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Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated
upon activation, normal T-expressed and secreted (RANTES) which is a cysteinecysteine chemokine that
mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque
are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration
in the skin and /or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence
of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced
the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with
eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression
of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA
sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein
of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that
of bovine and mouse RANTES genes, respectively. RTPCR analysis on RANTES mRNA in the skin of cats
with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in
the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline
eosinophilic plaque lesions.
Keywords: cat, cDNA, RANTES, RTPCR.
INTRODUCTION
Feline eosinophilic plaques are a common skin reaction pattern in cats. One of the most striking features
of these lesions is prominently eosinophilic infiltration
of the skin. These lesions are common in cats with hypersensitivities to inhalants, foods or insects, especially to
fleas and mosquitoes.1 However, the precise mechanism
of eosinophil infiltration in the skin and mucosa of cats
is unknown.
Regulated upon activation, normal T-expressed and
secreted (RANTES), a member of the cysteine cysteine
(CC) chemokine family is a potent chemoattractant
for various inflammatory cells such as eosinophils,
memory T cells and monocytes, and may be potentially important in recruiting these cells to the site of
Correspondence: Rui Kano.
Present address: Department of Pathobiology, Nihon University
School of Veterinary Medicine, 1866, Kameino, Fujisawa Kanagawa,
252-8510, Japan, Tel.: +81 466 84 3649; Fax: +81 466 84 3649; E-mail:
kano@brs.nihon-u.ac.jp
2003 European Society of Veterinary Dermatology
270
T. Kimura et al.
Table 1 Patients
No.
Breed
Sex
Age
Region
1
2
3
4
5
6
7
Cross-breed
Cross-breed
Cross-breed
Cross-breed
Cross-breed
Cross-breed
Abyssinian
M
M
F
UN
M
M
M
6
7
8
UN
11
14
8
Foreleg
Hindleg
Hindleg
Abdomen
Right ear
Abdomen
Hindleg
RESULTS
Full-length feline RANTES cDNA
Using cDNA of feline eosinophilic plaque as a template, a full-length feline RANTES gene was cloned
using 5- and 3-RACE methods. The full-length cDNA
sequence of the RANTES gene (742 bp) contained a
single open reading frame of 276 bp coding a protein
of 92 amino acids beginning with a putative initiating
methionine (ATG) (Fig. 1). The sequence reported
was deposited in the DDBJ database (Accession no.
AB083479, feline RANTES gene, complete cds).
The amino acid sequence of feline RANTES shared
6774% sequence identity with that of bovine (GenBank
Accession no. AJ007043),9 human (NM_002985)10 and
mouse (M77747)11 RANTES (Fig. 2).
RANTES mRNA analysis by RTPCR
After RTPCR, RANTES mRNA was detectable in feline
normal skin and skin lesions of eosinophilic plaque
(Fig. 3), suggesting that RANTES mRNA is expressed
in feline skin. Moreover, RANTES mRNA was more
strongly expressed in feline eosinophilic plaque lesions
than in the normal skin even from the same patient cats
(Fig. 3).
DISCUSSION
Feline eosinophilic plaques are histopathologically
characterized by prominent eosinophilic infiltration,
however, the precise mechanism of eosinophil infiltration into these lesions is unknown. In people, the production of RANTES from keratinocytes is believed
to be a partial explanation for the eosinophilic infiltration
of people with atopic dermatitis and allergic contact
dermatitis.1,3,4 The goal of this study was to determine
if RANTES might be involved in the pathogenesis of
feline eosinophilic plaques. If RANTES are present in
eosinophilic lesions of cats, treatment strategies aimed
at manipulating the production of RANTES may present
new therapeutic options for cats with these lesions or
for cats with other eosinophilic diseases.
The full-length of sequence of feline RANTES gene
was determined and the amino acid sequence identity
of RANTES genes was shown to be relatively conserved
among humans, cats, cattle and mice. In particular, the
CC structure in these RANTES gene was highly conserved
(Fig. 2).2 These results suggest that feline RANTES
might play the same functions as human and mouse.
RTPCR analysis revealed that RANTES mRNA
was highly expressed in skin lesions of eosinophilic
plaques when compared with normal skin. This
2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 269 273
271
Figure 2. Comparison of homologous regions of the predicted protein sequences of RANTES among cats, cattle, human and mouse. Bovine
RANTES gene (GenBank Accession no. AJ007043), human RANTES gene (NM_002985) and mouse RANTES gene (M77747). Asterisk
indicates identity with the amino acid of the feline RANTES. The signal peptide is underline. Four cysteine residues conserved among species
are boxed.
272
T. Kimura et al.
REFERENCES
1. Scott, D.W., Muller, W.H. Jr, Griffin, C.E. Feline eosinophilic granuloma complex. In: Small Animal Dermatology,
6th edn. Philadelphia: WB Saunders, 2001: 114853.
2. Schall, J., Thomas, Greaves, R.D. RANTES. In:
Oppenheim, J.J., Feldmann, M., eds. Cytokine Reference:
A Compendium of Cytokines and Other Mediators of Host
Defense. San Diego: Academic Press, 2001: 1161 6.
