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203 DISC

Veterinary Dermatology 2001, 12, 312

Sulphido-leukotriene production from peripheral


leukocytes and skin in clinically normal dogs and house
dust mite positive atopic dogs
ROSANNA MARSELLA and CONSTANCE F. NICKLIN
Blanche Saunders Dermatology Laboratory, Department of Small Animal Clinical Sciences, College of
Veterinary Medicine, University of Florida, Gainesville, FL, USA
(Received 16 June 1999; accepted 13 October 1999)

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

this test. They include poor correlation with IDST


results, high incidence of false positive reactions and
the lack of documentation of a correlation between
serum and skin IgE.5,8,9
Therapeutic options for canine atopic dermatitis are
also limited by our incomplete understanding of the
pathogenesis. Glucocorticoids, with potential serious
long-term side-eects, are still the primary therapy for
many patients. Immmunotherapy, antihistamines, and
fatty acids are other treatments that can be used with
variable success to decrease the use of glucocorticoids.10 Thus, a better understanding of the pathogenesis of canine atopy would help identify new adjunctive
diagnostic tests and therapeutic options.
In human medicine, several studies suggest that
leukotrienes (LT) play an important role in atopy1113
and LT inhibitors are often used as therapeutic
agents in atopic people for both respiratory and
dermatologic signs.14,15 Leukotrienes are divided into
two groups according to their chemical structure:
those having a sulphur linkage (also called sulphidoLT) and those that do not. The rst group includes
LTC4, LTD4 and LTE4 (previously known as slow
reacting substances of anaphylaxis) while LTB4
belongs to the second group. Increased production
of LT occurs in the skin of atopic patients after
allergen challenge16,17 and, of all the LT, LTC4 is the
one released in the largest quantity.18 In human skin,
LTC4 concentration correlates with severity of late
phase cutaneous reactions.12,19 In addition, synthesis of sulphido-LT (s-LT) after allergen challenge
3

203 DISC
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R. Marsella and C. F. Nicklin

correlates with the history and the severity of


clinical signs.20
In humans, LT synthesis by peripheral leukocytes
correlates with histamine release and these mediators
peak during the pollen season.21 Based on these
ndings, a new diagnostic test called Cellular Antigen
Stimulation Test Enzyme Linked Immunosorbent
Assay (CAST-ELISA) has been developed in human
medicine to measure s-LT release by peripheral
leukocytes after allergen stimulation.22,23 This method appears to be reliable in determining the allergic
status of patients, and its sensitivity is higher than the
determination of allergen-specic IgE.22,23 The objectives of the present study were: (1) to investigate
the production of s-LT (from peripheral mononuclear cells and skin) in dogs with atopic dermatitis
and to determine the correlation between s-LT
synthesis and severity of pruritus; and (2) to
determine if there is a correlation between serum
allergen specic IgE and allergen-stimulated release
of s-LT by peripheral leukocytes.
MATERIALS AND METHODS
Animals
Clinically normal dogs and atopic dogs were selected
for this study. Normal dogs were determined to be
healthy based on history and physical examination.
They had no evidence (clinically normal) or history of
skin disease. Diagnosis of atopy was based on
compatible history, suggestive clinical signs, exclusions of other pruritic skin diseases (e.g. sarcoptic
mange, food allergy), and at least three positive IDST
reactions using 57 common allergens (Greer, Lenoir,
NC, USA) of the geographical area.24 Food allergy
was excluded by an appropriate food trial prior to the
study.25 Sarcoptic mange was excluded by appropriate treatment, which included either three injections of ivermectin (Ivomec, Merial Limited, Iselin,
NJ, USA) at 300 mg kg71 at 2-week intervals, or
weekly lime sulphur dips (LymDip, DVM Pharmaceuticals Inc., Miami, FL, USA) for seven treatments,
depending on the breed of the dog.26 None of the
dogs included in the study had evidence of concurrent
ea allergy. Secondary skin infections (e.g. supercial
pyoderma, Malassezia dermatitis) were treated before
dogs were entered into the study. Antibiotic treatment for supercial pyoderma consisted of oral
administration of cephalexin at 22 mg kg71 twice
daily and antifungal therapy consisted of oral
ketoconazole (Nizoral, Janssen, Titusville, NJ,

