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VDE244.

fm Page 149 Thursday, April 26, 2001 4:55 PM

Veterinary Dermatology 2001, 12, 149154

Blackwell Science, Ltd

Intradermal skin test reactivity to histamine and substance


P is blunted in dogs with atopic dermatitis
ROSANNA MARSELLA and CONSTANCE F. NICKLIN
Blanche Saunders Dermatology Laboratory, Department of Small Animal Clinical Sciences, PO Box 100126,
College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0126, USA
(Received 30 December 1999; accepted 7 September 2000)

Abstract Skin reactivity to intradermal injections (0.1, 0.5 and 1 n) of substance P (SP) was evaluated in 20
clinically normal dogs and 20 dogs with atopic dermatitis (AD). Saline and histamine were used as negative and
positive controls, respectively. Wheal diameters were measured. Reactions were evaluated for erythema and induration and a subjective score, on a scale from 0 to 4+, was given. Evaluations were performed at 3, 5, 10, 15
and 30 min after the injections. Wheal diameters for histamine and SP injections were significantly smaller in dogs
with AD compared with clinically normal dogs. In both groups, reactions to the various concentrations of SP
were not significantly different from each other and were always smaller than histamine reactions. Erythema was
not seen with SP injections. In addition, subjective scores for SP injections were significantly lower in dogs with
AD compared with controls. The results of this study are similar to those reported in human medicine, where a
role for SP in AD is proposed and desensitization of receptors to both SP and histamine is hypothesized. Further
studies are needed to investigate the role of SP in the pathogenesis of canine AD.
Keywords: atopic dermatitis, canine, intradermal skin test, neuropeptides, substance P.

INTRODUCTION
Allergies are an extremely common problem in dogs.
Atopic dermatitis (AD) is the second most common
allergy in dogs (10% of canine population).1 It manifests with erythema and pruritus on face, feet and ears
and with an increased predisposition to skin infections.2
It has been defined as a genetically inherited type I
hypersensitivity,3 but confusion still exists regarding
the exact pathogenesis of this disease in the dog and the
role of various inflammatory mediators. Thus, despite
the fact that AD is extremely common, few therapeutic
options are currently available. Investigation of mediators involved in the pathogenesis of canine AD is crucial to offer alternative treatments, especially for cases
in which traditional therapy with glucocorticoids is
ineffective or side-effects are unacceptable.
In humans there is increasing evidence that neuropeptides, such as substance P (SP), are involved in the
pathogenesis of AD.4,5 Substance P is an undecapeptide belonging to the family of tachykinins. In the past,
interest in SP focused largely on its role as a neurotransmitter released by some primary sensory fibres
at their central endings in the spinal cord. However,
SP is also present in the peripheral endings of some
primary sensory nerves, and attention is now focused
on its role as a mediator of neurogenic inflammation.
Substance P is synthesized in the dorsal root ganglion

Correspondence: Rosanna Marsella. Tel.: +1-352-392-4700 (ext. 5756),


Fax: +1-352-392-6125, E-mail: Marsellar@mail.vetmed.ufl.edu
2001 Blackwell Science Ltd

