ARTICLE
Auteur(s) : Laetitia Furio1, Anne
Guezennec2, Blandine Ducarre1, Joelle
Guesnet2, Josette
Peguet-Navarro1
1EA 41-69, Université Lyon 1, Dermatology Unit,
Pavillon R, Hôpital E. Herriot, 69437, Lyon 03, France
2YSL Beauté, Neuilly/Seine, France
accepté le 15 Novembre 2007
Contact hypersensitivity (CHS) is a delayed type T-cell mediated
immune reaction occurring after epicutaneous application and
challenge with reactive haptens. It represents one of the most
common skin diseases [1] and the need to develop predictive tests
that could identify potential allergens has been recognized for
many years. Traditionally, sensitization tests have been conducted
in guinea pigs [2, 3] but the increasing number of new
commercialized chemicals, as well as the animal welfare movement,
has prompted the development of new testing methodologies. The
principles of alternative methods are now based on the three Rs,
i.e. all approaches that lead to the Refinement of animal
experimentation methods, the Reduction in the number of animals
used and the Replacement of such practices by in vitro models. The
murine local lymph node assay (LLNA), a reducing testing approach,
has become the preferred method for assessing skin sensitization
potential [4]. However, the current requirements concerning the
banning of animal testing for the assessment of cosmetic
ingredients in 2009 and the application of the new REACH
(registration, evaluation, authorization and restriction of
chemicals) legislation since June 2007, now implies the development
of in vitro models to assess the sensitizing potential of new
products.
There is strong evidence that cutaneous dendritic cells (DC),
i.e. epidermal Langerhans cells (LC) and dermal DC, play a key role
in CHS, by picking up the allergens and migrating to draining lymph
nodes where they trigger specific T cell activation and
proliferation [5, 6]. Upon hapten application, LC acquire a mature
phenotype characterized by increased expression of HLA-DR, CD83 and
CD54, CD40, CD86 co-stimulatory molecules [7, 8]. Moreover LC
exhibit decreased expression of CD1a and E-cadherin, which mediates
LC attachment to keratinocytes [9] and increased expression of
CCR7, the receptor for the CCL19/MIP3β chemokine that governs their
migration to lymph nodes [10]. All these changes correlate with a
higher capacity of migrating LC to stimulate naïve T cells [8].
The functional and phenotypic LC alterations induced in vivo
after hapten treatment have provided the basis for in vitro
alternative approaches that could differentiate potential
sensitizers from irritants [11-14]. The finding that cutaneous DC
could be generated in vitro, either from human monocytes [15] or
CD34+ cord blood progenitors [16], has largely
facilitated this task. Thus, peripheral blood monocytes cultured
for 6 days in the presence of granulocyte-macrophage stimulating
factor (GM-CSF) and interleukin 4 (IL-4) differentiate into
immature interstitial DC, while the addition of transforming growth
factor β (TGFβ) skews the cells towards a LC-like phenotype [17].
Following hapten treatment, several biological endpoints were
evaluated with these cells, such as cytokine production, migration,
activation of autologous T cells or transcript analysis [18-23].
Finally, most workers proposed measuring changes in cell surface
expression of some antigens as the easiest in vitro model for
contact sensitization. Thus, Aiba et al. [20] first reported
up-regulation of HLA-DR, CD86 and CD54 on monocyte-derived DC
(MoDC) following 24-hour treatment with 2,4-dinitrochlorobenzene
(DNCB) and NiCl2. In contrast, the irritants sodium
lauryl sulfate (SLS), benzalkonium chloride or ZnCl2,
had no effect. Many other groups have confirmed these findings,
when the same representative strong allergens were used, i.e. DNCB,
NiSO4 or NiCl2 [21, 22, 24]. The results were
far less convincing, however, when other strong allergens or,
especially, chemicals with only moderate or mild sensitizing
properties, were considered. In a recent study [25], we found that
discriminating mild or moderate allergens requires not only
consideration of many antigens on MoDC but also a prolonged time of
incubation with the chemicals.
In all the studies reported so far, DC treatment with chemicals
has been carried out after 5 to 6 days of monocyte culture. Here,
we first analyzed the kinetics of expression of several antigens on
MoDC cultured, or not, in the presence of TGFβ (TGFβ-MoDC), so that
the chemicals could be added to MoDC that were as immature as
possible. The dynamic range of marker expression by MoDC cultured
in the presence of TGFβ justified the addition of chemicals at day
3 of culture.
Materials and methods
Culture medium and cytokines
Culture medium was RPMI-1640 containing GlutamaxTM and
25 mM Hepes (Gibco, Cergy Pontoise, France), 1% gentamycin
(Sigma, L’isle d’Abeau Chesnes, France), 10% heat-inactivated fetal
bovine serum certified (Gibco, Cergy Pontoise, France), thereafter
referred to as complete medium. Recombinant human GM-CSF was a
generous gift from Schering-Plough Research Institute (Kenilworth,
NJ). IL-4 and TGFβ were from R&D System (Lille, France).
