Professional Documents
Culture Documents
Abstract Pathogenesis of canine atopy has not been completely elucidated. In humans, sulphido-leukotrienes
(s-LT) play a role in atopy, and increased production of s-LT occurs in the skin and peripheral leukocytes
after allergen challenge. The study population included 16 clinically normal and 13 atopic dogs. All atopic
dogs had in common a positive reaction (4+) to the intradermal injection of house dust mite (allergen of
reference). Blood samples and skin biopsies were collected. Sulphido-LT synthesis by peripheral leukocytes
after stimulation was measured, and no statistically signicant dierence was found between clinically normal
and atopic dogs. Sulphido-LT concentrations in skin samples from stimulated and unstimulated sites were
measured, and no statistically signicant dierence was detected between clinically normal and atopic dogs or
between lesional and nonlesional skin within the atopic group. Clinical signs of atopic dogs were graded by
owners and no correlation was found between their severity and cutaneous concentrations of s-LT. In this
study there was no increase in s-LT synthesis in atopic dogs.
Keywords: canine atopy, leukotrienes, peripheral mononuclear cells, skin.
INTRODUCTION
Canine atopic disease is a common disease in
veterinary dermatology aecting up to 10% of
animals presented with skin disease.1,2 It has been
dened as a genetically programmed disease in which
the patient develops IgE antibodies against environmental allergens.3 It is becoming increasingly evident,
however, that the pathogenesis of this disease is more
complicated than originally thought, as total and
allergen-specic IgE levels may not be always
elevated in atopic dogs.4,5 Thus, despite the fact that
canine atopic dermatitis is so common, controversy
still exists concerning its pathogenesis.6
Diagnosis of atopic disease is clinical, but various
tests have been advocated to help identify the
allergens to be used for immunotherapy. Intradermal
skin testing (IDST) is still considered the method of
choice, but disadvantages exist, including the expense
of maintaining a supply of allergens, clipping
animal's hair coat, the potential for adverse reactions
to sedation or to the skin test itself, and problems
associated with the withdrawal of drugs that can
interfere with the test.3,7 Serology testing for allergenspecic IgE can be used in cases where IDST can not
be done. However, several problems limit the value of
Correspondence: Dr Marsella. Current address: Department of
Small Animal Clinical Sciences, College of Veterinary Medicine,
University of Florida, PO BOX 100126, Gainesville, FL 32610
0126, USA.
# 2001 Blackwell Science Ltd
203 DISC
4
Table 1. Criteria for the evaluation and scoring of pruritus by the owner
Score
Denition
1
2
3
4
5
mild pruritus (scratching, rubbing, chewing or licking for less than 10% of day)
mild-moderate pruritus (scratching, rubbing, chewing or licking for 1030% of day)
moderate pruritus (scratching, rubbing, chewing or licking for 3050% of day)
moderate-severe pruritus (scratching, rubbing, chewing or licking 5075% of day, but still able to sleep at night)
severe pruritus (scratching, rubbing, chewing or licking all the time, even at night and during a meal)
203 DISC
Leukotrienes in normal and atopic dogs
that of human plasma (1.33mPas) and based on pilot
studies done in the authors' laboratory, the methodology suggested in the kit was used in this study;
though this protocol was designed for human
blood.31,32 The tubes were incubated for 90 min at
25 8C to separate erythrocytes (bottom fraction) from
leukocytes and thrombocytes (plasma fraction). The
upper phase was then transferred to a propylene tube
and spun in a centrifuge (200 g, 15 min, 25 8C) to
form a leukocyte pellet. The platelet rich supernatant
was decanted and a cell pellet containing leukocytes
was suspended in 3 mL of stimulation buer
containing recombinant Interleukin 3 (IL-3) at 1 ng
mL (ALPCO). The purpose of IL-3 was to increase
LT release.33 This cytokine by itself cannot induce
mediator release but, when used in the pre-incubation
step, it enhances the response to anti-IgE antibodies
and other stimuli.
Small aliquots (200 mL) of the re-suspended pellet
were placed in separate tubes and dierent positive
controls were used. They included bacterial lipopolysaccharide (LPS puried from Escherichia coli J5 Rc
mutant, 50 mL at 2 ng mL, Sigma, St. Louis, MO,
USA), Fab fragments of IgG monoclonal antibodies
against canine IgE (50 mL at 100 mg mL71, Clone no.
7715, Biodesign International, Kennebunk, ME,
USA) and monoclonal antibodies against human
high anity IgE receptor (FceRI, ALPCO).34,35 These
controls were used to evaluate both IgE-independent
and IgE-dependent mediator release of s-LT.
Prior to the addition of LPS to the cell pellet, the
LPS was incubated in homologous serum for 45 min.