USA) at 5 mg kg71 twice daily for 2 weeks. Both


treatments were discontinued at least 14 days before
the beginning of the study. Also, glucocorticoids and
antihistamines were discontinued 2 months and 2
weeks, respectively, prior to inclusion in the study
because they can interfere with IDST and LT
synthesis.3,7 Specically, glucocorticoids inhibit phospholipase A2 (PLA2) which is the enzyme responsible
for arachidonic acid release and ultimately LT
synthesis, while histamine stimulates this enzyme.27
Intradermal skin test reactions were evaluated for
erythema, induration and size of the wheal at 15 min
post-injection and graded subjectively on a scale from
0 to 4+. A score of 0 was assigned to the saline
reaction (negative control) and a score of 4+
assigned to the histamine (positive control). A
positive reaction was considered a 2+ or greater
reaction. A 4+ reaction to the intradermal injection
of house dust mite at 15 min post injection (HDM,
Dermatophagoides farinae at 25 PNU ml71, Greer,
Lenoir, NC, USA) was a criteria for inclusion in the
study for the atopic dogs. The concentration of
HDM was selected to minimize the potential for
irritant reactions.28,29 This allergen was selected as
the allergen of reference for this study because it is
less aected by seasonality and geographical variation than other allergens. The owners of atopic dogs
graded pruritus on a scale from 0 to 5 on the day of
the study (Table 1).30 Normal dogs had no reaction
on IDST to HDM at 15 min post-injection. A
complete IDST was not carried out in these dogs.
Blood collection
Peripheral blood (13 mL) was drawn from all dogs
and 6 mL was placed into ethylenediamine tetraacetic
acid (EDTA) tubes (Vacuette CAST venipuncture
tubes, Greiner Labortechnik Ltd, Glos, UK), while 7
mL was collected into serum separator tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA).
The serum separator tubes were centrifuged (1371 g,
15 min, 25 8C) to obtain serum. 1 mL of serum was
used in the stimulation experiments, while the
remaining serum was submitted (Heska, Fort Collins,
CO, USA) to measure D. farinae-specic IgE.
Sulphido-leukotrienes were measured following the
methodology recommended for the commercial
CAST ELISA (ALPCO, Windham, NH, USA).
3 mL of blood was taken from the EDTA tubes
and mixed with 0.75 mL of dextran solution to
increase the blood viscosity and isolate leukocytes, as
recommended by CAST ELISA kit. Considering that
the viscosity of canine plasma (1.22mPas) is similar to

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)

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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

and all the concentrations were calculated subtracting


the background.
All stimulations were incubated for 40 min and
centrifuged (3 min, 1000 g, 5 8C). Supernatants were
collected and s-LT measured using an ELISA
technique.20 Colour absorbence was measured at
405 nm wavelength in a microtitre plate reader
(Microtiter Absorbance Detection System, MRXHD, Dynex Technologies, Chantilly, VA, USA). The
minimal detectable dose of s-LT with this ELISA was
45 pg mL71 according to the kit insert. The following
cross-reactivity of the monoclonal antibody used in
the assay have been calculated at 50% binding and
are 109.7% for LTC4, 100% for LTD4, and 103.4%
for LTE4.
Intradermal injections
An area on the lateral thorax was clipped on all dogs.
All dogs received four intradermal injections (0.05
mL site71). They included one negative control
(phosphate buered saline), two positive controls
(histamine phosphate and LPS) and HDM (D.
farinae, 25 PNU ml71, Greer). Histamine was
injected at a dilution of 1/100 000 w/v. Lipopolysaccharide was used as positive control (0.4 mg mL71)
to trigger LT release.38 Saline was used as a negative
control because it is used as a diluent for preparation
of the allergen and LPS.
Full thickness 6 mm skin biopsies were taken from
all dogs. They were obtained using local anaesthetic
(0.5 mL site71 of lidocaine hydrochloride 2%,
Phoenix Pharmaceutical Inc, St. Joseph, MI, USA)
and a disposable skin biopsy punch (Miltex Instrument Company, Lake Success, NY, USA). Sites were
sutured routinely. In all dogs, biopsies were taken
from injected sites (saline, LPS, and HDM) 90 min
after injection and from sites that were not injected.
The purpose of injecting the sites prior to biopsy was
to reduce variation and increase reproducibility of the
data since duplicate biopsies were not possible due to
the fact that privately owned animals were used in this
project. The interval of time after injection was
selected based on previous studies.38,39 The sites that
were not injected included one lesional and one
nonlesional area for the atopic group and one area
of normal skin for the other dogs (lateral thorax).
Skin samples were blotted on gauze to remove
excess blood and subcutaneous fat was removed with
scissors. The specimens were placed in a capped
polypropylene tube and immediately frozen in liquid
nitrogen. The sample vials were ushed with argon
gas to displace oxygen and prevent oxidation of s-LT
present in the samples prior to storage at 70 8C.
Sulphido-LT extraction and measurement from the
skin
Sulphido-LT was extracted from the skin as previously described.38,39 Briey, frozen skin samples
were weighed, minced with scissors and placed in
2 mL ethyl acetate (pH 2.0). Minced skin samples
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203 DISC
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R. Marsella and C. F. Nicklin