Chemicals
The strong allergen 2, 4-dinitrochlorobenzene (DNCB; CAS number:
97-00-7), the moderate allergens NiSO4 (CAS number:
7786-81-4) and cinnamic aldehyde (CAS number: 104-55-2) and the
irritant sodium lauryl sulfate (SLS; CAS number: 68485-47-7) were
from Sigma Chemicals (L’isle d’Abeau Chesnes, France). The strong
sensitizer, balm of Peru (CAS number: 8007-00-9), was from COOPER
(Melun, France) and the moderate allergen, isothiazolinone (CAS
number: 55965-84-9), from Spectrum Chemicals and Laboratory
Products (Gardena, CA, USA). For phenotypic analysis, all chemicals
were used at non-toxic concentration, as assessed by trypan blue
staining of DC suspensions after an 18h treatment with the
chemicals. DNCB, cinnamic aldehyde and balm of Peru were initially
prepared in DMSO (Sigma, L’Isle d’Abeau Chesnes, France) and
subsequently diluted in complete medium to give a final
concentration of DMSO of 1/10 000. NiSO4,
isothiazolinone and SLS were prepared in complete medium.
Monocyte purification and culture
Mononuclear cells were obtained from the peripheral blood of
healthy donors by centrifugation on Lymphoprep (Pharmacia, St
Quentin en Yvelines, France). Monocytes were depleted of T and B
cells using hapten-conjugated anti-CD3, CD7, CD19, CD45RA, CD56
mAbs and anti-hapten Ig coupled to magnetic microbeads according to
the manufacturer’s instructions (Monocyte Isolation Kit, Miltenyi
Biotec, Germany). The technique routinely resulted in more than 80%
purity, as assessed by flow cytometry. In all the reported
experiments, monocytes were frozen in liquid nitrogen and thawed
before beginning the culture.
Depending on the experiments, monocytes (0.5 × 106
cells/mL) were cultured for one to 5 days in 12-well tissue culture
plates (Costar Corp., Cambridge, MA), in complete medium
supplemented with rhGM-CSF (200 U/mL), IL-4 (50 U/mL) and TGFβ
(0.5 ng/mL), or with rhGM-CSF (200 U/mL) and IL-4 (50 U/mL),
only.
DC treatment with chemicals
Chemicals were added at day 3 of the monocyte culture, for four
days and at the following concentrations: DNCB (33 μM),
NiSO4 (50 μg/mL), balm of Peru (10 μg/mL),
isothiazolinone (10 μg/mL), cinnamic aldehyde (10 μg/mL) and SLS
(10 μg/mL). Control cells received either DMSO or medium alone. As
assessed by cell numeration in trypan blue after the 4-day
treatment, the cell viability in the presence of the chemicals was
not different from that observed with control cells, and averaged
85-90%.
Immunofluorescence staining and flow cytometry analysis
For phenotypic analysis, cells were incubated for 30 min at 4
°C with affinity purified mouse mAbs at the appropriate
concentration or with irrelevant isotype-matched mouse Igs at the
same concentration. Cells were washed and, for indirect staining,
further incubated for 30 min at 4 °C with FITC-conjugated
F(ab’)2 fragments of goat anti-mouse Ab. The following
monoclonal antibodies were used: anti-HLA-DR-FITC (B8.12.2),
anti-CD54-FITC (84H10), anti-CD83-FITC (HB15A) and anti-E-cadherin
(67A4) all from Immunotech (Marseille, France); anti-CD40-FITC
(mAb89) from Biosource international (Nivelles, Belgium);
anti-CD1a-FITC (NA/134) and anti-CD14-FITC (TÜK 4) from Dako
(Trappes, France); anti-CD86-FITC (FUN-1) from BD Biosciences (Pont
de Claix, France); anti-CCR7-FITC from R&D System (Lille,
France).
HLA-DR intracellular staining was carried out using the fix and
perm cell permeabilization kit (Caltag Laboratories, San Francisco,
CA), according to the manufacturer’s instructions.
Flow cytometry was performed with a FACScan and data were
analyzed using the Cell Quest software (Becton Dickinson, Le Pont
de Claix, France).
Statistical analysis
Statistical analysis was carried out using the paired Student’s t
test. Only p-values < 0.05 were considered as statistically
significant.
