ORIGINAL ARTICLE
Evaluation of immunologic profile in patients
with nickel sensitivity due to use of fixed
orthodontic appliances
Marcelo Marigo, PhD,a Darcy Flávio Nouer, PhD,b Marisa Cristina Santos Genelhu, MS,a Luiz Cosme Cotta
Malaquias, PhD,a Virgı́nia Ramos Pizziolo, MS,a Alexandre Sylvio Vieira Costa, PhD,c Olindo Assis MartinsFilho, PhD,d and Lucia Fraga Alves-Oliveira, PhDa
Governador Valadares, São Paulo, and Belo Horizonte, Brazil
The aim of this study was to develop a new approach to testing the impact of nickel antigen on in vitro
cell-proliferation assay, to identify adverse reactions to casting alloys among orthodontic patients. Cellproliferation assay in vitro was used as the basic methodology to assess the influence of such variables as
source of nickel antigen, type of serum used to supplement the culture medium, and number of cells in the
culture. We selected 35 orthodontic patients who were classified as nickel sensitive and non–nickel sensitive,
based on their clinical records. Our results showed that hexahydrated nickel sulfate at 10 ␮g/mL, 10% of
autologous sera, and 2 ⫻ 105 cells was the best condition for inducing the most marked nickel proliferation
response in vitro. This optimized method was able to distinguish nickel-sensitive from non–nickel-sensitive
dental patients and also to discriminate those with positive skin tests. Our data suggest that continuous
exposure to nickel casting alloys might lead to oral tolerance mechanisms that modulate nickel sensitivity,
as evidenced by the lower cell proliferation index in patients undergoing orthodontic treatment over 24
months. Finally, our findings demonstrated a known nickel-induced type 2 immune response and a marked
lack of type 1 immunity (interferon ␥) as the hallmarks of nickel-sensitive patients. Further studies are needed
to clarify the major cell phenotype associated with this type 2 immune response and the lack of type 1
immunity observed in nickel-sensitive people. (Am J Orthod Dentofacial Orthop 2003;123:000-00) (Am J
Orthod Dentofacial Orthop 2003;124:46-52)
M
any objects contain nickel and can cause
nickel sensitivity, and many people are in
continuous contact with items that contain
nickel.1 Nickel-containing objects are common in dentistry, especially in dental implants and compounds
used in crowns, bridgework, and orthodontic appliances. Nickel-titanium alloys are widely used in orthodontic treatment, and all these devices can induce skin
and mucosal reactions, leading to tissue inflammation.2-4 An immune response induced by nickel appliances is generally called contact dermatitis and, from
a
Núcleo de Pesquisa em Imunologia, Faculdade de Ciências da Saúde,
Universidade Vale do Rio Doce, Governador Valadares, Minas Gerais, Brazil.
b
Faculdade de Odontologia de Piracicaba, UNICAMP, São Paulo, Brazil.
c
Faculdade de Ciências Agrárias, Universidade Vale do Rio Doce, Governador
Valadares, Minas Gerais, Brazil.
d
Centro de Pesquisas René Rachou, Fundaçáo Oswaldo Cruz, Belo Horizonte,
Minas Gerais, Brazil.
Supported in part by Fundação de Amparo à Pesquisas do Estado de Minas
Gerais, Brazil.
Reprint requests to: Dr Marcelo Marigo, Rua João Pinheiro, 610 Centro,
Governador
Valadares,
MG
CEP
35020-270,
Brazil;
e-mail,
[email protected].
Submitted, July 2002; revised and accepted, October 2002.
Copyright © 2003 by the American Association of Orthodontists.
