Systemic Nickel Allergy: Oral Desensitization and Possible Role of Cytokines Interleukins 2 and 10

Abstract

Nickel ingested with food can elicit either systemic cutaneous or gastrointestinal symptoms causing a systemic nickel allergy syndrome (SNAS) that can be treated with tolerance by oral ingestion of the metal. It has been suggested that interleukins 2 (IL-2) and 10 (IL-10) are involved in the mechanisms underlying oral tolerance. We evaluated the clinical efficacy of oral desensitization therapy in SNAS consisting in the administration of nickel sulphate. Because nickel allergy prevalently affects women, only female subjects (N = 22) were recruited. Oral nickel desensitizing therapy was associated with low-nickel diet for three months. Before and after therapy, clinical conditions were evaluated, and circulating cytokines IL-2 and IL-10 were measured. After the two-year treatment, visual analogue scale (VAS) scores for symptoms were significantly reduced (P less than 0.001). Patients were released by either cutaneous or gastrointestinal symptoms and by tolerating nickel-containing food. At the end of the treatment, nickel oral challenge test was negative in 18 patients, and IL-2 level in the serum was significantly reduced while IL-10 was increased, although this datum was not statistically significant. Our study confirms the clinical efficacy of nickel oral immunotherapy and focuses on the mechanisms triggered by oral tolerance indicating that reduction of IL-2 can be associated with success of oral nickel desensitizing therapy.

Full text

INTERNATIONALJOURNAL OF IMMUNOPATHOLOGYAND PHARMACOLOGY Vol. 26, no.
1,251-257
(2013)
LETTER TO THE EDITOR
SYSTEMIC
NICKEL
ALLERGY: ORAL DESENSITIZATION AND POSSIBLE
ROLE
OF
CYTOKINES INTERLEUKINS 2 AND 10
L.
RICCIARDI"
A. CARNI2, G. LOSCHIAVOI, S. GANGEMJI,
V.
TIGANOI,
E.
ARENA
I, C.
MANNUCCF
and
G.
CALAPAF
'Allergy
and
Clinical Immunology Unit, Azienda Ospedaliera Universitaria "Policlinico G.
Martino, "University
of
Messina, Italy; 2Department
of
Clinical
and
Experimental Medicine
and
Pharmacology, Section
of
Pharmacology, School
of
Medicine, University
of
Messina, Italy
ReceivedApril 26, 2012 -AcceptedDecember 14, 2012
Nickel ingested with food can elicit either systemic cutaneous or gastrointestinal symptoms causing a
systemic nickel allergy syndrome (SNAS)
that
can be treated with tolerance by oral ingestion of the metal.
It
has been suggested
that
interleukins 2 (IL-2)
and
10 (IL-10) are involved in the mechanisms underlying
oral tolerance. We evaluated the clinical efficacy of oral desensitization therapy in SNAS consisting in the
administration of nickel sulphate. Because nickel allergy prevalently affects women, only female subjects
(N =22) were recruited.
Oral
nickel desensitizing
therapy
was associated with low-nickel diet for three
months. Before
and
after therapy, clinical conditions were evaluated,
and
circulating cytokines IL-2
and
IL-10 were measured.After the two-yeartreatment, visual analogue scale (VAS)scores for symptoms were
significantly reduced
(p
<0.001). Patients were released by either cutaneous or gastrointestinal symptoms
and
by tolerating nickel-containing food. At the end of the treatment, nickel oral challenge test was
negative in 18 patients,
and
IL-2 level in the serum was significantly reduced while IL-10 was increased,
although this
datum
was not statistically significant.
Our
study confirms the clinical efficacy of nickel oral
immunotherapy
and
focuses on the mechanisms triggered by oral tolerance indicating
that
reduction of
IL-2 can be associated with success of oral nickel desensitizing therapy.
Allergic contact dermatitis (ACD) is a cutaneous
reaction caused by an increased hypersensitivity
after a direct contact with chemicals involving
cell-mediated immunological mechanisms (1).
According to recent data, prevalence
of
ACD in
the adult general population is approximately 30%,
whereas that in children is approximately 20% (2, 3).
Qualitative and quantitative environmental exposure
to sensitizing compounds varies from region to
region. Nickel sulphate is generally considered one
of
the most common among substances causingACD
(4). Epidemiological studies indicate that it is the
number one allergen in frequency
of
positive patch
test reactions both in Europe and in North America
(5-7). Prevalence
of
nickel allergy in the United
States has almost tripled in the last two decades,
and young patients had significantly higher rates
of
nickel sensitivity compared with older patients;
women also had significantly higher rates
of
nickel
allergy than men (8). Recent data from the European
Surveillance System
of
Contact Allergies showed
that 20%
of
9871 tested patients were sensitized to
nickel, with the highest prevalence in Italy (32.2%)
(9).
Key words: nickel, systemic allergy, oral tolerance, oral desensitizing therapy, cytokines
Mailing address: Dr Gioacchino Calapai,
Department
of
Clinical and Experimental Medicine and Pharmacology,
Section
of
Pharmacology, School
of
Medicine,
University
of
Messina, Via Consolare Valeria 5,
98100 Messina, Italy
Tel.: +39
902213646
Fax: +39 90 2213300
e-mail: gcalapai@unime.it 251
0394-6320 (2013)
Copyright ©by BIOLlFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties
DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF
INTEREST RELEVANT TO THIS ARTICLE.

