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Photodermatol Photoimmunol Photomed 2003; 19: 203212

Blackwell Munksgaard

Copyright r Blackwell Munksgaard 2003

Immunomodulatory effects of low-intensity near-infrared laser irradiation on


contact hypersensitivity reaction
Lidija Kandolf-Sekulovic, Milena Kataranovski, Milos D. Pavlovic
Department of Dermatology and Institute for Medical Research, Military Medical Academy, Belgrade, Serbia and Montenegro

Background/Purpose: Contact hypersensitivity (CHS)


reaction is a useful model for studying the skin immune
system and inammatory reactions in the skin. In this
study, an experimental model of CHS reaction was
employed to assess immunomodulatory effects of nearinfrared (near-IR) low-intensity laser (LIL) irradiation, which is used as adjuvant therapy in dermatology,
physical medicine, rheumatology, etc., because of its
declared anti-inammatory, biostimulative and analgesic effects.
Methods: The effects of near-IR LIL irradiation
(k 5 904 nm, irradiance 60 mW/cm2, uence 3.6 J/
cm2) on CHS reaction to 1-chloro-2,4-dinitrochlorobenzene (DNCB) in Albino Oxford rats were
examined by irradiating experimental groups of
animals before the induction phase of CHS reaction,
while nonirradiated animals and animals that received
vehicle instead of hapten served as controls. Earswelling assay, histopathological examination of H&E
preparations of ear skin, computer-assisted image
analysis of dermal inltrate, ear skin organ culture
with the determination of cutaneous production of
tumour necrosis factor-a (by ELISA assay) and nitric
oxide (by Griess assay) were used for measuring the
effects of LIL in the elicitation phase of CHS reaction.
Cellularity, dendritic cell content, ow cytometry and
proliferation assays (spontaneous and in the presence
of IL-2 and concanavalin A) of the draining lymph
node cells (DLNC) were performed for the assessment
of LIL irradiation effects in the induction phase.
Results: In the irradiated group of animals, ear
swelling was signicantly diminished compared to
control animals (101711.5% vs. 58711.6%, Po0.01).
This was accompanied by a highly signicant decrease

in the density of dermal inltrate (2270.81 vs.


14.271.75 cells per unit area, Po0.01) and a
signicant decrease in nitrite levels in the medium
conditioned by organ-cultured ear skin (17.6371.91
vs. 3.1671.69 lM NaNO2; Po0.01), while TNF-a
concentration was not changed. Cellularity and
dendritic cell content in DLNC population, as well
as the expression of TCR-a, CD4, CD8 and CD25,
were not changed between irradiated and nonirradiated animals. Proliferation rates of DLNC cultured
for 72 h were signicantly lower in irradiated animals
(17.374.1 vs. 13.970.9  103 c.p.m.; Po0.01). In
cultures of DLNC with added rIL-2 or 0.5 lg/ml of
concanavalin A, proliferation rates were also signicantly decreased in irradiated animals (34.773.5 vs.
31.272.9  103 c.p.m. in IL-2-supplemented culture,
Po0.01; 70.976.4 vs. 58.379.1  103 c.p.m. in concanavalin A-supplemented culture, Po0.01). However, this effect was overcome in the presence of the
higher concentration of concanavalin A (2.5 lg/ml)
(nonirradiated 38.773.1, irradiated 123.177.3 
103 c.p.m., Po0.01).
Conclusion: LIL irradiation showed a systemic
immunomodulatory effect on CHS reaction to DNCB
in rats. Decreased ear swelling observed in the
elicitation phase was associated with diminished
proliferative responses of the DLNC in the induction
phase of CHS reaction. Further experimental work is
needed to examine the possible mechanisms of these
effects.

effects), analgesia and reduction of inammation. A


few studies in the past decades examined the antiinammatory, biostimulative and analgesic effects of

ear-infrared irradiation (l 5 8001000 nm) is


used in low-intensity laser (LIL) therapy for
the promotion of wound healing (biostimulative

Key words: contact hypersensitivity reaction; draining


lymph node cells proliferation; low-intensity laser; nearinfrared irradiation.