0889-5406/2003/$30.00 ⫹ 0
doi:10.1016/S0889-5406(03)00239-7
46
the immunologic standpoint, is considered type IV
hypersensitivity. In this context, nickel binding to
endogenous macromolecules can stimulate macrophages and cytotoxic cells, up-regulating the expression of adhesion molecules.5-7 It has also being reported that low-dose exposure can alter the metabolism
of human monocytes.8 Additionally, nickel induces T
lymphocytes to produce several cytokines, including
interferon IFN-␥, interleukin IL-2, IL-5, and IL-10, and
stimulates cellular proliferation.9 The pattern and level
of cytokines secreted are critical to triggering differential immune responses, which can lead to different
degrees of tissue damage. The temperature, microorganisms, enzymatic compounds, and ions normally
present in the oral cavity particularly favor the breakdown and release of metallic elements from dental
casting alloys.10
Contact dermatitis generally appears clinically as
eczema. Reactions of the mucous membranes, such as
stomatitis, also occur, and gum hyperplasias, cheilitis,
labial desquamation, and multiform erythemas are frequently noted. Nickel hypersensitivity has increased in
the last 10 years, and it is estimated that 15% to 30% of
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 124, Number 1
Europeans and Americans, in a proportion of 1 to 8,
men to women, show symptoms.11 A prevalence of 7%
to 28% of the general population has been also reported.12,13
Nickel sensitivity has been diagnosed through biocompatibility testing,14,15 including cutaneous sensitivity tests,13 and also through clinical observations and
family history. However, major problems regarding
specificity of the cutaneous tests are frequently reported. Therefore, a great challenges in the diagnosis of
nickel sensitivity has been to discover and develop
alternative in vitro assays to improve specificity and
minimize false-positive results. Lymphocyte stimulation assays with peripheral blood mononuclear cells
(PBMC) and different concentrations of nickel salts
have contributed to advances and show promising
results.16 In one study, lymphocytes from all suspected
nickel-sensitive patients showed a significantly greater
response than did those of healthy controls.17 Lymphocyte transformation, as measured by increased deoxyribonucleic acid (DNA) synthesis, seems to be an important tool for investigating the problems arising from
false-positive or false-negative patch tests.18
The purpose of this research was to develop a new
approach to testing the effect of nickel antigen on
lymphoproliferative response in vitro, by using PBMC
from patients with orthodontic appliances. Using this
strategy, we evaluated whether lymphoproliferative
response is a useful method to discriminate nickelsensitive from non–nickel-sensitive dental patients. We
also studied patients wearing orthodontic appliances
(either with or without clinical symptoms) to determine
the influence of certain variables on in vitro lymphoproliferation assay, including source of antigen, type of
serum used to supplement the medium, and the number
of cells used.
MATERIAL AND METHODS
Thirty-five patients, aged 10 to 21 years (mean
14.3), all undergoing orthodontic treatment with fixed
appliances, were selected based on their clinical health
records and classified into 2 groups. The first group
comprised 26 patients (8 males, 18 females, aged 10 to
21 years) with clinical manifestations of contact dermatitis, including angular cheilitis, labial desquamation, or gum hyperplasia resulting from metallic compounds, especially earrings, bracelets, and other
jewelry. The second group comprised 9 patients (3
males, 6 females, aged 11 to 21 years) who did not have
clinical signs or histories of nickel sensitivity. All
patients underwent cutaneous nickel sensitivity tests,
and those selected for the control group had negative
results. We could not use all 26 patients for all tests
Marigo et al 47
owing to the limited quantity of PBMC we were able to
extract from their blood samples. Informed consent was
obtained from all subjects and their parents. The studies
were approved by the Ethical Committee of Universidade Estadual de Campinas and Universidade Vale do
Rio Doce.
The cutaneous sensitivity test was performed as
described by Carvalho.19 Briefly, a 5% nickel sulfate
and petroleum jelly substrate (nickel sulfate 5%, Alergofar, Rio de Janeiro, Brazil) was kept in direct contact
with the skin of the forearm for 48 to 72 hours. The
results were classified as positive or negative, based on
the reaction.
Nickel sources used as stimulating agents for the
cellular proliferation assays in vitro were obtained from
solutions containing nickel from in vitro corrosion of
orthodontic appliances (nickel extract) and from hexahydrated nickel sulfate solutions (NiSO4.6H2O, Sigma
Chemical, St. Louis, Mo). The nickel extracts were
obtained from 4 different brands of orthodontic appliances. Alloys were kept in a sterile 0.85% saline
solution at 37°C for 7, 30, and 60 days. After centrifugation to remove the particles produced during the
corrosion process, the solutions were sterilized by
filtration (0.2 ␮m, Filter Millex-HA Millipore Products
Division, Bedford, Mass). Then the dosage of nickel in
this solution was measured with an atomic absorption
spectrophotometer (Hitachi Z-8200, Tokyo, Japan).