and transported peripherally to the sensory nerve endings


of myelinated C fibres functioning as a neurotransmitter for the communication of pain and pruritus.
In humans, SP release is triggered by various stimuli,
including histamine.6 In humans, receptors for SP have
been identified on a variety of cells, including mast
cells.7,8 Stimulation of mast cell SP receptors leads to
cell degranulation and histamine release9 11 Tissue
specificity exists in humans, and cutaneous mast cells
appear to be particularly sensitive to stimulation by
SP.12 Intradermal injection of SP causes wheal and
flare reactions in humans.13,14 Substance P is thought
to cause these reactions via both histamine-dependent
and -independent pathways.15
Substance P also has numerous pro-inflammatory
actions.16 It is chemotactic for neutrophils, monocytes,
T cells and eosinophils.1720 It induces the expression
of adhesion molecules by post-capillary venules of the
skin.21 It induces the proliferation of T lymphocytes
(especially the helper phenotype) and of keratinocytes.22,23 It stimulates the secretion of pro-inflammatory cytokines from monocytes and keratinocytes.24 26
Cytokines or second messenger signalling mediates
some of SP actions. Interleukins, prostaglandins,
tumour necrosis factor (TNF)- and intracellular calcium have all been implicated as messengers for SP.27
Substance P acts via neurokinin 1 receptors to cause
vasodilation2831 and may be the neurohumoral factor
responsible for axon reflex vasodilation.32
Substance P is released after allergen challenge
in humans with atopic disease33,34 and contributes significantly to the recruitment of eosinophils in allergic
149

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150

R. Marsella and C.F. Nicklin

rhinitis.35 Abnormal reactivity to injections of SP has


been reported in human patients with AD.36 Humans
with AD develop smaller flares and the onset of pruritus after the injection of SP is delayed compared with
healthy controls.37 To explain this abnormal reaction
a decreased sensitivity of SP receptors has been postulated.38,39 It has also been hypothesized that this
altered sensitivity might be a consequence of the
increased availability of neuropeptides in the skin
of patients with AD. The purpose of this study was
to evaluate the intradermal skin test reactivity to SP
in clinically normal dogs compared with dogs with
AD.

MATERIALS AND METHODS


Animals
Twenty clinically normal dogs and 20 dogs with AD
were selected for this study. Normal dogs were determined to be healthy based on history and physical
examination. The diagnosis of AD was based on compatible history, suggestive clinical signs, exclusion of
other pruritic skin diseases (e.g. sarcoptic mange, food
hypersensitivity or adverse reactions to food), and at
least three positive intradermal skin test (IDST) reactions using 57 common allergens (Greer, Lenoir, NC)
of the geographical area.40 Reactions were graded subjectively as detailed below. A reaction was considered
positive if the score was 2 or higher.
In animals with a history of nonseasonal pruritus,
food allergy was excluded by an appropriate food trial
prior to the study.41 Sarcoptic mange was excluded by
appropriate treatment, which included either three
injections of ivermectin (Ivomec, Merial Limited,
Iselin, NJ) at 300 g kg1 at 2-week intervals, or weekly
lime sulfur dips (LymDip, DVM Pharmaceuticals Inc.,
Miami, FL) for seven treatments.42 None of the dogs
included in the study had evidence of concurrent flea
allergy. Secondary skin infections (e.g. superficial pyoderma, Malassezia dermatitis) were treated before
dogs were entered into the study. Antibiotic treatment
for superficial pyoderma consisted of oral administration of cephalexin at 22 mg kg1 twice daily for 4 weeks
and antifungal therapy consisted of oral ketoconazole
(Nizoral, Janssen, Titusville, NJ) at 5 mg kg1 once
daily for 2 weeks. Both treatments were discontinued at
least 14 days before the beginning of the study. Also,
glucocorticoids (both systemic and topical) and oral
antihistamines were discontinued 2 months and
2 weeks, respectively, prior to inclusion in the study.
Intradermal injections
Intradermal (ID) injections (0.05 mL) of saline, histamine at 1/100 000 w/v (Greer) and SP (Sigma Chemicals, St. Louis, MO) at three concentrations (0.1, 0.5,
1 n) were performed on the shaved skin of the lateral
thorax. Concentrations of SP were selected based on
previous studies in humans36,37 and pilot studies in
dogs (Marsella, unpublished data). Buffered saline was
2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 149 154