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Kandolf-Sekulovic et al

LIL light (13). However, the results of these studies


were often controversial, and possible mechanisms of
the observed photobiological effects are not clear.
This is the reason why this treatment modality is still
not widely accepted, although in use since the 1960s.
Most of the studies on biostimulative and antiinammatory effects of low-intensity near-IR irradiation were performed on cell cultures (49). However, a
few studies examined anti-inammatory effects of
near-infrared irradiation in animal models. In the
present study, the effects of near-IR irradiation were
evaluated in an animal model of contact hypersensitivity (CHS) reaction.
A commonly used model in testing the ability of an
animal or a human to mount a cutaneous immune
response is CHS reaction. In the induction phase
(sensitisation or afferent phase), epicutaneous application of hapten to the dorsal or the abdominal skin
induces activation and proliferation of hapten-specic
T lymphocytes in regional lymph nodes. In the
elicitation (efferent) phase, repeated contact with the
hapten applied on the ear skin induces recruitment of
the primed T lymphocytes to the site of challenge.
They produce a variety of inammatory mediators,
amplifying the background inammatory response
into a more vigorous process (10, 11). As it starts and
ends in the skin, the experimental model of CHS
reaction is suitable for studying immune and inammatory reactions in the skin. It is a widely accepted
model in studies on immunomodulatory and antiinammatory effects of various physical and chemical
agents (1215). For instance, UV-induced immunosuppression was largely evaluated in experimental
models of CHS reaction in rodents (1618).
Local immunomodulatory effects on CHS reaction
were observed by LIL irradiation at 635 nm wavelength, before and immediately after the elicitation of
the reaction (19, 20). Systemic effects of the LIL
irradiation were noticed in a wound healing model,
where it was found that LIL irradiation of the wound
shortens healing time not only on the irradiated but
also on the distant, nonirradiated site (21). LIL
irradiation suppressed delayed hypersensitivity reaction to the intradermally applied antigen in guineapigs not only on irradiated but also on distant sites,
which also points to possible systemic effects of the
irradiation in this wavelength range (22). However,
there were no studies that examined possible systemic
effects of near-IR LIL irradiation on CHS reaction. In
this study, we tested the hypothesis that near-IR LIL
irradiation has systemic immunomodulatory effects
on CHS reaction. An animal model of CHS reaction
to 1-chloro-2,4-dinitrochlorobenzene (DNCB) in Al-

204

bino Oxford (AO) rats, developed in our laboratory,


was used (23). Animals were irradiated before the
induction phase of CHS reaction, while the intensity
of CHS reaction was measured and expressed as ear
swelling after elicitation of the reaction. To further
document the observed effects, histopathological
analysis and quantitative histology were employed,
in addition to the skin organ culture technique, with
determination of cutaneous production of TNF-a and
nitric oxide release. Phenotypic characterisation and
proliferation assays were performed in a population
of draining lymph node cells (DLNC), in order to
examine the possible effects of LIL irradiation in the
induction phase.
In previous experimental and clinical studies,
various doses of LIL light were used, from 0.01 J/
cm2 to more than 50 J/cm2, and opposite effects were
observed with different doses (2426). In this study, a
uence of 3.6 J/cm2 and an irradiance of 60 mW/cm2
were used, which is in line with doses used in previous
studies on anti-inammatory effects of LIL irradiation (22, 27, 28).