252 L. RICCIARDI ET AL.
In the last decades, researchers focused not only
on local cutaneous symptoms caused by nickel
but also on systemic symptoms provoked by the
absorption
of
the metal. It has been observed that
in subjects with nickel ACD wearing dental or
orthopedic implants containing nickel, systemic
eczemaor chronic urticaria may occur. This condition
is known as systemic contact dermatitis (SCD) (10).
It has also been observed that in some subjects with
nickel ACD, the metal ingested with food is able to
elicit either systemic cutaneous or gastrointestinal
symptoms. This condition can be associated to
recurrent infections (11, 12) and has been defined
as systemic nickel allergy syndrome (SNAS) (13).
SNAS can be misdiagnosed as an adverse drug
reaction, delaying the correct diagnosis and leading
to inappropriate treatment (14).
Because nickel sensitization is a hapten-specific
immunological process, it is possible to induce
immune tolerance to this metal. The induction
of
tolerance has been proposed for the treatment
of
SNAS. Nickel desensitization in humans was first
performed in Sweden in 1987, by oral administration
of
5 mg
of
NiS04
once a week for six weeks,
obtaining areduction
of
patch test reaction. Positive
results were confirmed by a multicenter study carried
out in Italy through the administration
of
lower
doses
of
NiS04
(0.1 ng) per day for three years
(15). Some authors support the view that oral nickel
desensitization can be useful in nickel allergy and,
through in vitro studies, suggest that cytokines IL-2
and
IL-l
0 are involved in the mechanisms underlying
oral tolerance (16, 17). The aim
of
this study was to
evaluate the effects
of
nickel oral desensitization on
clinical symptoms and serum concentration
of
the
cytokines IL-2 and
IL-l
O.
MATERIALS AND METHODS
Study design
We recruited 24 female subjects with nickel allergic
contact dermatitis, positive to nickel patch testing, and
those reporting systemic cutaneous and gastrointestinal
symptoms, such as chronic urticaria-angioedema,
bloating, constipation, and recurrent abdominal pain, after
ingestion of nickel-containing food (Table I). Patients
positive to nickel patch test and reporting at least either
one cutaneous or one gastrointestinal symptom were
asked to follow a diet for one month, allowing only food
with known low nickel content and avoiding food with
known high nickel content or high nickel bio-availability
(15, 18) (Table II).
The study design was authorized by the ethics
committee of the Azienda Ospedaliera Universitaria
"Policlinico G. Martino" of Messina. Subjects
participating in the study signed an informed consent
authorized by the same committee.
In each subject, diagnosis of SNAS was confirmed
Table I. Systemic nickel allergy symptoms (SNAS) reported by the patients recruited before oral nickel desensiti-
zation therapy.
S:-
mptorns Patients 1111
N
Patients N= 22.
Urticaria
and/or
angioedema
Systemic
contact
dermatitis
Bloating
and
meteorism
Recurrent
abdominal
pain
Costipation
Diarrhea
Dyspepsia
Sickness
and/or
vomiting
Gastro
esophageal
reflux
Itching
Headache
Iron
deficiency
anemia
13
13
13
12
8
7
'6
5
4
3
3
1
59
59
59
54.5
36.3
31.8
27.2
22.7
18.1
13.6
13.6
4.5