Rsum L'un des mcanismes expliquant l'infiltration par les osinophiles est la prsence de chemoattractants
comme le RANTES (Regulated Upon Activation, Normal T Expressed and Secreted), qui est une CC chmokine
qui provoque la chmotaxie et l'activation des osinophiles chez l'homme et la souris. Les lsions cutanes de
plaques osinophilique fline sont caractrises par une infiltration massive d'osinophiles. Le(s) mcanisme(s)
de l'infiltration osiniophilique de la peau et des muqueuses chez le chat est (sont) inconnu(s). Il est possible que
le RANTES soit en cause. Afin d'tudier la prsence de RANTES dans la peau de chats prsentant une plaque
osinophilique, nous avons clon et squenc la totalit du gne du cDNA RANTES. Le RANTES a t recherch
au niveau des lsions et au niveau de la peau saine des chats atteints. La squence de cDNA du gne du RANTES
(742bp) contenait une sonde simple de 276bp codant pour une protine de 92 acides amins. La squence en acides
amins du RANTES flin partageait 67 et 74% d'identit avec celles du RANTES de boeuf et de souris. L'analyse
par RT-PCR de l'ARNm du RANTES dans la peau des chats plaque osinophilique a montr que cette expression tait plus importante en peau lse qu'en peau saine. Ces rsultats suggrent que le RANTES pourrait jouer
un rle dans l'infiltration osinophilique dans les lsions de plaque osinophilique chez le chat.
Resumen Uno de los mecanismos de infiltracin de ls eosinfilos es la induccin mediante quimioatractores
como el Regulado por Activacin, T Normal Expresado y Secretado (Regulated Upon Activation, Normal T
Expressed and Secreted, RANTES) que es una quemoquina CC que media la quimiotaxis y activacin de los
eosinfilos en humanos y ratones. Las lesiones cutneas de la placa eosinoflica felina se caracterizan por un
infiltrado con predominio de eosinfilos. Se desconoce el o los mecanismo(s) de la infiltracin eosinoflica en la
piel y/o mucosa de los gatos. Es posible que RANTES se encuentre implicado. Para investigar la presencia de
RANTES en la piel de gatos con placas eosinoflicas y en piel no afectada, en este estudio, se clon y secuenci
la totalidad del gen cDNA de RANTES felino, con el fin de determinar si se encuentra presente en la piel de gatos
con placas eosinoflicas y/o si se encuentra presente en piel adyacente normal. En este estudio pudimos documentar la expresin de mRNAs de RANTES en piel con placa eosinoflica felina as come en piel felina normal..
La secuencia completa de cDNA del gen de RANTES (742bp) contena una nica ventana de lectura de 276bp
que codifica una proteina de 92 aminocidos. La secuencia de aminocidos de RANTES felino comparta un
67 y 74 % de la identidad de la secuencia de genes de RANTES bovino y murino, respectivamente. El anlisis
de RT-PCR sobre mRNA de RANTES en la piel de gatos con placa eosinoflicas mostr que su expresin era
mayor que en piel normal. El resultado sugera que RANTES puede jugar un papel importante al inducir la
infiltracin de eosinfilos en las lesiones de placa eosinoflica felina
2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 269 273
273
Zusammenfassung Einer der Mechanismen bei der Infiltration mit Eosinophilen ist die Auslsung durch chemoattraktive Substanzen wie RANTES (Regulated Upon Activation, Normal T Expressed and Secreted), ein
CC-Chemokin, welches Chemotaxis und Aktivierung von Eosinophilen bei Menschen und Musen vermittelt.
Hautvernderungen bei felinen eosinophilen Plaques sind vorrangig durch die Infiltration von Eosinophilen
charakterisiert. Mechanismen, nach denen die Infiltration mit Eosinophilen in die Haut oder/und Mukosa von
Katzen erfolgt, sind nicht bekannt. Es ist mglich, dass RANTES mitbeteiligt ist. In dieser Studie haben wir die
ganze Lnge des felinen RANTES cDNA Gens geklont und sequenziert, um herauszufinden, ob es in der Haut
von Katzen mit eosinophilen Plaques und/oder in der normalen angrenzenden Haut vorhanden ist. In dieser
Studie konnten wir die Expression von RANTES mRNS in der Haut mit felinen eosinophilen Plaques wie auch
in normaler Katzenhaut nachweisen. Die ganze Lnge der cDNA Sequenz des RANTES Gens (742bp) enthlt
einen einzelnen Open-reading-frame mit 276bp, der ein Protein mit 92 Aminosuren kodiert. Die Aminosuresequenz von felinem RANTES war zu 67, bezw. 74% mit Rinder- bezw. Maus-RANTES identisch. RT-PCRAnalyse von RANTES mRNA in der Haut von Katzen mit eosinophilen Plaques zeigte, dass seine Expression
in Haut mit eosinophilen Plaques hher als in normaler Haut war. Das Ergebnis weist darauf hin, dass RANTES
bei der Induktion der Eosinophilen-Infiltration von felinen eosinophilen Plaques vielleicht eine Rolle spielt.