Methods
All experiments were performed on inbred male AO
rats (Farm for Experimental Animals, Military
Medical Academy, Belgrade, Serbia and Montenegro)
in adherence to the NIH guidelines for the use of
experimental animals, with the permission of the
Ethical Committee of our institute.
CHS reaction
Groups of 68 rats received 100 ml of 2% w/v of
DNCB (BDH Chemicals Ltd., England) dissolved in
vehicle (acetone : olive oil 4 : 1, AOO) or an equal
volume of only vehicle, on the shaved dorsum for 2
consecutive days as described (23). Three days
following sensitisation, the rats were challenged by
application of 50 ml of a suboptimal dose (0.66%) of
DNCB to the outer half of the left ear. Ear thickness
was measured with an engineer micrometer 24 h after
the challenge. The intensity of the response was quantied as the difference in the thickness between the
challenged and nontreated ears of the same animal and
expressed as a percentage of increase in ear thickness.
LIL irradiation
Two groups of animals, one treated with vehicle and
the other treated with DNCB as described before,
were irradiated with near-infrared LIL irradiation.
Animals were irradiated by a single exposure for 60 s
on the shaved back immediately before the rst

Immonomodulatory effects of low-intensity near-infrared laser irradiation on contact hypersensitivity reaction

application of vehicle/DNCB in the induction phase


of CHS reaction. A gallium-arsenide laser
(l 5 904 nm; max. power output 4  15 mW) was
used, which has a laser probe with four apertures of
0.25 cm2, giving an irradiation area of 1 cm2. The
power density (irradiance) was 60 mW/cm2; the
energy delivered per irradiation point was 0.9 J with
an energy density (uence) of 3.6 J/cm2; the total
energy dose delivered was 3.6 J; and the frequency was
adjusted to 5000 Hz. The experimental protocol is
shown in Fig. 1.
Histology
Cartilage-free halves of exposed ears were xed, and
stained with haematoxylin and eosin for histological
evaluation. The density of the dermal inltrate was
measured by the computer-assisted image analysis
system using software Mikro (Laboratory for the
Computer Systems, Institute Mihajlo Pupin, Belgrade).
Skin organ culture
Exposed ears were taken 24 h after elicitation of the
CHS, split into ventral and dorsal halves and
submerged in 1.5 ml of culture medium in wells of
24-well culture plates. After 24 h at 37 1C, the
conditioned medium (CM) was collected (29). TNFa concentrations in CM were determined by a
commercially available ELISA for rat TNF-a (Biosource Int., Camarillo, CA, USA).
The level of nitrites, which reects the amount of
nitric oxide release, was determined by Griess reaction
(30). Briey, 50 ml of CM and 50 ml of Griess reagent,
consisting of naphthylethylenediamine dihydrochloride in water and sulphanilamide in phosphoric acid,
were incubated for 10 min at room temperature.
Absorbance was measured at dual wavelength
570 nm/650 nm by an ELISA 96-well plate reader
(Behring, Germany). Sodium nitrite was used as a
standard for quantication of nitrites in CM.
Preparation of DLNC, DLNC proliferation assays and
ow cytometry
DLNC were obtained from the axillary and subscapular lymph nodes, on the third day after
sensitisation with 2% DNCB, and were pooled for
each experimental group. DLNC from vehicle-treated
animals served as a control group for the DNCBsensitised DLNC. A single-cell suspension of lymph
node cells was prepared under aseptic conditions by
mechanical and enzymatic disaggregation by collagenase and passed through sterile nylon gauze. The cells
were washed three times and resuspended in RPMI1640 culture medium (ICN Flow, ICN Biomedicals