This was to provide LPS-binding protein that is
essential in the LPS-mediated activation of mononuclear cells.36,37 This protein allows binding of LPS
to the cell surface-expressed receptor CD14 and
increases cell response to LPS stimulation. Concentration of LPS and incubation times were selected
based on pilot studies carried out by the authors
(data not shown).
Fab fragments were prepared from monoclonal
anticanine IgE antibodies using the Fab preparation
kit (Immunopure, Pierce, Rockford, IL, USA)
according to the manufacturer's instructions. These
antibodies were specic for the heavy chain. The
rationale for using only the Fab fragments of the
monoclonal antibodies against canine IgE was to
ensure that the observed reactions would only be
due to cross-linking of IgE molecules without any
binding of the Fc portion of the antibody. If such
binding had occurred, it could have skewed results
and prevented a clear dierentiation between atopic
and normal individuals.
House dust mite (D. farinae, BAG-D2, at 100 ng
mL71, ALPCO) was the allergen of reference for the
in vitro stimulations. This concentration was selected
based on pilot studies carried out by the authors
(data not shown). This concentration is comparable
with the one used in humans.20,21 Background release
of LT (without stimulation) was the negative control
203 DISC
6
Statistics
Data were analysed by least squares analysis of
variance (LSANOVA) with the main eects and all
interactions included in the model. The SAS System
for Windows version 6.12 (SAS Institute Inc, Cary,
NC, USA) was used. Dierences among groups were
analysed using orthogonal contrast analysis. P-values
Table 2. Suldo-LT concentrations (pg mL71) after stimulation of peripheral leukocytes isolated from normal and atopic dogs. Data are
presented as mean + std err. P-values have been calculated comparing normal and atopic dogs. A P-value less than 0.05 was considered
statistically signicant
Stimulant
Normal group
Atopic group
Background
Fab fragment of IgG against canine IgE
IgG against human FceRI
LPS
HDM
6.32 + 17.68
76.94 + 17.68
160.51 + 17.68
81.56 + 17.68
26.09 + 17.68
23.16 + 19.62
34.48 + 19.62
180.37 + 19.62
74.02 + 19.62
12.28 + 19.62
0.52
0.11
0.45
0.78
0.60
Table 3. Suldoleukotriene concentration (pg mL71) after stimulation of peripheral leukocytes. Values are expressed as mean + SE
combining the atopic and normal dogs. Comparisons considered signicantly dierent are indicated with the * symbol
Suldoleukotriene concentration over all dogs (mean + SE)
Background release
(14.74 + 3.2)
0.03*
0.0001*
0.001*
0.81
0.0001*
0.24
0.05*
0.0001*
0.0001*
0.002*
203 DISC
Leukotrienes in normal and atopic dogs
between normal and atopic dogs was detected. No
signicant correlation was found between concentrations of s-LT released by peripheral leukocytes after
allergen stimulation and serum allergen-specic IgE
for both the normal (Fig. 1) and atopic (Fig. 2) dogs
(r = 0.14 and r = 0.26, respectively). No signicant
dierence was found in the concentrations of HDMspecic IgE between normal and atopic dogs
(P = 0.42).
Sulphido-LT from skin
No statistically signicant dierences were detected in
s-LT concentrations between the two groups for all
the injections, including the allergen (P = 0.065,
P = 0.29, P = 0.065 for saline, LPS and HDM
injection, respectively) (Table 4). Thus, there was no
correlation between positive IDST for HDM (which
implies the presence of cutaneous IgE) and s-LT
release in the skin. Also, there was no signicant
dierence in s-LT concentrations between normal
and nonlesional atopic skin (P = 0.75) and, within
the atopic group, between lesional and nonlesional
skin (P = 0.54).
203 DISC
8
Table 4. Suldo-LT concentrations (pg mL71) from the skin of atopic and normal dogs. Data are presented as mean + SE. P-values have
been calculated comparing normal and atopic dogs. There were no signicant dierences between the injection sites over all dogs. A P-value
less than 0.05 was considered statistically signicant
Injected areas
Non-injected areas
Site of biopsy
Normal group
Atopic group
P-value
Saline
LPS
HDM
Lesional
Non-lesional
50.94 + 9.73
51.06 + 9.73
62.20 + 9.73
Not applicable
58.40 + 9.73
78.33 + 10.79
66.45 + 10.79
90.36 + 10.79
76.29 + 16.54
62.83 + 10.79
0.06
0.29
0.056
0.76
203 DISC
Leukotrienes in normal and atopic dogs
There was no dierence between atopic and
clinically normal dogs in any of the injected sites
and, within the atopic group, there was no dierence
between lesional and nonlesional atopic skin. These
results are also in contrast with what has been
reported in human atopic dermatitis. In humans, sLT are released after allergen challenge12,17,50 and their
concentrations are signicantly higher in lesional
atopic skin than normal and nonlesional atopic skin.17
All the atopic dogs selected in this study had a
strong positive reaction to the intradermal injection
of HDM, implying that they had cutaneous allergenspecic IgE. In addition, all the normal dogs were
intradermally tested with the same allergen to ensure
that they had no positive reaction to it. However, sLT concentrations released after allergen stimulation
were not dierent between groups. This suggests that
there is poor correlation between cutaneous allergenspecic IgE and s-LT in the skin (at least for HDM).