The extracts thus obtained were diluted in culture
media (RPMI-1640, Gibco BRL, Grand Island, NY)
containing a 3% solution of antibiotic-antimycotic
(stock solution: 10,000 IU penicillin, 10,000 IU streptomycin/mL, and 25 ␮g amphotericin B/mL, Sigma
Chemical) and 1.6% L-glutamine (stock solution: 200
mmol/L, Gibco), labeled incomplete RPMI. Extracts
containing nickel at concentrations of 1.25, 2.5, and 5
␮g/mL were used in the cellular proliferation assays.
The spectrophotometer readings of saline extract from
the 4 appliances (A, B, C, and D) showed similar levels
of nickel after 60 days of spontaneous corrosion. No
significant differences on the yield of nickel between
the 4 different brands were observed (A, 40.23 ␮g/mL;
B, 39.58 ␮g/mL; C, 40.03␮g/mL, and D, 40.23 ␮g/
mL). Solution A was used in cell culture. The nickel
sulfate (NiSO4.6H2O, Sigma) was also diluted in the
above-described incomplete RPMI medium and used at
concentrations of 2.5, 5, and 10 ␮g/mL in the cellular
proliferation assays.
In vitro cellular proliferation assays were performed
with the procedure described by Gazzinelli et al.20 In
96-well, flat-bottom, tissue-culture plates, 2 different
concentrations of PBMC (2.0 ⫻ 105 and 3.0 ⫻ 105 cells
per well) were incubated with 100 ␮L of 2 different
48 Marigo et al
culture mediums (RPMI supplemented with 5% heatinactivated AB⫹ human serum and with 5% autologous serum) with several concentrations of the antigenic preparations, including nickel extract (1.25, 2.5,
and 5 ␮g/mL) and nickel sulfate (2.5, 5, and 10
␮g/mL). Triplicate cultures were incubated at 37°C
(Queue Systems, Parkersburg, WVa), 95% humidity,
5% CO2 for 6 days. Six hours before harvesting the
cells, 1.0 ␮Ci of tritiated thymidine (3H) was added to
each well. After this stage, the cells were collected on
glass fiber paper (Whatman, Clifton, NJ) with an
automatic cell harvester (Skatron Instruments, Sterling,
Va). The incorporated radioactivity was determined
with a Beckman LS 100 C scintillator (Beckman,
Scientific Instruments Division, Irvine, Calif). The
results obtained from the proliferation assays were
expressed in counts per minute. The cellular stimulation index was calculated by taking the average value of
the triplicate series of the stimulated cultures (E)
divided by the average values from the triplicate series
of controls (C). We chose E/C values greater than 2.0 to
indicate significant enhanced PBMC proliferation,
based on values described in previous studies.17,21-23
The analysis of cytokines, IFN-␥, and IL-5 in the
supernatants from nickel-stimulated and control cultures were assayed with enzyme-linked immunosorbent
assays, as described by Lunde et al.24 Briefly, a 1 ⫻ 106
PBMC sample from each patient undergoing orthodontic treatment was stimulated with nickel sulfate (10
␮g/mL) for 24 hours in medium containing autologous
sera. Controls of nonstimulated cells were tested. For
cytokine analysis in the supernatant, initially, 60 ␮L per
well of anti-IL-5 monoclonal antibodies (5 ␮g/mL;
DNAX, Palo Alto, Calif), and 60 ␮L per well of
anti-IFN-␥ (2 ␮g/mL; R&D Systems, Minneapolis,
Minn) were added to the 96-well plates (Imunolon 2,
Dynatech Laboratories, Alexandria, Va) for 12 hours at
room temperature. After washing, the plates were
blocked with phosphate-buffered saline (PBS) solution
supplemented with 1% of bovine serum albumin, 5% of
sucrose, and 0.05% of sodium azide. After blockage, 50
␮L of culture supernatants were added to the test wells.