Results
Kinetics of antigen expression on differentiating MoDC
We first compared the kinetics of expression of several cell
surface markers on a pool of monocytes cultured for five days with
GM-CSF and IL-4, in the presence or absence of TGFβ. As shown in
figure 1, the
percentage of stained cells was quite comparable under the two
culture conditions. Monocytes gradually lost expression of the
monocyte marker CD14 while acquiring that of CD1a, which is
characteristic of DC. A significant proportion of
CD1a+/CD14– DC could be observed as early as
after 3 days of culture. The percentage of HLA-DR+ cells
remained stable throughout the monocyte culture, although the
staining intensity strongly increased as early as the first days of
culture. An increasing number of cells expressed the co-stimulatory
molecules CD40 and CD54, with CD86 being an exception. For the
three markers, however, the mean fluorescence intensity (MFI)
increased throughout the culture and was globally lower in the
presence of TGFβ. At day 3, the levels of CD40, CD54 and CD86 were
consistently lower in the presence of TGFβ, as assessed by a
difference of more than 20% in the MFI values. Moreover, in the
presence of the cytokine, the MFI for the three markers was
significantly lower at day three than at day five of culture.
E-cadherin expression was very low from day 0 to day 5 monocyte
culture (not shown). It increased thereafter, only on the
TGFβ-treated cells and, depending on experiments, was present on 60
to 90% of TGFβ-MoDC at day 7 of culture, as described earlier [25].
These cells did not express langerin, the Langerhans cell specific
marker, however (not shown). As expected, in any cytokine
conditions, few or no cells expressed the maturation markers CD83
and CCR7. Note, however, that a significant percentage of positive
cells could be observed on day 1 of the culture. This most probably
reflects a non-specific activation of the cells. Indeed, at that
time only, the cells slightly adhered to the cell support and had
to be repeatedly agitated to be recovered from the cell
support.
Phenotypic alterations induced by the chemicals on day
3-cultured MoDC
Inasmuch as immature MoDC could be already obtained at day 3 of
culture, and as the expression of most cell-surface activation
markers was lower than that observed at day 5, especially in the
presence of TGFβ, the chemicals were added on day 3-TGFβ-MoDC and
the cells were stained 4 days later. The mean results from eight
experiments carried out with different donors are shown in figure 2. They are
expressed as the relative mean fluorescence intensity (MFI) of each
antigen relative to that of non-treated cells. Overall, a large
variation was observed among the experiments, as illustrated by the
high SD values. Treatment with NiSO4 induced the most
striking effect with significant modulation of most surface
markers, i.e. reduced MFI for CD1a and E-cadherin as well as
up-regulated MFI for surface HLA-DR, CD54 and CD86 antigens. These
results were highly significant. Furthermore, for these antigens,
the MFI differed by at least 20% from control values in virtually
all the experiments, as summarized in table
1. In the presence of DNCB, the intensity of CD83
expression was significantly up-regulated and exceeded 120% of the
control value in five out of eight experiments, while the
expression of CD86 and HLA-DR only tended to increase. Moreover,
the intensity of CD1a expression was significantly decreased and
the relative MFI was below 0.6 in five out of eight assays.
TGFβ-MoDC treated with the other strong allergen, balm of Peru,
showed no significant sign of DC activation. On the contrary, the
intensity of HLA-DR expression was decreased at the cell surface
while intra-cellular staining of the antigen was significantly
increased. Isothiazolinone, a moderate allergen, induced
significant up-regulation of only CD86 MFI. Interestingly, cinnamic
aldehyde, classified as a moderate allergen, induced significant
down-regulation of both E-cadherin and CD1a and up-regulation of
CD83 MFI.
Regarding the percentage of stained cells, only NiSO4
and DNCB allowed consistent variations with a significant decrease
in CD1a and E-cadherin positive cells and a significant increase in
CD83 and CD86 positive cells. It should be noted that cell
treatment with SLS did not induce significant modulation of any
marker (figure
2).
Table 1 Variation frequency of the mean fluorescence
intensity (MFI) for each antigen. TGFβ-MoDC were treated at day
three with the different chemicals and analyzed, four days later,
for phenotypic alterations by flow cytometry. The variation in the
expression of a given antigen was considered as significant when
the MFI differed by at least 20% from the control value. The data
indicate the frequency of significant Ag variation in eight
experiments
|
CD1a
|
HLA-DR extra
|
CD54
|
CD86
|
CD40
|
E-cad
|
CCR7
|
HLA-DR intra
|
CD83
|
|
NiSO4
|
8/8
|
8/8
|
8/8
|
7/8
|
1/8
|
7/8
|
2/8
|
4/8
|
4/8
|
|
DNCB
|
6/8
|
3/8
|
1/8
|
3/8
|
1/8
|
3/8
|
4/8
|
2/8
|
5/8
|
|
0/8
|
5/8
|
1/8
|
0/8
|
3/8
|
2/8
|
5/8
|
1/8
|
4/8
|
|
Isothiazolinone
|
0/8
|
1/8
|
0/8
|
4/8
|
1/8
|
1/8
|
2/8
|
1/8
|
2/8
|
|
Cin ald
|
6/8
|
0/8
|
1/8
|
0/8
|
3/8
|
4/8
|
2/8
|
3/8
|
6/8
|
|
SLS
|
0/8
|
0/8
|
0/8
|
2/8
|
2/8
|
1/8
|
2/8
|
1/8
|
1/8
|
Discussion
The activating effect of sensitizers on MoDC phenotype has been
largely explored to develop in vitro alternative assays for
discrimination of contact allergens from irritants. However, the
sensitivity of the assay remains controversial [26], especially for
mild or moderate sensitizers. In a previous study using 5-day
cultured MoDC [25], we showed that 4-day, but not 2-day treatment
of MoDC with chemicals allows discrimination of moderate allergens
from irritants, provided several surface antigens were considered.