were homogenized in an ice water bath for 90 s using


a tissue homogenizer (Omni International, Warrenton, VA, USA) equipped with a microtip probe. The
probe was washed with 2 mL of ethyl acetate. The
wash was combined with the initial homogenate and
each mixture was centrifuged at 500 g for 10 min at
4 8C to pellet the protein and cellular debris. The
pellets were stored at 70 8C for Lowry protein
determination (Sigma Chemical Co). The supernatants were pipetted into plastic tubes, while the
volume was measured. The supernatants were blown
to dryness with nitrogen and re-dissolved with 200 mL
of ethanol, ushed with argon and stored at 70 8C.
Prior to the ELISA analysis, the ethanol used to redissolve the supernatants was blown to dryness with
nitrogen, and the residues were re-suspended in 100
mL ELISA buer. A standard curve was generated
and s-LT concentrations in the samples measured.
The ELISA measurements were then divided by the
volume of the supernatant, the protein content and
the weight of the sample to calculate the concentration of s-LT in each skin sample.38,39
Final concentration of s-LT (pg mL71) =
ELISA recording/volume of the supernatant

Weight of the skin biopsy/Protein content of skin sample

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

5 0.05 were considered signicant. Pearson Product


Moment Correlation was used to evaluate correlation
between the severity of pruritus and the concentration
of s-LT in the skin, as well as correlation between
serum HDM-specic IgE and concentrations of s-LT
released by peripheral leukocytes for both normal
and atopic dogs. An r-value 4 0.85 was considered
signicant. If the s-LT concentration in a sample was
below the limit of detection for the ELISA kit (45 pg
mL71), a value of zero was used for that sample in
calculating group means.
RESULTS
Animals
Sixteen clinically normal and 13 atopic dogs were
used in this study.
Sulphido-LT from peripheral leukocytes
Lipopolysaccharide was an eective positive control,
triggering signicantly higher concentration of s-LT
than both the background (P = 0.001) and HDM
(P = 0.002) (Tables 2 and 3). Fab fragments of
monoclonal antibodies against canine IgE were also
an eective positive control, triggering higher concentrations of s-LT than both the background
(P = 0.030) and HDM (P = 0.05). Antibodies
against human FceRI, however, triggered the highest
concentration of s-LT, which was signicantly higher
than LPS (P = 0.0001) and the Fab fragments of
antibodies against canine IgE (P = 0.0001).
No statistically signicant dierence in s-LT
synthesis in response to HDM and positive controls

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)

Fab fragment of IgG against canine IgE (55.71 + 13.2)


IgG against human FceRI (170.45 + 13.2)
LPS (77.79 + 13.2
HDM (19.18 + 13.2)

0.03*
0.0001*
0.001*
0.81

Fab fragment of IgG


against canine IgE
(55.71 + 13.2)

IgG against human FceRI (170.45 + 13.2)


LPS (77.79 + 13.2)
HDM (19.18 + 13.2)

0.0001*
0.24
0.05*

IgG against human FceRI


(170.45 + 13.2)

LPS (77.79 + 13.2


HDM (19.18 + 13.2)

0.0001*
0.0001*

LPS (77.79 + 13.2)

HDM (19.18 + 13.2)

0.002*

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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).