Inc., Costa Mesa, CA, USA) supplemented with 5%


heat-inactivated fetal calf serum (FCS) (ICN Flow),
1% gentamycin and 5  10  5 M 2-mercaptoethanol.
Viable cell counts were performed by excluding cells
that were stained with 0.5% trypan blue. The cell
concentration was adjusted to 5  106 per ml of
RPMI-FCS medium prepared as described above.
Dendritic cell content was identied in freshly isolated
DLNC suspensions by their distinct dendritic morphology under a light microscope at  400 magnication as described by Gerberick et al. (31).
For the proliferation assays, DLNC were cultured
at 37 1C with 5% CO2 for 72 h in the medium only or
in the presence of 50 IU/ml of recombinant human
IL-2 (Genzyme, MA, USA) (for spontaneous proliferation and IL-2-stimulated proliferation, respectively) and for 72 h in the presence of concanavalin A
(2.5 or 5 mg/ml). Proliferation was estimated by
incorporation of 3H-thymidine (1 mCi/culture, Amersham, UK) added at the onset (spontaneous proliferation) or during the last 1618 h of culture (IL-2stimulated and concanavalin A-stimulated proliferation). Results are expressed as counts per minute
(c.p.m.) measured by a b-radioactivity scintillation
counter (LKB RACK-Beta).
For ow cytometry, suspensions of DLNC were
incubated in phosphate-buffered saline with monoclonal antibodies R73 (anti-rat TCR a/b), W3/25
(anti-rat CD4), OX-8 (anti-rat CD8) and OX-39 (p55
anti-IL-2 receptor, CD25), all purchased from Serotec
Ltd. (Bicester, UK), followed by rabbit anti-Ig FITCconjugated antibody (INEP, Zemun, Serbia and
Montenegro). Cells were analysed for uorescence
intensity on a FACScan ow cytometer (Becton
Dickinson, Germany). The results are expressed as
percentages of uorescence positive cells.
Statistical analysis
Data are expressed as mean value7SD for each
experimental animal group (68 animals) for the
determination of ear swelling and DLNC phenotype.
In the DLNC proliferation assays, data represent
means7SE for triplicates or quadruplicates from
single experiments representative out of three. Signicance was dened by MannWhitney and Students t-test. P values less than 0.05 were considered to
be signicant, while values less than 0.01 were
considered highly signicant.

Results
Groups of animals received near-infrared LIL irradiation immediately before the application of vehicle or
DNCB on the dorsal region according to an experi-

205

Kandolf-Sekulovic et al

mental protocol for CHS reaction (Fig. 1). Nonirradiated animals who received only vehicle/DNCB
using the same experimental protocol served as
controls. The effects of near-infrared irradiation were
evaluated by the classical ear swelling assay in vivo,
pathohistological analysis with quantitative histology
and skin organ culture with determination of TNF-a
and nitrite levels.
Ear swelling assay
Ear swelling, measured 24 h after application of the
hapten/vehicle to the ear skin, was signicantly
diminished in irradiated animals who received 2%
DNCB in the induction phase, and 0.66% DNCB in
the elicitation phase, compared to the corresponding
nonirradiated group of the animals (Fig. 2). In
animals receiving vehicle, 4% or 8% DNCB in the
induction phase, and vehicle, 1.33% or 2.66% DNCB
in the elicitation phase, no differences in the ear
swelling between irradiated and nonirradiated animals
were noticed.
Pathohistological analysis of ear skin samples and
quantitative histology
Ear skin samples taken in the elicitation phase of
reaction were prepared for pathohistological analysis

Fig. 1. Protocol for investigation of near-IR irradiation effects on contact hypersensitivity (CHS) reaction. Two groups of animals, one treated with vehicle
and the other treated with 1-chloro-2,4-dinitrochlorobenzene (DNCB) as described before, were irradiated with near-infrared low-intensity laser
irradiation (l 5 904 nm, irradiance 60 mW/cm2, uence 3.6 J/cm2) immediately before the rst application of vehicle/DNCB in the induction phase of CHS
reaction (see text).

206

(H&E staining). In the irradiated group of animals


challenged with the lowest concentration of DNCB,
hyperkeratosis and vacuolar degeneration of the basal
layer induced by DNCB were less pronounced as well
as dermal oedema and dermal inltrate density,
compared with the nonirradiated group of animals
(Fig. 3A). In groups that received vehicle, 1.33% or
2.66% DNCB, there were no signicant changes
between irradiated and nonirradiated groups of
animals (not shown).
In the irradiated group of animals that received
0.66% DNCB in the elicitation phase, the dermal
inltrate density was signicantly lower compared to
the corresponding nonirradiated group of animals
(Fig. 3B). Again, in other groups of animals (that
received vehicle, 1.33% or 2.66% DNCB), there were
no signicant changes between irradiated and nonirradiated animals.
Since a signicant decrease in the CHS reaction
intensity was observed only in animals treated with
0.66% DNCB in the elicitation phase, the following
experiments were performed in these animals.
Skin organ culture
Skin organ culture technique is a suitable and useful
model for the investigation of various aspects of
normal and abnormal skin biology (29). In previous
studies, we demonstrated the utility of the skin organ
culture system in the detection of NO and TNF-a
production during the expression of CHS (29, 32). In
this study, this technique was used for the determination of levels of inammatory mediators in the
elicitation phase of CHS reaction and to evaluate
effects of near-infrared irradiation. Levels of TNF-a
were determined by a commercial ELISA assay in the