used as the diluent for all injections. Skin reactions


were evaluated at 3, 5, 10, 15 and 30 min. Evaluation
times for the skin reactions were selected based on previous studies in humans.36,37 Erythema and induration
were graded subjectively on a scale of 0 to 4+, where 0
was the score assigned to the negative control (saline)
and 4+ was the score assigned to the positive control
(histamine) at 15 min. The diameter of the wheal was
measured, in mm, using a ruler and values were recorded. One investigator performed all the injections,
scoring and recording of the areas (RM). Injections
were not randomized and the investigator was not
blinded.
Statistics
Data were analysed using the least square analysis
of variance (LS) with all the main effects and
interactions included in the model (SAS for Windows,
version 6.12, Cary, NC). Differences among group
responses at various injection sites and measurement
times were analysed using orthogonal contrast analysis. A P-value < 0.05 was considered significant.
Data are presented as least squared mean SEM.

RESULTS
Animals
Twenty clinically normal dogs and 20 dogs with AD
were selected. In the control group, 15 dogs were
spayed females and five were neutered males. In the
atopic group, 11 dogs were spayed females and nine
were neutered males. The average age was 5.8 years in
the control group and 4.3 years in the atopic group.
Twenty different breeds were represented in this study.
Because of the small number of dogs in this study it was
not possible to statistically evaluate the influence of
age, breed and sex on the results of this study.
Wheal diameters
There was no difference in wheal diameter (Table 1) for
SP injections between the two groups (P = 0.06). However,
significant differences were detected between groups
for individual injection sites at various times. This difference was observed not only for SP, but also for the
histamine reactions. The peak reaction for histamine
was observed at 15 min, whereas the peak reaction to
SP injections was reached at 5 min in the dogs with AD
and 10 min in the normal dogs. No significant difference was detected between the three concentrations of
SP. Reactions to SP were always significantly larger than
those for saline for both dogs with AD and normal
dogs (P 0.0005). Histamine reactions were always
significantly larger than SP reactions (P = 0.0001) at
all times for both normal dogs and dogs with AD.
Subjective scores
Subjective scores for SP injections (Table 2) were significantly lower in the dogs with AD at 10 and 15 min
compared with normal dogs. Substance P scores were

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Substance P and histamine skin test reactivity in canine atopic dermatitis


Table 1. Wheal diameters after intradermal injections of saline,
histamine and substance P (SP) in dogs with atopic dermatitis (AD)
and in clinically normal dogs

Histamine
3 min
5 min
10 min
15 min
30 min
SP (0.1 n)
3 min
5 min
10 min
15 min
30 min
SP (0.5 n)
3 min
5 min
10 min
15 min
30 min
SP (1 n)
3 min
5 min
10 min
15 min
30 min
Saline
3 min
5 min
10 min
15 min
30 min

Atopic (n = 20)
Mean
SEM

Normal (n = 20)
Mean
SEM

P-value

0.91
0.99
1.10
1.14
1.13

0.069
0.069
0.069
0.069
0.069

0.87
1.04
1.19
1.27
1.24

0.069
0.069
0.069
0.069
0.069

0.0826
0.0509
0.0001
0.0001
0.0001

0.72
0.76
0.71
0.65
0.57

0.069
0.069
0.069
0.069
0.069

0.75
0.80
0.79
0.78
0.69

0.069
0.069
0.069
0.069
0.069

0.1928
0.0509
0.0005
0.0001
0.0001

0.72
0.77
0.74
0.66
0.59

0.069
0.069
0.069
0.069
0.069

0.75
0.80
0.81
0.78
0.70

0.069
0.069
0.069
0.069
0.069

0.1928
0.1928
0.0024
0.0001
0.0001

0.72
0.75
0.70
0.62
0.59

0.069
0.069
0.069
0.069
0.069

0.75
0.81
0.85
0.79
0.67

0.069
0.069
0.069
0.069
0.069

0.1287
0.0049
0.0001
0.0001
0.0005

0.60
0.62
0.57
0.52
0.49

0.069
0.069
0.069
0.069
0.069

0.59
0.63
0.60
0.58
0.53

0.069
0.069
0.069
0.069
0.069

0.6640
0.6640
0.1287
0.0093
0.0826

Reactions in the AD group are significantly smaller than in the


normal group, especially when observations are made after 10 min.
P-values represent comparisons between normal dogs and dogs with
AD. A P-value 0.05 was considered significant.