Several hypotheses could explain these results. IgEmediated release of s-LT has been demonstrated from
mast cells, basophils and eosinophils.33,34 It is
possible, however, that functional heterogeneity of
canine IgE51 could play a role and that some IgE
would be more responsible for histamine release
(positive reaction on IDST at 15 min) while others
would trigger more s-LT release. Alternatively, HDM
could cause s-LT release in a non-IgE mediated way,
which would be the same for both normal and atopic
individuals. It is interesting to note that a cysteine
protease that has the potential of generating bradykinin has been recently puried from D. farinae.52,53
Bradykinin in turn has the potential to activate PA254
and LT synthesis, and this process would be
independent from aggregation of IgE receptors and
histamine release.34,55 Strong evidence suggests that
this protease might be Der f 1 itself, which is also one
of the major allergenic fractions for humans. Thus, in
humans, the fractions that are highly allergenic
(which trigger IgE release) are the same ones that
have a high content of protease. In dogs, controversy
exists on the identity of the major allergenic portion
of HDM. However, evidence exists that the fractions
that are allergenic in dogs are dierent from the ones
that are important in humans.56 Thus, the fractions
that could be responsible for LT release are not the
same ones against which IgE are directed and that
would help explain the dierent results.
In this study, the atopic dogs have consistently
higher values of cutaneous s-LT, even though such
dierence is not statistically signicant. Based on the
results obtained in this preliminary study and
applying the power of analysis, 35 dogs per group
might be a more appropriate number to detect
signicant dierences. Further studies should be
carried out before the hypothesis of increased
synthesis of s-LT in the skin atopic dogs is dismissed.
Another consideration is that the interval between
injection and biopsies might not have been optimal to
detect a signicant dierence between groups, espe-
cially when the number of dogs is small. The 90 mininterval between injections and time of biopsy was
elected based on previous studies,38,39 but the kinetics
on the release of LT at various times after challenge
have never been studied. The kinetics of the
cutaneous inltrate during IgE and allergen mediated
late phase reactions in atopic dogs have been
evaluated in one study. Leukocyte accumulation
seemed to peak between 6 and 12 h, however, no
skin biopsies were collected between 0 and 6 h
following intradermal injections. It is reasonable to
assume that LT peak should occur prior to leukocyte
accumulation, as LTs are among the mediators
responsible for it.57
Finally, no correlation was found in this study
between the severity of pruritus (as perceived by
owners) and cutaneous s-LT concentrations. This
may be partially due to the diculty of scoring
clinical signs like pruritus because perception of its
severity is very subjective. Future studies to evaluate
the role of s-LT in canine pruritus may need to
include the investigator's evaluation and scoring of
clinical lesions.
In conclusion, this study does not support the
hypothesis that in vitro measurement of s-LT release
after allergen stimulation would be a useful adjunctive test in canine dermatology and it does not
support a role for s-LT in the pathogenesis of
pruritus in canine atopic dermatitis. However, considering the peculiar nature of HDM and the limited
number of dogs, further studies using various pollens
and a larger number of animals are needed before
more denite conclusions can be made and before LT
are discarded as mediators of inammation in canine
atopic dermatitis.
ACKNOWLEDGEMENTS
This project was funded by the American Academy of
Veterinary Dermatology.
Authors would like to thank Cathy McCall and
Kim Stedman at Heska for helping in the measurement of serum IgE.
REFERENCES
1. Scott, D.W. Observations on canine atopy. Journal of the
American Animal Hospital Association 1981; 17: 91100.
2. Scott, D.W., Paradis, M. A survey of canine and feline
skin disorders seen at a University practice. Small
Animal Clinic, University of Montreal, Saint
Hyacinthe, Quebec 198788. Canadian Veterinary
Journal 1990; 31: 8305.
3. Grin, C.E. Atopic disease. Seminars in Veterinary
Medicine and Surgery 1991; 6: 2905.
4. Hill,
P.B.,
Moriello,
K.A.,
DeBoer,
D.J.