lot
J. IrnrnuoopathoL Pharrnacol.
Table II. Low nickel diet:
food
allowed
and
not allowed.
Food allowed Food not allowed
SPICES AND Paprika, pepper, VEGETABLES Asparagus, broccoli,
DRESSINGS olive oil, vinegar, artichokes, carrots, cabbage,
mayonnaise, cauliflower, onions, green
tomato sauce, beans, peas, fennel,
salt. mushrooms, lettuce,
tomatoes, rocket salad, celery,
spinach, Brussels sprouts
DRINKS Freeze drinks, FRUIT Apricots, pineapples,
beer (not canned), watermelon, avocado,
coffee, wine. cherries, figs, berries, kiwi,
pears, plums, grapes.
CEREALS Plain flour, bread, HERBS AND Bay leaves, basil, marjoram,
pasta, pizza, rice DRESSINGS parsley, ketchup, seed oil,
mustard
VEGETABLE Citrus fruits, CEREALS wholemeal products,
AND FRUITS bananas, apples, buckwheat oat, barley, maize,
melons, peaches, malt, rye, soy.
cucumbers,
aubergines,
peppers, potatoes,
garlic, pumpkins,
courgettes.
FISH Sword-fish, tuna. SEAFOOD Cod, shell-fish, salmon,
octopus, squid, plaice.
SWEETS Choux-pastry, CHOCOLATE All chocolate sweets.
biscuits, custard,
honey, pastry,
sponge cakes,
sugar
OTHERS Meat, eggs, NUTS Everyone.
salami, ham,
dairy products DRINKS Tea.
[modifiedfrom Sharma, 2007(18) J
253
through the observation
of
improvement
of
symptoms after
the thirty days low-nickel diet and worsening consequent
to oral challenge with nickel sulphate
of
up to 3.75 mg.
The
maximum
cumulative dose
of3.75
mg
of
nickel sulphate
was chosen on the basis
of
previous studies showing
that it is an amount approximately 10 times greater than
the estimated normal daily dietary intake
of
nickel,
and
therefore, it is expected to result in flare-up reactions
in
most
of
the nickel-sensitive individuals (17). Nickel
sulphate was given at a dose
of
1.25 mg on the first day; in
the absence
of
symptoms, two further single doses
of
1.25
mg were given on the second day. Symptoms
of
allergy
were considered: eruptions
of
previously unaffected skin,
flare-up at previous sites
of
contact dermatitis, including
the flare-up reactions
of
previously positive nickel patch
test, urticaria, and other systemic symptoms such as
headache, marked fatigue, and gastrointestinal symptoms
(bloating, abdominal pain, and diarrhea).
Twenty-four
women
aged between 20 and 48 years
(median age 30.81) having ahistory
of
nickel systemic
allergy underwent the nickel challenge test. Two subjects
who
were
not
showing symptoms after the oral nickel
challenge were excluded
and
considered non-responders.
Twenty-two
women
were included in the study; 14 nickel-
sensitive
women
(63.6%)
had
systemic symptoms upon
oral exposure to 1.25 mg
of
nickel
and
8
women
(36.3%)
to 3.75 mg
of
nickel.
For the oral nickel hyposensitizing treatment, the