significantly higher than the saline at 3, 5, 10 and


15 min (P 0.01) for both normal dogs and dogs with
AD. Histamine scores were always significantly higher
than SP scores for both groups (P = 0.0001). The highest scores for histamine were recorded at 15 min,
whereas the peak for SP scores was at 5 min. One dog
in the control group and two in the atopic group had
suppressed histamine reactions (subjective scores at
15 min were < 4). This was attributed to the nervous
nature of the animals and caused the mean values at
15 min for histamine reactions to be < 4. No significant
difference was detected when various concentrations of
SP were compared with each other in both groups. No
erythema was detected for any of the SP reactions.

DISCUSSION
In this study, intradermal skin test reactivity to SP and
histamine injections was significantly lower in dogs
with AD than normal dogs. These findings are similar
to those reported in human literature, in which decreased
reactions to histamine34,4345 and SP injections34,35,37
have been documented in patients with AD.

151

Table 2. Subjective scores for intradermal injections with saline,


histamine and substance P (SP) in dogs with atopic dermatitis (AD)
and in clinically normal dogs

Histamine
3 min
5 min
10 min
15 min
30 min
SP (0.1 n)
3 min
5 min
10 min
15 min
30 min
SP (0.5 n)
3 min
5 min
10 min
15 min
30 min
SP (1 n)
3 min
5 min
10 min
15 min
30 min
Saline
3 min
5 min
10 min
15 min
30 min

Atopic (n = 20)
Mean
SEM

Normal (n = 20)
Mean
SEM

P-value

2.15
2.95
3.65
3.80
3.10

0.085
0.085
0.085
0.085
0.085

1.80
2.65
3.60
3.95
3.55

0.085
0.085
0.085
0.085
0.085

0.0041
0.0138
0.6809
0.2175
0.0002

0.90
1.00
0.65
0.40
0.05

0.085
0.085
0.085
0.085
0.085

0.70
1.30
1.15
0.80
0.20

0.085
0.085
0.085
0.085
0.085

0.1003
0.0138
0.0001
0.0011
0.2175

0.90
1.05
0.80
0.35
0.05

0.085
0.085
0.085
0.085
0.085

0.65
1.15
1.10
0.75
0.15

0.085
0.085
0.085
0.085
0.085

0.0401
0.4109
0.0138
0.0011
0.4109

0.85
0.90
0.50
0.30
0

0.085
0.085
0.085
0.085
0.085

0.70
1.20
0.95
0.70
0.20

0.085
0.085
0.085
0.085
0.085

0.2175
0.0138
0.0002
0.0011
0.1003

0
0
0
0
0

0.085
0.085
0.085
0.085
0.085

0.10
0
0
0
0

0.085
0.085
0.085
0.085
0.085

0.4109
1.0000
1.0000
1.0000
1.0000

A P-value 0.05 was considered significant.

Histamine mediates many, but not all, effects of


SP.9,10,46 This is confirmed by the finding that pretreatment with an H1 histamine antagonist, or by a histaminedepleting agent (e.g. 48/80), decreases, but does not
completely abolish, the wheal and flare reactions to SP
injections in people.5 The hyporesponsiveness to SP
injections in humans with AD has been hypothesized
to be either related directly to decreased sensitivity to
SP, or to histamine. Both hypotheses are likely to apply.
Patients with AD have increased concentrations of SP
in affected skin,2,3 thus, it is reasonable to believe that
the higher availability of this neuropeptide may lead to
a decreased sensitivity of the SP receptors. In addition,
patients with AD have enhanced releasability of histamine upon stimulation47,48 and increased concentrations of histamine in the skin,49 which leads to a
downregulation of the histamine receptors.
The results of this study suggest that similarities between the human and canine disease exist. Dogs with
AD may also have increased cutaneous SP concentrations and this could affect the reactivity to SP injections.
The authors are currently working on the extraction
and measurement of SP concentrations in the skin of
dogs to better elucidate this hypothesis. Alternatively,
it is possible that the decreased response to SP injections in dogs with AD may be due to a decreased
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R. Marsella and C.F. Nicklin