Blank and standard wells were prepared with 50 ␮L of
PBS, recombinant IFN-␥ (25-0.78 ng/mL; R&D Systems), and recombinant IL-5 (50-1.56 ng/mL; DNAX),
respectively. The plates were then incubated for 2 hours
at room temperature. After washing with PBS 0.05% of
Tween 20 (polyoxyethylene sorbitan monolaurate, Sigma), 60 ␮L per well of biotinylated anti-IFN-␥ polyclonal antibody (0.3 ␮g/mL; R&D Systems) and antiIL-5 (0.3 ␮g/mL; DNAX) were added and the plates
incubated for 1 hour at room temperature. After incubation, the plates were washed with PBS 0.05% of
American Journal of Orthodontics and Dentofacial Orthopedics
July 2003
Fig 1. Effect of antigen source on in vitro lymphoproliferation assay. Results are expressed as mean cell
proliferation index (E/C) ⫾ standard error. E/C ⬎ 2.0
was adopted as arbitrary cutoff to signify significant cell
proliferation index.
Tween 20 and 100 ␮L of peroxidase-conjugated
streptavidin (1 ␮g/mL, Sigma) and reincubated for 20
minutes at room temperature. After this step, 60 ␮L of
the substrate (2,2 azino-bis 3 ethylbenz-thiazolidine-6sulfonic acid, Sigma, 1 mg/mL) were added to all wells.
After color development, the reaction was stopped by
adding 50 ␮L of 1 M sulfuric acid. The optical density
was read with an automatic reader (Benchmark Microplate Reader, Bio-Rad Laboratories, Hercules, Calif)
with a 405-nm filter.
Data analysis was performed by one-way analysis
of variance followed by Student t tests. Significance
was set at P ⬍ .05.
RESULTS
To improve specificity of nickel sensitivity diagnosis, we tested the effect of nickel antigen source on the
in vitro lymphoproliferative response by using PBMC
from 22 patients wearing orthodontic appliances. Using
the basic blastogenesis method described by Gazzinelli
et al,20 we tested 3 different concentrations of nickel
extract and nickel sulfate (Fig 1). Data analysis demonstrated that exposure of PBMC to the nickel extract
did not lead to significant proliferation (E/C ⬍ 2.0).
Concentrations of 2.5 and 5.0 ␮g/mL of nickel sulfate
did not give significant results. On the other hand, a
concentration of 10 ␮g/mL of nickel sulfate significantly stimulated cell proliferation (E/C ⬎ 2.0), measured by 3H-thymidine incorporation. Therefore, the
antigen source chosen for further study was nickel
sulfate.
The responses of PBMC from 21 subjects to nickel
sulfate 10 ␮g/mL was evaluated in the presence of
autologous and AB⫹ type sera with 2 ⫻ 105 and 3 ⫻
105 cells per well (Fig 2). The in vitro responses of 2 ⫻
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 124, Number 1
Fig 2. Influence of serum-type supplement and number
of cells on in vitro lymphoproliferation assay. Results
are expressed as mean cell proliferation index (E/C) ⫾
standard error. E/C ⬎ 2.0 was adopted as arbitrary
cutoff to signify significant cell proliferation index.
Fig 3. Comparative analysis of cell proliferation index
between nickel sensitive and non–nickel-sensitive patients. Results are expressed as mean cell proliferation
index (E/C) ⫾ standard error. E/C ⬎ 2.0 was adopted as
arbitrary cutoff to signify significant cell proliferation
index.
105 PBMC in the presence of autologous serum differed significantly (E/C ⬎ 2.0) from 3 ⫻ 105 cells in
autologous serum and AB⫹ serum. Additional comparative studies were performed with 2 ⫻ 105 PBMC in
the presence of autologous serum to derive E/C values.
The PBMC responses of 21 subjects, 12 nickelsensitive and 9 non–nickel-sensitive, were measured
after in vitro stimulation with nickel sulfate in medium
containing autologous serum (Fig 3). The nickel-sensitive group had significantly higher cell proliferation
indexes than did the non–nickel-sensitive group. No
differences were observed when nickel-sensitive patients were subdivided into groups based on morbidity
of oral disease (data not shown).