Correlation between pruritus and s-LT in the skin


No correlation (r = 0.38) was found between severity
of pruritus (as graded by owners) and s-LT concentration in the skin of atopic dogs (Fig. 3).
DISCUSSION
In this study, no increased s-LT synthesis was seen in
atopic dogs. Specically, s-LT release by peripheral
leukocytes after stimulation was not signicantly
dierent between atopic and normal dogs. These
ndings are in contrast to what has been reported in
human medicine. Peripheral leukocytes from atopic
people have increased activity of the enzyme LTA4
hydrolase and up-regulation of LT synthesis.18,40 In
these patients the release of s-LT after allergen
challenge correlates with the history and the severity
of clinical signs, and the CAST-ELISA test is considered a diagnostic indicator of the allergic status of the
patient.15,16 In humans, s-LT synthesis from peripheral
leukocytes after allergen stimulation is signicantly

Figure 1. Correlation between serum


allergen-specic IgE and sLT
concentrations in the blood of clinically
normal dogs. No signicant correlation
was detected (r = 0.14).

Figure 2. Correlation between serum


allergen-specic IgE and sLT
concentrations in the blood of atopic
dogs. No signicant correlation was
detected (r = 0.26).
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R. Marsella and C. F. Nicklin

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

Figure 3. Correlation between sLT


concentrations in the skin of atopic
dogs and the severity of pruritus. No
signicant correlation was detected
(r = 0.38).

dierent between sensitive individuals and controls and


correlates with histamine release.41 Both histamine and
s-LT peak during the pollen season.42,43
In this study, all the positive controls used for the
in vitro stimulations triggered a signicantly higher sLT release than both the background and the allergen
of reference, but no signicant dierence was found
between groups. The strongest positive control in our
system was represented by the antibodies against
human FceRI, suggesting that the receptor is well
conserved among species. Its amino acid sequence
has been identied in humans, mice and rats, and
69% of residues are identical.44
One consideration when evaluating the various
positive controls in this study is the role of native
occupancy of the receptor by IgE (native content of
IgE) and its role on releasability of inammatory
mediators and how this factor could inuence the
results of this study. In humans it seems that receptor
occupancy with IgE is not signicantly involved in
the mechanism of increased releasability of histamine
and leukotrienes from atopic leukocytes.45,46 In rats,
however, a positive correlation exists between histamine release and both the number of IgE receptors
and the native IgE content.47 In dogs very little
information is available.48 When atopic and clinically
normal dogs were compared, an increased number of
mast cells and an increased concentration of histamine were found in the skin of atopic dogs. However,
no correlation between histamine and serum IgE was
detected. Unfortunately, cutaneous deposition of IgE
was not evaluated in that study.
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 312

Peripheral leukocytes of HDM positive atopic


dogs, in this study, do not release a signicantly
higher amount of s-LT after allergen challenge when
compared to clinically normal dogs. One possible
explanation is that the clinically normal dogs also had
circulating allergen-specic IgE antibodies. In this
study, no signicant dierence was found in the
concentration of allergen-specic IgE between clinically normal and atopic dogs. Clinically normal dogs
had fairly high concentrations of D. farinae-specic
IgE and these antibodies might have interfered with
the results. All these dogs were indoor/outdoor pet
dogs with no skin disease and the presence of
antibodies indicated exposure to HDM. The nding
of high concentrations of IgE in clinically normal
dogs, though, is not a new nding and has been
reported by various authors in the past.4,5,48,49
In humans, when HDM (D. pteronyssinus positive)
allergic patients were examined, Ferrer et al. reported
a `weak correlation' (r = 0.39) between serum allergen-specic IgE and s-LT release.41 In this study, a
similar r-value was found (r = 0.38) but r-values less
than 0.85 were considered an indication of poor
correlation. Interpretation of these results, however,
is complicated by the fact that the source of HDM
used for serology testing (IgE measurement) and one
used for the in vitro stimulations and measurement of
s-LT were dierent. Heska's HDM was used for
serology testing and ALPCO's HDM was used for
the in vitro stimulations and s-LT measurement. This
choice was the result of technical considerations and
represents a limitation of this study.