responsiveness to histamine. It has been documented


that dogs with AD have increased cutaneous histamine
concentrations50 52 and this may lead to an altered sensitivity of the receptors. Also, the dogs with AD in this
study had decreased histamine reactions compared
with the normal dogs.
It is interesting to note that SP reactions peaked
faster in dogs with AD, compared with normal dogs.
This might be interpreted either as a form of altered
sensitivity to SP or as a form of increased releasability
of histamine subsequent to SP stimulation in dogs with
AD. The first hypothesis is supported by the observation that SP reactions occurred faster than histamine
reactions (in both groups) leading to the belief that SP
reactions, in the dog, may be mediated primarily by a
direct action of SP on blood vessels rather than being
histamine mediated. The second hypothesis is in line
with a previously documented increased releasability
of mast cells in dogs with AD.48
The peak of subjective scores for SP was at 5 min,
whereas the peak of wheal diameter was reported at
10 min. This is very similar to what has been reported
in people, where the maximum flare response was
observed at 3 min and the maximum wheal diameter
was reached at 12 min.53 In this study, no significant
difference in wheal diameter was found between the
various SP concentrations, and increasing concentrations of SP did not cause larger wheals or higher reaction scores. This might be due to a saturation of the
receptors available.
In both groups, reactions to SP were always smaller
than histamine reactions suggesting that, in the dog,
this neuropeptide may not be as strong an inflammatory mediator as histamine. This seems to be confirmed
by the lack of erythema and pruritus for SP injections
in both groups. It is possible that wide species variation
exists in the sensitivity to SP and that higher concentrations of SP are needed in the dog compared with
humans. In rats, concentrations 100 times higher than
those used in people are required to induce reactions
comparable with the ones seen in people.5
In conclusion, skin reactivity to SP and histamine
was decreased in dogs with AD compared with clinically normal dogs in this study. A desensitization of
receptors is proposed. Further studies evaluating concentration of SP in the skin of normal dogs and dogs
with AD are necessary to better evaluate the role of SP
in the pathogenesis of this disease.

ACKNOWLEDGEMENT
This study was funded by Morris Animal Foundation.