It has been proposed that long-term exposure to
nickel-containing devices can induce immunologic tol-
Marigo et al 49
Fig 4. Down-regulation of nickel sulfate–induced lymphoproliferation in vitro associated with exposure time
to orthodontic appliances. Results are expressed as
mean cell proliferation index (E/C) ⫾ standard error. E/C
⬎ 2.0 was adopted as arbitrary cutoff to signify significant cell proliferation index.
erance.25 To investigate this hypothesis, PBMC from
21 dental patients with different times of exposure to
orthodontic appliances were submitted to the lymphoproliferation protocol described above. Evidence of cell
proliferation index inhibition due to long-term exposure to orthodontic appliances was found when patients
were divided into 2 groups based on the time of
exposure to nickel-containing devices (Fig 4). Patients
with less than 24 months of exposure (n ⫽ 10) showed
significant cell proliferation indexes (E/C ⬎ 2.0) in
contrast to those with more than 24 months of oral
nickel exposure (n ⫽ 11).
To validate the use of cell proliferation assay in
vitro in clinical trials, we conducted a parallel study of
PBMC nickel sulfate-induced proliferation in vitro with
skin tests (patch test) for nickel sensitivity. Our findings
demonstrated that a positive skin test is associated with
a higher proliferation index, leading to significant
results compared with patients with a negative patch
test (Fig 5).
In our system, the variables of sex and allergic
reaction were also evaluated, but they had no impact on
cell proliferation index (data not shown).
To further investigate the impact of in vitro nickel
stimulation on PBMC from 19 subjects (13 nickelsensitive and 6 non–nickel-sensitive), IFN-␥ and IL-5
were measured after 24 hours of stimulation with nickel
sulfate in medium containing autologous sera (Fig 6).
Our data demonstrated a significant difference in IL-5
production between nickel-stimulated and nonstimulated cultures from nickel-sensitive patients. This difference was not detected in control cultures. On the
other hand, nickel stimuli significantly inhibit in vitro
IFN-␥ production by PBMC from nickel-sensitive patients but not non–nickel-sensitive subjects (Fig 6).
50 Marigo et al
Fig 5. Parallel study of in vivo and in vitro cellular
immune response to nickel. Results are expressed as
mean cell proliferation index (E/C) ⫾ standard error. E/C
⬎ 2.0 was adopted as arbitrary cutoff to signify significant cell proliferation index.
DISCUSSION
Induction of nickel hypersensitivity has been studied extensively.13,26-34 Orthodontic appliance therapy
could enhance the liberation of nickel directly into the
oral cavity, and therefore it is a potential source of
antigenic stimulation for the human immune system.
Orthodontic treatment is available to many more people. In general, the appliances and archwires used in
treatment are composed of metal alloys that are more
than 55% nickel, representing a potential source of this
heavy metal in the buccal cavity.11 The biodegradation
of orthodontic appliances has been studied by various
authors, and the allergic response to nickel-containing
dental alloys has been reported in several publications.12,35,36 Because nickel can sensitize certain people
and cause severe allergic reactions, the safe use of these
alloys is still under study.
The assay most commonly reported to evaluate
cellular immune response in vitro is the measure of
lymphoproliferative activity of PBMC in the presence
of specific antigenic materials of interest. From the
dental care standpoint, it has been demonstrated that
lymphocytes from nickel-sensitive dental patients
showed significantly greater responses compared with
those of controls.17 Several methods are available to
assess cell proliferation in vitro, including protocols
with whole blood and purified PBMC.20 Despite its
potential use for evaluating immunologic status, the in
vitro lymphocyte transformation test is not standardized for nickel sensitivity studies. Questions regarding
nickel source and concentration for antigenic stimulation are still topics of research.21,22 In attempting to
evaluate whether the lymphoproliferative response in
vitro could be a useful method for discriminating
American Journal of Orthodontics and Dentofacial Orthopedics
July 2003
nickel-sensitive from non–nickel-sensitive patients, we
performed a parallel study of lymphocyte transformation in vitro with different concentrations of 2 distinct
sources of nickel: nickel released from orthodontic
appliances and nickel sulfate. Our data demonstrated
that nickel sulfate at 10 ␮g/mL was the best solution to
induce lymphocyte transformation in vitro. We observed that nickel extract in 3 different concentrations
(1.25, 2.5, and 5.0 ␮g/mL) could not induce significant
lymphocyte proliferation, and high doses of nickel
extract were cytotoxic for PBMC cultures in vitro (data
not shown). These data agree with those reported by
Silvennoinen-Kassinen,37 showing that higher concentrations of nickel can induce lymphocyte death in vitro.