254 L. RICCIARDI ET AL.
product Tio niche1from Lofanna (Milan, Italy) was used.
Each patient was treated with an up-dosing
of
nickel
sulphate for nine weeks as follows: first week, 0.1 ng three
times per week; second week, 0.2 ng three times per week;
third week, 1.0 ng three times per week; fourth week, 2.0
ng three times per week; fifth week,
lOng
three times per
week; sixth week, 20 ng three times per week; seventh
week, 100 ng three times per week; eighth week: 200 ng
three times per week; ninth week: and 500 ng three times
per week. After the ninth week, the dose
of
500 ng was
kept three times per week for two years. Doses were taken
by fasting subjects always on Mondays, Wednesdays, and
Fridays.
Patients were instructed to start a gradual re-
introduction
of
nickel-containing food starting from
the tenth week
of
therapy. For all the treatment period,
information on the appearance
of
side effects or more
severe adverse reactions and the need for antiallergicdrugs
(corticosteroids, antihistamine drugs) were collected. At
the end
of
the two-year period
of
oral nickel desensitizing
treatment, the nickel oral challenge test was performed
again.
Symptoms
To evaluate the effects of therapy, each patient was
shown a visual analogue scale (VAS) represented by
a horizontal line, 10 em in length, going from zero
(maximum malaise) to ten (no symptoms) corresponding
to maximum perceived severity
of
SNAS symptoms and
absence
of
symptoms, respectively (19). Each patient was
asked to mark a point on the line corresponding to their
own perception
of
malaise correlated to SNAS symptoms,
immediately before the beginning
of
the therapy and after
the 2-year period
of
treatment.
Cytokine measurements
Whole blood samples
of
4.5 ml were collected using a
vacutainer blood-collecting system and sterile endotoxin-
free blood collection tubes containing 120 IV sodium
heparin. Serum samples were centrifuged and stored at
-80°C. ELISA measurement for cytokines IL-2 and IL-10
(Diaclone, Milan, Italy) was used. Each kit was a solid-
phase sandwich ELISA. Monoclonal antibodies specific
for each cytokine have been coated onto the wells
of
the
microtiter strips provided. Value for each cytokine was
expressed as pg/ml. Absorbance reading
of
each well on
the spectrophotometer was performed using 450 nm as the
primary wavelength.
Statistical analysis
Analysis
of
data obtained with measurements
of
blood
cytokines was performed using analysis
of
variance. Data
are expressed as the means ±standard error. Statistical
analysis
of
VAS scores was performed with SPSS for
Windows (version 17.0). Differences between paired
groups were analyzed using the Wilcoxon test. Statistical
significance was set at P <0.05.
RESULTS
In one case, treatment was interrupted after the
first year, as the patient had become pregnant and,
following gestation; had
no
complications. Statistical
analysis
of
VAS scores shows a highly significant
difference in all the patients between symptoms
collectedbefore (2.71±0.23) and after (7 .86±0.25) the
two-year treatment with oral nickel desensitization
therapy (P <0.001). Patients were released by either
cutaneous or gastrointestinal symptoms, and data
obtained using VAS indicate that two-year nickel
oral treatment was overall perceived efficacious by
the patients according to the variation
of
VAS score
before and after nickel oral treatment. After the two-
year oral nickel treatment, patients also were made
to tolerate nickel-containing food; the reintroduction
was total in 85.7% (N =18/21)
of
the patients and
partial in 14.3% (N =3/21)
of
the patients. Precisely,
all vegetables, fruits, fish, and cereals were generally
well tolerated after one year
of
nickel oral treatment,
whereas some patients still did not tolerate the intake
of
chocolate, crustaceans, and nuts, which not only
contain nickel but also histamine-releasing food.
At the end
of
the two-year period
of
oral nickel
desensitizing treatment, nickel oral challenge test
was negative in 18 patients even after a cumulative
dose
of
3.75 mg, whereas 3 patients presented mild
systemic itching, cutaneous rash, and mild abdominal
pain. Patients generally well tolerated the nickel oral
treatment. Antihistamine tablets were taken by 54%
(N =12/22)
of
the patients during the up-dosing
phase especially when starting the 100- and the 500-
ng dose. No drugs were needed elsewhere.
Measurements
of
cytokines IL-2 and IL-lO
produced the following results: statisticallysignificant
reduction ofIL-2level in theserum after the two-year
period
of
oral desensitization (JL-2 was unchanged
after the first year
of
treatment) (Fig. 1). Basal IL-
10 mean level
(TO)
was 2.1O±0.97 and 2.08±0.85 pgl
ml after the first year (Tl); IL-lO increased after the
second year
of
treatment (T2 =2.52±1.20 pglml),
although these data were not statistically significant