Concentrations of total serum IgE, IgA, and IgG in
atopic and parasitized dogs. Veterinary Immunology
and Immunopathology 1995; 44(2): 10513.
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 312
203 DISC
10
203 DISC
Leukotrienes in normal and atopic dogs
38. Marsella, R., Kunkle, G.A., Vaughn, D., MacDonald,
J. Double-blind pilot study on the eects of
ketoconazole on intradermal skin test and
Leukotriene C4 concentration in the skin of atopic
dogs. Veterinary Dermatology 1997; 8(1): 310.
39. Vaughn, D.M., Reinhart, G.A., Swaim, S.F., Lauten,
S.D. Evaluation of eects of dietary n-6 to n-3 fatty acid
ratios on leukotriene B synthesis in dog skin and
neutrophils. Veterinary Dermatology 1994; 5(4): 16373.
40. Okano-Mitani, H., Ikai, K., Imamura, S. Leukotriene
A4 hydrolase in peripheral leukocytes of patients with
atopic dermatitis. Archives of Dermatological Research
1996; 288(4): 16872.
41. Ferrer, M., Sanz, M.L., Prieto, I., Oheling, A. In vitro
antigen-specic sulphido-leukotriene production in
patients allergic to Dermatophagoides Pteronyssinus.
Clinical Experimental Allergy 1998; 28(6): 70914.
42. Bircher, A.J., Haldiman, D., Hampl, K., Languer, S.
Lymphocyte proliferation and suldo-leukotriene
release test (CAST) in hypersensitivity to lactam
antibiotics. Allergy 1995; 50(Suppl. 26): 53.
43. Siridipoulos, J., Papastavrou, T.H., Charissoulis, S.,
Chamalidou, C.H., Lioliou, M., Panteliadis, C.H. The
cellular allergen stimulation test (CAST) New
perspective in allergy diagnosis. Allergy 1995;
50(Suppl. 26): 245.
44. Kuster, H., Zhang, L., Brini, A.T., MacGlashan, D.W.,
Kinet, J.P. The gene and cDNA for the human high
anity immunoglobulin E receptor beta chain and
expression of the complete human receptor. Journal of
Biological Chemistry 1992; 267(18): 127827.
45. Bisho, S.C., Zwalhlen, R., Stucki, M. et al. Basophil
histamine release and leukotriene production in
response to anti-IgE and anti-IgE receptor antibodies.
Comparison of normal subjects and patients with
urticaria, atopic dermatitis or bronchial asthma.
International Archives of Allergy and Immunology
1996; 110(3): 26171.
46. Ring, J., Sedlmeier, F., Dorsch, W., Hermann, K. In
vitro IgE eluation and histamine releasability from
peripheral leukocytes of atopics and normals. Journal
of Dermatological Science 1991; 2(6): 41321.
47. Chen, X.J., Enerback, L. Inuence of genetics and
environmental factors on surface expression and
occupancy of IgE receptors and on histamine
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
11
Resume La pathogenie de l'atopie canine n'a pas ete completement elucidee. Chez l'homme, les sulphidoleucotrienes (s-LT) jouent un role dans l'atopie: une production augmentee de s-LT est observee au niveau de
la peau et des leucocytes peripheriques apres une stimulation allergenique. 16 chiens normaux et 13 chiens
atopiques ont ete inclus dans cette etude. Tous les chiens atopiques presentaient une reaction positive (4 +) a
l'injection intradermique d'un extrait d'acarien de la poussiere de maison (allergene de reference). Des
prelevements sanguins et des biopsies cutanees ont ete realises. La synthese de s-LT par les leucocytes
peripheriques a ete mesuree apres stimulation: aucune dierence statistiquement signicative n'a ete observee
entre les chiens atopiques et les chiens normaux. Les concentrations en s-LT ont ete mesurees dans les biopsies
cutanees obtenues au niveau de zones stimulees et non stimulees. Aucune dierence statistiquement
signicative n'a ete detectee entre les chiens normaux et les chiens atopiques, ni entre les zones lesees et les
zones saines chez les atopiques. La severite des signes cliniques, evaluee par les proprietaires, n'etait pas
correlee avec les concentrations cutanees de s-LT. Dans cette etude, aucune augmentation de la synthese de sLT n'a ete observee chez les chiens atopiques. [Marsella, R. et Nicklin, C. F. Sulphido-leukotriene production
from peripheral leukocytes and skin in clinically normal dogs and house dust mite positive atopic dogs.
(Production de sulphido-leucotrienes par les leucocytes peripheriques et la peau chez des chiens normaux et
des chiens atopiques allergiques aux acariens des poussieres.) Veterinary Dermatology 2001; 12: 312.]
203 DISC
12