Moreover, we found that optimization of lymphoproliferation assay in vitro can be achieved by substituting
autologous serum for AB⫹ and by using fewer cells per
culture (2.0 ⫻ 105 cells per well). We believe that
several factors, such as the cytokines’ microenvironment in cultures with autologous sera, were responsible
for the differences observed. By using this improved
methodology, it was possible to discriminate nickelsensitive from non–nickel-sensitive patients (Fig 3).
Our study also found a correlation between nickelinduced proliferation in vitro and nickel cutaneous
sensitivity test results (Fig 5). Because skin tests have
been routinely used not just to evaluate nickel hypersensitivity, but also for diagnostic purposes,26,27,29,32,34
lymphocyte activation and proliferation measured by
the DNA synthesis test is a method for diagnosing
nickel sensitivity that does not expose patients to the
hazards of patch testing.
A question that remained without a clear answer is
whether long-term intraoral exposure to nickel from
orthodontic appliances might result in a higher incidence of allergic reactions or lead to oral tolerance. In
this study, we found that patients who had been
undergoing orthodontic treatment for more than 24
months showed lower nickel-induced cell proliferation
indexes than those with less than 24 months of exposure (Fig 4). These data suggest that the longer the
treatment continues, the lower the nickel-induced
PBMC proliferation index; this in turn suggests that
mechanisms of oral tolerance might develop in this
context. Immunologic tolerance to nickel was described
by Vreebur et al25 in 1984, when oral administration of
nickel induced partial tolerance in guinea pigs with a
splint fixed to their teeth or receiving nickel in their
food. According to these authors, this state of partial
tolerance could contribute to reducing the incidence of
allergic reactions in patients undergoing orthodontic
treatment in which nickel alloys are used. In agreement
with this hypothesis, it has also been reported that
Marigo et al 51
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 124, Number 1
Fig 6. Effect of nickel stimuli on cytokine profile. Results are expressed as cytokine concentration
in the cell culture supernatant (ng/mL) for control cultures (white bars) and nickel-stimulated cultures
(black bars) ⫾ standard error. Statistical analysis was performed by analysis of variance followed by
Student t test. Significance was considered when P ⬍ .05. Differences are marked by distinct letter.
treatment with nickel-containing appliances before sensitization to nickel (ear piercing) can lead to reduced
frequency of nickel hypersensitivity.30 Tolerance induction might be a possible benefit of using intraorally
placed alloys.3
We have also begun to investigate the role of type
1 and type 2 immune responses in the pathogenesis of
nickel sensitivity. It seems that nickel stimuli can elicit
IL-5 production in nickel-sensitive patients. There was
a significant difference between their cytokine levels
and those observed in the control cultures. Moreover,
we showed that our optimized method for lymphocyte
transformation in vitro is a useful tool for identifying
the lack of type 1 (IFN-␥) immune response in nickelsensitive compared with non–nickel-sensitive patients
(Fig 6). Further studies are needed to elucidate the
major cell phenotype associated with the type 2 immune response, as well as the lack of type 1 immunity
observed in nickel-sensitive people. Moreover, phenotypic studies will help define the target cells for
nickel-specific activation that could lead to immunologic interventions in dental patients having orthodontic appliances.
CONCLUSIONS
● Optimization of lymphoproliferation assay in vitro
was achieved by using fewer cells per culture (2.0 ⫻
105cells per well), substituting autologous for AB⫹
sera, and stimulating cells with 10 ␮g/mL of nickel
sulfate. With this ideal culture, it was possible to
assess the nickel-induced cell proliferation index and
thus distinguish nickel-sensitive from non–nickelsensitive patients.
●
●
●
This optimized methodology for assessing cellular
immune status could enable diagnostic testing of
nickel sensitivity without exposing patients to the
hazards of patch testing.
The exposure to nickel alloy castings for more than
24 months resulted in lower cell proliferation indexes, suggesting that the development of oral tolerance mechanisms might play a role in modulating the
cellular response to nickel.
A known nickel-induced type 2 immune response
and a marked lack of type 1 immunity (IFN-␥) were
the hallmarks of nickel-sensitive patients.
We thank Marlucy Rodrigues Lima, Maria de
Fátima da Silva, and Lilia Cardoso Moreira for technical assistance.
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