Fig. 2. Effects of near-IR low-intensity laser irradiation on contact hypersensitivity (CHS) reaction. Earswelling assay. Values are given as means7SD for
each treatment group. nPo0.06 vs. 0.66% 1-chloro2,4-dinitrochlorobenzene (DNCB).

Immonomodulatory effects of low-intensity near-infrared laser irradiation on contact hypersensitivity reaction

Fig. 3. Effects of near-IR low-intensity laser irradiation on contact hypersensitivity (CHS) reaction. (A)
Histopathologic picture of H&E stained samples of ear skin (H&E,  400). I: vehicle; II: 0.66% 1-chloro-2,4dinitrochlorobenzene (DNCB). a: nonirradiated animals; b: irradiated animals. In irradiated animals epidermal
and dermal changes induced by DNCB are less pronounced compared with the nonirradiated group of animals.
Hyperkeratosis and vacuolar degeneration of the epidermal basal layer are milder; dermal oedema and inltrate
density are present to a lesser extent. In vehicle-treated animals, no signicant changes are seen before and after the
near-IR laser irradiation. (B) Dermal inltrate density measured by computer-assisted image analysis. Values are
given as means7SD for each treatment group. nPo0.05 vs. DNCB.

CM of the organ-cultured ear skin. No signicant


differences were noticed between irradiated and
nonirradiated animals (43.972.6 vs. 47.173.2), while
signicantly higher levels were present in the haptentreated animals compared to the vehicle-treated
animals (43.972.6 vs. 25.271.1).
In contrast to the results that showed no changes in
TNF-a levels after LIL irradiation, nitrite levels
determined by Griess assay were signicantly lower
in irradiated animals, showing a lower release of the
nitric oxide by the resident and inammatory cells
(Fig. 4).

Proliferation rates and phenotypic characteristics of


DLNC after near-infrared irradiation
In the induction phase of CHS reaction, after
epicutaneous application of hapten, Langerhans cells
with characteristic dendritic morphology migrate to
the draining lymph nodes, where they present antigen
to hapten-specic lymphocytes. Subsequent activation
and proliferation of DLNC takes place, with their
recirculation and migration to the skin at the site of
repeated contact with hapten. To examine the possible
mechanisms underlying the observed effects of nearinfrared irradiation on CHS reaction, cellularity of

207

Kandolf-Sekulovic et al

draining lymph nodes and proliferation rates of


DLNC taken during the induction phase were
determined. DLNC were sampled 3 days after the
laser irradiation and application of DNCB, when a
DLNC proliferation peak was documented (23) and
at the time of reaction elicitation. Basic phenotypic
characteristics of DLNC in the induction phase were
determined using ow cytometry as well as the
dendritic cells content.
Cellularity, dendritic cell content and phenotypic
characteristics of DLNC
Cellularity of the draining lymph nodes and dendritic
cell content were not changed in irradiated and
nonirradiated groups of animals, except for a
signicant increase in the TCR-ab expression in
irradiated vehicle-treated animals. Flow cytometry
did not show any signicant changes in the expression
of CD4 and CD8 between irradiated and nonirradiated groups of animals (Table 1).