0.17
0.14
0.11
~
01
a. 0.08
C)I
::!
0.05
0.02
0.00
Int. J. Immunopatbol. Pbarmacol. 255
TO
T1 12
Fig. 1. Interleukin 2(IL-2) in the serum
of
patients affected by systemic nickel allergy syndrome (SNAS) before
(TO)
de-
sensitizing therapy with oral nickel
and
after one (T1) or two years (T2). Each value is the mean ±error standard
of
the
means
of21
patients. IL-2 was evaluated by ELISA method. *=p<0.01 vs
TO.
in respect to the basal mean level.
DISCUSSION
Becausenickelispresentinmostofthe constituents
of
a normal diet, one
of
the possible therapies for
nickel allergy is represented by a low-nickel diet.
However, to measure precisely the daily intake of
nickel is extremely difficult because its content in
food can vary widely. Moreover, extremely rigorous
and prolonged nickel restriction in diet may have
nutritional consequences (20). Consequently, nickel
diet restriction alone is not considered a definitive
solution for the care
of
SNAS.
Studies indicate that oral nickel desensitizing
treatment associated with a low-nickel diet can be
effective in inducing clinical tolerance to nickel-
containing foods (21).
SCD seems to be mediatedby type IV mechanism.
Evidence suggests that tolerance to nickel and
also to other antigens are associated with more
immunoregulatory mechanisms (22). Through the
repeated administration
of
increasing doses
of
the
causative allergen, allergen-specific immunotherapy
induces a state
of
tolerance associated with
constitution
of
T regulatory (Treg) cells, including
allergen-specific interleukin (IL)-1O-secreting Treg
type 1 cells and CD4(+)CD25(+)Treg cells, and
induction
of
suppressive cytokines such as IL-IO
(26 23). Evidence shows that oral nickel tolerance
is mediated by CD4+CD25+ and CD8+ Treg cells
(24), and it has also been observed that nickel-
sensitive individuals reacting to oral high nickel
doses had significantly elevated levels
of
interleukin
5 in the serum, indicating an activation
of
type 2 T
lymphocytes (25).
Our study shows that oral nickel desensitizing
therapy, associated with low-nickel diet, produce
a strong attenuation
of
SNAS symptoms after the
second year
of
treatment.
Statistically significant difference
of
IL-2 level
in the serum was observed after the two-year period
of
oral desensitization, whereas IL-2 circulating
level was unchanged after the first year of treatment.
Not statistically significant increase
of
IL-lO was
observed after the treatment.
Some regimens
of
feeding induce tolerance
of
IL-2-producing T cells in vitro or in vivo.
Anergic T cells could function as regulatory
cells in orally tolerized mice by releasing such
mediators (26). Regulatory T cells are able to
restore immunoregulatory function and have

256 L. RICCIARDI ET AL.
been characterized as CD4+ T cells that express
the high affinity receptor for IL-2 (IL-2RA or
CD25) (27). The presence
of
IL-2RA, a membrane
protein regulating homeostatic T-cell proliferation,
is required for the proper function
of
Treg cells;
although it is not known whether IL-2 acts as a
peripheral differentiation factor or as an expansion
factor or whether it triggers their effector activity
(28). Other types
of
T cells with regulatory function
(such as Th3 cells and Trl cells) can be triggered by
oral or nasal administration of antigens. These cells
carry out their suppressive function by the secretion
of
anti-inflammatory cytokines such as IL-IO, and
they have been referred to as "adaptive" regulatory
T cells as opposed to the naturally occurring or
"innate" thymic Tregs (CD4+CD25+ T cells) (29).
Presence in oral tolerance
of
a state
of
immune
unresponsiveness defined as "anergy" was first
suggested by experiments in which T-cell tolerance
could be reversed in vitro by exogenous IL-2 (26).
On this basis, it could be believed that the decrease
of
serum IL-2 following oral desensitizing therapy
provokes anergy in T cells responsible for allergy
reaction to nickel, thus leading to improvement of
symptoms.
It
has been observed that increased IL-
10 production by SIT also causes specific anergy
in peripheral T cells (30). Therefore, it could be
hypothesized that it is the interplaying balance
between the two cytokines IL-2 and IL-lO, promoted
by oral tolerance, which plays a key role among the
factors responsible for the beneficial results obtained
with oral nickel desensitizing therapy.
Finally, our study confirms the clinical efficacy
of nickel oral immunotherapy after two years of
treatment and highlights the possible mechanisms
triggered by oral tolerance. Although other studies
are warranted to confirm this view, data indicate that
reduction
of
IL-2 in the serum is associated with
success of oral nickel desensitizing therapy.
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