histiocytosis treated with photochemotherapy. Journal of Dermatology 1997; 24: 546.
26. Laihia, J.K., Jansen, C.T. Up-regulation of human epidermal Langerhans' cell B71 and B72 co-stimulatory
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Resume Une femelle Teckel agee de 8 mois est presentee pour la presence de nombreux nodules et de
plaques, prurigineux et generalises. Une exerese chirurgicale d'un histiocytome cutane present sur le pavillon
auriculaire gauche avait ete eectuee deux mois auparavant. Un mois apres l'intervention, un nodule est
reapparu au meme endroit et, peu apres, d'autres nodules se sont developpes. L'examen histopathologique a
montre une proliferation diuse de cellules histiocytaires, qui ont fortement reagi vis a vis des anticorps
vimentine et lysozyme. Une analyse immunohistochimique a montre que la plupart des cellules exprimaient les
marqueurs CD1a, CD1c, CD11c, CD18, CD45 et le CMH de classe II. Un examen en microscopie
electronique a revele des lopodes et des structures paracrystallines cytoplasmiques. Ces elements indiquent
que ces cellules sont d'origine langerhansienne. La maladie a evolue malgre l'administration de
glucocortico des, et de la griseofulvine a ete prescrite comme agent immunomodulateur. Toutes les lesions
ont alors totalement semaines de traitement. Cependant, le chien est mort trois mois plus tard. L'autopsie n'a
pas ete realisee. Ce cas pourrait correspondre a une forme grave d'histiocytose langerhansienne comme decrit
chez l'homme, plutot qu'a un histiocytome cutane canin. [Nagata, M., Hirata, M., Ishida, T., Hirata, S. et
Nanko, H. Progressive Langerhans' cell histiocytosis in a puppy. (Histiocytose langerhansienne progressive
chez un chiot.) Veterinary Dermatology 2000; 11: 241246.]
Resumen Un Dachshund miniatura hembra de 8 meses se presento con un cuadro de prurito y multiples
nodulos y placas generalizadas. Dos meses atras, se hab a extirpado un nodulo en el pabellon auricular
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M. Nagata et al.
izquierdo y hab a sido diagnosticado como histiocitoma cutaneo. Un mes despues de la excision, reaparecio
un nodulo en la misma localizacion y, al cabo de poco, aparecieron otros nodulos. El estudio histopatologico
revelo una proliferacion difusa de celulas histioc ticas, que reacciono fuertemente a anticuerpos para
vimentina y lisozima. El analisis inmunofenot pico mostro que la mayor a de las celulas expresaban
marcadores CD1a, CD1c, CD11c, CD18, CD45 y MHC clase II. El examen ultramiscroscopico revelo
lopodios citoplasmaticos y estructuras paracristalinas. Estos hallazgos indicaban que las celulas se
originaban en celulas de Langerhans. La enfermedad progreso a pesar de la terapia glucocorticoidea y con
griseofulvina administrada como farmaco inmunomodulador. Todas las lesiones se resolvieron
completamente despues de 7 semanas de la terapia con griseofulvina. El perro, sin embargo, murio al cabo
de tres meses de la retirada de la terapia con griseofulvina y no se realizo necropsia. Se considera que esta
dermatosis canina se corresponde mas con una histiocitosis severa de celulas de Langerhans en humanos que
con un histiocitoma cutaneo canino. [Nagata, M., Hirata, M., Ishida, T., Hirata, S. e Nanko, H. Progressive
Langerhans' cell histiocytosis in a puppy. (Histiocitosis progressiva de celulas de Langerhans en un cachorro.)
Veterinary Dermatology 2000; 11: 241246.]
Zusammenfassung Ein acht Monate alter, weiblicher Zwergdackel wurde wegen generalisiert auftretender,
juckender, multipler Knoten und Plaques vorgestellt. Zwei Monate zuvor war ein Knoten von der linken
Pinna entfernt und als Histiozytom diagnostiziert worden. Einen Monat nach der Exzision war erneut ein
Knoten an derselben Stelle aufgetreten und kurze Zeit spater traten zusatzliche Knoten auf. Die
Histopathlogische Untersuchung ergab eine diuse Proliferation histiozytischer Zellen, die stark mit
Antikorpern gegen Vimentin und Lysozym reagierten. Immunophanotypische Analysen zeigten, da die
meisten Zellen CD1a, CD1c, CD11c, CD18, CD45 und MHC-Klasse-II Molekule exprimierten.
Elektronenmikroskopische Studien demonstrierten zytoplasmatische Filopodien und parakristalline
Strukturen. Diese Ergebnisse weisen darauf hin, da die Zellen von Langerhanszellen abstammen. Die
Erkrankung schritt trotz Glukokortikoidtherapie weiter fort, und Griseofulvin wurde als
immunomodulierende Substanz verabreicht. Alle Eoreszenzen verschwanden vollstandig nach 7 Wochen
Behandlung mit Griseofulvin. Drei Monate nach Beendigung der Griseofulvintherapie verstarb der Hund,
und eine postmorale Sektion wurde nicht durchgefuhrt. Moglicherweise handelt es sich bei der vorliegenden
caninen Dermatose eher um das Aquivalent einer schweren Form von Langerhanszellhistiozytose des
Menschen als dem caninen Histiozytom. [Nagata, M., Hirata, M., Ishida, T., Hirata, S. und Nanko, H.
Progressive Langerhans' cell histiocytosis in a puppy. (Progressive Langerhanszellhistiozytose in einem
Welpen.) Veterinary Dermatology 2000; 11: 241246.]

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