Proliferation rates of DLNC after near-infrared


irradiation
For the DLNC proliferation assays, DLNC were
cultured for 72 h in culture medium or in the presence
of concanavalin A (policlonal lymphocyte activator),
and proliferation rate was determined by 3H-thymidine incorporation. Proliferation rates in culture
medium were signicantly lower in irradiated animals
treated with hapten compared to the hapten-treated
nonirradiated animals (Fig. 5). Proliferation rates of
DLNC cultured for 72 h in the presence of 0.5 mg/ml
of concanavalin A were also decreased. However,
cultivation of DLNC in the presence of 2.5 mg/ml of
concanavalin A led to increased proliferation rates in
the irradiated group of animals (Fig. 6).
Lymphocyte response to IL-2 added to the culture
medium is considered to be the measure of their
activation level, as only activated cells express CD25
(a subunit of the IL-2 receptor), which is necessary for
the transduction of activation signals into lymphocytes. To further elucidate the functional state of the
DLNC in the irradiated animals, DLNC proliferation
rate in the presence of IL-2 was determined, together
with ow cytometric analysis of the CD25 expression
on DLNC (a subunit of the IL-2 receptor). Proliferation rates of DLNC in the presence of rIL-2 were
signicantly lower in the irradiated animals, while the
expression of IL-2R was not altered when compared
to nonirradiated animals (Table 2).

Discussion
Fig. 4. Effects of near-IR low-intensity laser irradiation on contact hypersensitivity (CHS) reaction.
Nitrite concentration in conditioned medium of
organ-cultured ear skin sampled in elicitation phase.
n
Po0.01 vs. nonirradiated animals treated with 1chloro-2,4-dinitrochlorobenzene (DNCB).

Near-infrared LIL irradiation was found in some


studies to be effective in the physical therapy of
rheumatoid disorders, and in dermatology for the
promotion of wound healing (2, 33, 34). In patients
with rheumatoid arthritis, near-IR LIL irradiation
reduced inammation and the levels of acute-phase

Table 1. Cellularity, dendritic cells content and phenotypic characteristic of DLN cells

Cellularity
Dendritic cells content (%)
TCR-abw
n

CD4w
CD8w
CD4/CD8z

Vehicle

Vehicle1near-IR laser

DNCB

DNCB1near-IR laser

16.674.3
0.0870.05
84.473.7
(11.270.5)
55.176.6
(7.270.6)
21.874.7
(2.971.1)
2.5770.31

17.971.1
0.0670.06
88.373.2
(17.375.0)
56.175.4
(11.073.4)
22.272.3
(4.371.3)
2.5270.09

41.8713.3
0.3670.09
79.772.8
(53.678.2)
44.771.5
(30.174.3)
31.271.0
(21.073.1)
1.4370.02

35.373.2
0.3670.045
81.571.2
(56.9721.19)
44.670.3
(31.4711.9)
32.771.6
(23.078.9)
1.3670.056

n
Results are expressed as mean value of DLNC number/dendritic cells content for six animals 7SD.
wResults are expressed as mean value of percentage of positive DLNC for six animals7SD, and as mean value7SD of absolute cell number of
positive DLNC.
zResults are expressed as ratio of the absolute number of CD4- and CD8-positive cells 7SD for six animals.
Po0.05 vs. vehicle.

208

Immonomodulatory effects of low-intensity near-infrared laser irradiation on contact hypersensitivity reaction

Fig. 5. Effects of near-IR low-intensity laser (LIL)


irradiation on contact hypersensitivity (CHS) reaction. Spontaneous proliferation rate of DLNC in the
induction phase of CHS reaction, after irradiation
with near-infrared LIL. Values are given as means7SE for each treatment group. nPo0.01 vs.
nonirradiated animals treated with 1-chloro-2,4-dinitrochlorobenzene (DNCB).

Fig. 6. Effects of near-IR low-intensity laser irradiation on contact hypersensitivity (CHS) reaction.
Proliferation rate of DLNC in the induction phase
of CHS reaction, cultured for 72 h in the presence of
concanavalin A (0.5 or 2.5 mg/ml). Tritiated thymidine
was added during the last 1618 h of culture. Values
are given as means7SE for each treatment group.
n
Po0.05 and nnPo0.01 vs. nonirradiated animals
treated
with
1-chloro-2,4-dinitrochlorobenzene
(DNCB).

proteins and circulating immune complexes (35).