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Rsum Cette tude a valu la ractivit cutane aprs linjection intradermique de substance P (0.1, 0.5 et
1 n) chez 20 chiens normaux et 20 chiens prsentant une dermatite atopique (DA). Lhistamine et un solut sal
ont t utiliss comme contrle positifs et ngatifs. Les diamtres des ractions ont t mesurs. Lrythme et
linduration ont t mesures subjectivement (0 4+). Les mesures ont t ralises 5, 10, 15 et 30 minutes aprs
les injections. Les diamtres des ractions lhistamine et la SP taient significativement plus faibles chez les
chiens avec une DA que chez les chiens sains. Dans les deux groupes, aucune diffrence na t observe avec les
diffrentes concentrations de substance P. Les ractions la SP taient systmatiquement plus faibles que celles
lhistamine et ne prsentaient pas drythme. En outre, les scores obtenus au niveau des ractions la SP taient
significativement plus faibles dans le groupe DA que chez les chiens sains. Les rsultats de cette tude sont semblables ceux rapports chez lhomme. Il a t suggr que la SP pourrait jouer un rle dans la DA chez lhomme
et une drgulation des rcepteurs la SP et lhistamine est galement suspecte. Des tudes supplmentaires
sont ncessaires pour valuer le rle de la SP dans la pathognie de la DA chez le chien. [Marsella R., Nicklin C. F.
Intradermal skin test reactivity to histamine and substance P is blunted in dogs with atopic dermatitis. (La ractivit
des tests cutans intradermiques lhistamine et la substance P est diminue chez les chiens prsentant une
dermatite atopique.) Veterinary Dermatology 12: 149154.]
Resumen Se evalu la reactividad cutnea a las inoculaciones intradrmicas (0.1, 0.5 y 1 n) de sustancia P (SP)
en 20 perros clnicamente normales y 20 perros con dermatitis atpica (DA). Se utiliz suero salino e histamina
como controles negativo y positivo, respectivamente. Se midi el dimetro de los habones. Se evalu en las reacciones el eritema y la induracin, y se dio una puntuacin subjetiva, en una escala de 0 a 4+. Las evaluaciones
se realizaron a los 3, 5, 10, 15 y 30 minutos despus de las inoculaciones. El dimetro de los habones por las
inoculaciones de histamina y SP fueron significativamente menores en perros con DA comparado con los perros
clnicamente normales. En ambos grupos, las reacciones a diferentes concentraciones de SP no fueron significativamente diferentes entre ellas y fueron siempre menores que las reacciones a la histamina. No se observ eritema
en las inoculaciones de SP adems, las puntuaciones subjetivas para las inoculaciones de SP fueron significativamente inferiores en perros con DA comparado con los controles. Los resultados de este estudio son similares
a los publicados en medicina humana, donde se propone un papel de la SP en la DA y se formula la hiptesis de
una desensitizacin de receptores de SP e histamina. Sern necesarios ms estudios para investigar el papel de
la SP en la patognesis de la DA canina. [Marsella R., Nicklin C. F. Intradermal skin test reactivity to histamine
and substance P is blunted in dogs with atopic dermatitis. (La reactividad de la prueba cutnea intradrmica a la
histamina y a la sustancia P se encuentra reducida en perros con dermatitis atpica.) Veterinary Dermatology 12:
149 154.]
Zusammenfassung Die Hautreaktivitt nach intradermalen Injektionen (0,1, 0,5 und 1 nM) von Substanz P
(SP) wurde bei 20 klinisch normalen Hunden und bei 20 Hunden mit atopischer Dermatitis (AD) bewertet.
Salzlsung und Histamin wurden als negative und positive Kontrolle verwendet. Der Durchmesser der Quaddeln
wurde gemessen. Reaktionen wurden auf Hautrtung und Verhrtung untersucht und subjektiv auf einer Skala
von 0 bis 4+ bewertet. Auswertungen wurden 3, 5, 10, 15 und 30 Minuten nach den Injektionen durchgefhrt.
Die Quaddeldurchmesser der Histamin- und SP-Injektionen waren bei den Hunden mit AD erheblich kleiner
als bei klinisch normalen Hunden. In beiden Gruppen waren keine unterschiedlichen Reaktionen auf die
verschiedenen SP-Konzentrationen festzustellen, die Reaktionen waren immer kleiner als die Histaminreaktionen.
Eine Hautrtung wurde nach SP-Injektionen nicht festgestellt. Zustzlich war die subjektive Bewertung fr SPInjektionen bei den Hunden mit AD erheblich niedriger als bei den Kontrollhunden. Die Resultate dieser Studie
hneln denen in der Humanmedizin, wo hypothetisch eine Desensibiliserung der SP- und Histamin-Rezeptoren
vorgeschlagen wird. Weitere Studien sind erforderlich, um die Rolle von SP in der Pathogenese der atopischen
Dermatitis beim Hund zu untersuchen. [Marsella R., Nicklin C. F. Intradermal skin test reactivity to histamine
and substance P is blunted in dogs with atopic dermatitis. (Die intradermale Hauttestreaktivitt gegen Histamin
und Substanz P ist bei Hunden mit atopischer Dermatitis abgeschwcht.) Veterinary Dermatology 12: 149154.]

2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 149 154