Indeed, as compared to the irritant SLS, the number of modified
antigens (as assessed by a MFI variation of at least 20% from
control value) was increased with nearly all the tested chemicals.
However, we failed to demonstrate significant variations in the MFI
of a given surface antigen, because of the high variability in the
identity of the modified antigens among experiments.
Here, our aim was to improve the sensitivity of the assay by
adding the chemicals to MoDC that were as immature as possible, in
order to underscore the activating effects of allergens. The
kinetic analysis of several marker expression on MoDC justified the
addition of chemicals at day 3 of culture and the use of TGFβ.
Indeed, at that time, most monocytes have differentiated into
CD1a+/CD14– DC. Moreover, in the presence of
TGFβ, a cytokine known to maintain the cells in a more immature
state [27], the MoDC expressed lower levels of CD40, CD54 or CD86
antigens than 5 day-cultured MoDC. Overall, early treatment of
TGFβ-MoDC with the chemicals for 4 days induced significant
phenotypic changes for all the strong and moderate sensitizers
tested, whereas the irritant had no effect. The results were highly
reproducible and significant with the strong and moderate allergens
DNCB and NiSO4, which validate the reactivity of early
MoDC to maturation stimuli. The results were also convincing with
the other moderate allergens tested, although significant
phenotypic modifications were sometimes observed in only half of
the experiments. To explore whether these variations depend on the
donor or experiment, some assays were carried out repeatedly with
thawed monocytes from the same donor (not shown). A similar
variability was observed, therefore demonstrating that the absence
of reproducibility does not reflect the individual sensitivity to
chemicals but rather the limits of the assay.
An interesting finding was the high variability in the number as
well as the identity of the modified activation markers, according
to the chemicals tested. Except for NiSO4 that induced
significant modulation of nearly all the tested antigens, the other
chemicals triggered significant modification of only one or two of
them. These results demonstrate the need to study many surface
antigens to detect the maturation potential of any chemical.
Moreover, they suggest that many and specific mechanisms may be
involved according to the chemicals, or category of chemicals, that
could not be identified with a universal marker.
Since Aiba et al. [20] reported up-regulation of CD86 on MoDC
following a 24-hour treatment with DNCB and NiCl2, CD86
remains the most studied activation marker in studies dealing with
DC activation by contact sensitizers. However, we found here that
strong or moderate allergens i.e. DNCB, balm of Peru and cinnamic
aldehyde failed to increase CD86 expression. The results extend
previous studies showing that only three out of eleven MoDC
cultures responded with increased CD86 expression after addition of
DNCB [28] and that no significant increase occurred after addition
of sub-toxic concentrations of either MCI/MI or DNFB [26]. Overall,
in our experiments, significant variations of CD1a, HLA-DR and CD83
were at least as frequently observed as variations of CD86.
The LC characteristic markers, CD1a and E-cadherin, have been
far less studied. Interestingly, however, significant
down-regulation of either or both antigens was observed with
NiSO4, and DNCB, as previously reported [29] and also
with cinnamic aldehyde. Balm of Peru, a brown viscous mixture of
more than two hundred compounds, did not induce significant
activation of cell-surface antigens. By contrast, it significantly
decreased the membrane expression of HLA-DR, while up-regulating
the intra-cellular content of the antigen. This most probably
reflects the very efficient cell uptake of the antigen, as
evidenced by the presence of multiple brown intracellular vesicles
under microscopic examination (not shown).
In conclusion, our results suggest that the set of markers
studied on early TGFβ-MoDC allowed us to discriminate allergens
from irritants. However, as only a few chemicals were used, a huge
list of chemicals should be further tested to validate the assay.
The results also demonstrate the high variability in the number and
nature of the modified markers, according to the chemicals and
experiments. This implies that many surface antigens must be
analyzed and many experiments carried out to use this assay as an
alternative screening method for contact sensitizers.
Acknowledgments
The work has been carried out with financial support from
YSL-Beauté (Neuilly/Seine, France). The authors have no conflict of
interest to declare.
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