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.
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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.]

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Resumen La patogenesis de la atop a canina no ha sido completamente aclarada. En los humanos, los
suldo-leucotrienos (s-LT) juegan un rol en la atop a, y ocurre un incremento en la produccion de s-LT en la
piel, y en los leucocitos perifericos despues de un desaf o alergenico. La poblacion estudiada incluyo 16 perros
clinicamente normales y 13 perros atopicos. Todos los perros atopicos ten an en comun una reaccion positiva
(+4) a la inyeccion intradermica de acaros de polvo domestico (alergeno de referencia). Se tomaron muestras
de sangre y biopsias de la piel. Se midio la s ntesis de suldo-leucotrienos por leucocitos perifericos despues de
ser estimulados, y no se encontro diferencia estad sticamente signicativa entre perros cl nicamente normales y
los atopicos. Se midieron las concentraciones de suldo-LT en las muestras de piel extra das tanto de las
partes estimuladas como de las no estimuladas, y no se detectaron diferencias estad sticamente signicativas
entre perros cl nicamente normales y los atopicos, y en el grupo de los atopicos, entre piel lesionada y no
lesionada. Los signos cl nicos de los perros atopicos fueron graduados por los duenos y no se encontro
ninguna correlacion entre la severidad y la concentracion cutanea de s-LT. En este estudio no hubo aumento
en la s ntesis de s-LT en perros atopicos. [Marsella, R. y Nicklin, C. F. Sulphido-leukotriene production from
peripheral leukocytes and skin in clinically normal dogs and house dust mite positive atopic dogs. (Produccion de
suldo-leucotrienos de leucocitos perifericos y piel en perros cl nicamente normales y en perros atopicos
alergicos pos tivos a acaros de polvo domestico.) Veterinary Dermatology 2001; 12: 312.]
Zusammenfassung Die Pathogenese atopischer Dermatitis beim Hund ist noch nicht vollstandig aufgeklart.
Beim Menschen spielen Suld-Leukotriene (S-LT) in atopischer Dermatitis eine Rolle und nach
Allergenexponierung werden S-LT vermehrt in der Haut und peripheren Leukozyten produziert. Sechzehn
klinisch normale und dreizehn atopische Hunde nahmen an der Studie teil. Alle atopischen Hunde reagierten
positiv auf die intradermale Injektion von Hausstaubmilben (Referenzallergen). Blutproben und Hautbiopsien
wurden genommen. Suld-LT Synthese peripherer Leukozyten wurden nach Stimulation gemessen, zwischen
klinisch normalen und atopischen Hunden wurde kein statistisch relevanter Unterschied festgestellt. Suld-LT
Konzentrationen in stimulierten und nicht stimulierten Hautproben wurden gemessen und ergaben keinen
statistisch relevanten Unterschied zwischen klinisch normalen und atopischen Hunden oder zwischen
betroener und nicht betroener Haut bei atopischen Hunden. Klinische Symptome atopischer Hunde
wurden von Besitzern bewertet, kein Zusammenhang zwischen Grad der Hautveranderungen und S-LT
Konzentrationen konnte festgestellt werden. In atopischen Hunden war in dieser Studie die S-LT Synthese
nicht vermehrt. [Marsella, R. und Nicklin, C. F. Sulphido-leukotriene production from peripheral leukocytes
and skin in clinically normal dogs and house dust mite positive atopic dogs. (Sulphid-Leukotrien Produktion
peripherer Leukozyten und der Haut bei normalen Hunden und atopischen Hunden mit positiven Reaktionen
auf Hausstaubmilben.) Veterinary Dermatology 2001; 12: 312.]

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