Anti-inammatory effects of LIL irradiation were
further documented by histopathological examination
of the rheumatoid synovia taken after the treatment
with LIL irradiation at 904 nm (27). However, there
are only few experimental data about the possible
immunomodulatory and anti-inammatory effects of
laser light in this wavelength range.
In this study, an experimental model of the CHS
reaction was employed to evaluate possible immunomodulatory effects of near-IR irradiation.
Ear swelling assay is a measure of CHS reaction
intensity, and it was found to be signicantly
diminished in the group of animals irradiated with
near-IR LIL light before the induction of CHS
reaction with 2% DNCB, and subsequent elicitation
with 0.66% DNCB. Near-IR LIL was employed
before the induction phase, while the effects were
observed in the elicitation phase, thus showing
systemic immunomodulatory effects of the irradiation. Systemic effects of the LIL were observed in
previous studies. Promotion of wound healing was
noticed not only on the irradiated wound but also on
the distant site (21). Suppression of the tuberculin
delayed hypersensitivity reaction in guinea-pigs was
also observed on distant sites after single LIL
irradiation (22).
The observed in vivo effect was further documented
by histopathological analysis of the ear skin samples
and examined in a skin organ culture system. Less
pronounced epidermal changes and signicantly lower
density of the dermal inltrate were found in
irradiated animals.
Nitric oxide is one of the most important mediators
of ear swelling in the elicitation of a CHS reaction.
The contribution of NO synthesis was documented in
CHS reaction to DNFB in mice, and the activity of
inducible nitric oxide synthase (iNOS) was suggested
as largely responsible for NO production during CHS
(36). A dose-dependent increase in nitrite levels was
found in our model of CHS reaction to DNCB in rats,

Table 2. Proliferation rate of DLNC in the presence of IL-2 and expression of CD25 on DLNC during the induction phase of CHS reaction

Proliferation rate of DLNC in the presence of IL-2w


CD25 (IL-2Ra) expressionz

Vehicle

Vehicle1laser

DNCB

DNCB1laser

ND
5.570.2
(2.370.2)

ND
ND

346 96673528
8.670.99
(72.1722.5)

311 64672998n
8.171.2
(39.7712.9)

n
Po0.01 vs. nonirradiated animals treated with DNCB. ND: not determined.
wDLNC taken in the induction phase of CHS reaction were cultured for 72 h in the presence of recombinant IL-2 (50 IU/ml). Tritiated thymidine
was added during the last 1618 h of culture. Values are given as means7SE for each treatment group.
zExpression of CD25 in DLNC during the induction phase of CHS reaction. Values are given as mean percentage of positive cells7SD, and as
mean value of absolute number of positive cells7SD  106 (in parentheses).

209

Kandolf-Sekulovic et al

with signicant correlation between ear swelling and


nitrite level in the skin organ culture system and
abrogation of reaction in the presence of aminoguanidine, a potent inhibitor of iNOS (32). In the present
study, signicantly lower nitrite levels in the CM of
organ-cultured ear skin were found in irradiated
animals, suggesting a decreased nitric oxide release.
This could be explained by a lower density of the
inammatory cells in the dermis as is shown by
quantitative histology, which may be responsible for
amplication of the reaction (32). There is also a
possibility of lowered capacity for NO production by
resident cells, keratinocytes and Langerhans cells,
which were suggested to be NO-producing cells in a
CHS reaction (36).
TNF-a is also one of the important mediators of
ear swelling in the elicitation of the CHS reaction (10,
37, 38). However, measurements of TNF-a levels did
not show signicant differences between irradiated
and nonirradiated groups of animals. It seems that
other mediators are responsible for the diminished ear
swelling after LIL irradiation. It was shown that
chemokines, particularly Gro-a, are responsible for
lymphocyte migration to the site of challenge of a
CHS reaction (39). Furthermore, it was shown that
nitric oxide modulates the expression of some
chemokines (IL-8 and MCP-1) in the skin (40).
Decreased nitric oxide production in irradiated
animals could be responsible for the diminished ear
swelling.
CHS reaction starts in the skin, but amplication of
the response by proliferation of hapten-specic
lymphocytes takes place in the draining lymph node
(11). In order to examine possible mechanisms of the
near-IR light effect observed in the elicitation phase,
DLNC were prepared and functional and phenotypic
characterisation was performed.
Cellularity and dendritic cell content were not
changed in irradiated animals. There were no
differences in the expression of TCR-a, CD4 and
CD8 between irradiated and nonirradiated animals.
However, proliferation rates of the DLNC were
signicantly lower in the irradiated group of animals.
This is in line with the results of in vitro studies that
showed diminished lymphocyte proliferation rates in
cell cultures after LIL irradiation (28, 41).
It can be speculated that lower DLNC proliferation
rates in irradiated animals could be due to an
alteration in the availability of cytokines responsible
for lymphocyte proliferation, i.e. IL-6 and IL-2 (42).
Funk et al. (43, 44) showed that LIL irradiation
lowers the production of IL-2 in peripheral monocyte
cell cultures. Modulation of IL-1a, TNF-a and IFN-g

210

production by LIL irradiation was also found at the


protein and mRNA level. Changes in cytokine
production in DLNC could be responsible for
diminished proliferation rates.
Proliferation rates were also diminished in DLNC
cultures with added IL-2, while the expression of
CD25 was not changed. These ndings, together with
the nding of lower proliferation rates of DLNC
cultured in the presence of 0.5 mg/ml of concanavalin
A, suggest that DLNC activity in irradiated animals is
impaired. However, this effect can be overcome in the
presence of the higher concentration of concanavalin
A, a potent polyclonal lymphocyte stimulant. Diminished proliferation rates in the presence of IL-2,
together with the unchanged expression of CD25,
point to the unresponsiveness of DLNC to IL-2,
possibly mediated by an alteration in signal transduction pathways induced by near-IR irradiation, as was
observed in a previous study (8).
Cellularity, dendritic cell content and phenotypic
characteristics of DLNC were not changed by LIL
irradiation, except for a signicant increase in the
TCR-ab expression in irradiated vehicle-treated animals, compared to nonirradiated animals. It is
possible that LIL irradiation changed the expression
of TCR-ab at the mRNK or protein synthesis level, as
was observed in previous studies for IL-1a, IL-2,
TNF-a, IFN-g, ICAM-1 and IL-2R expression in
peripheral blood mononuclear cells (44).
The observed effects of near-IR LIL light on
draining lymph nodes can be explained by direct
effects of light on DLNC and cytokine production, as
it penetrates up to 50 mm into the tissue (45). On the
other hand, it can be speculated that near-IR light
could exert effects on Langerhans cells and keratinocytes, which can cause insufcient antigen presentation in draining lymph nodes, and subsequent
impaired activation and proliferation of lymphocytes.
Migration of lymphocytes to the site of challenge
could also be changed. In our preliminary experiments of adoptive transfer of lymphocytes from
irradiated animals into recipient rats, a subsequent
elicitation of CHS reaction showed that ear swelling
was signicantly lower in recipient animals
(8.472.1%), compared to control animals who
received lymphocytes from the nonirradiated group
(19.0374.0%). Changes in the expression of adhesion
molecules and alterations in the level of circulating
mediators altered by irradiation can be responsible for
these effects.
In conclusion, near-IR LIL irradiation at 904 nm
and a uency of 3.6 J/cm2 suppress CHS reaction
induced by the lowest examined concentration of

Immonomodulatory effects of low-intensity near-infrared laser irradiation on contact hypersensitivity reaction

DNCB. Suppressive effects of LIL irradiation observed in the elicitation phase and documented
histologically were found together with the diminished
proliferative response of DLNC. Further experimental work is needed to examine the possible mechanisms of these effects.

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Accepted for publication 23 April 2003


Corresponding author:
e-mail: svitac@eunet.yu