Basic inve.~tigation Nitric oxide in contact hypersensitivity 
Skin-organ culture as an approach in 
evalun,tion q/nitric oxide (JVO) 
involvement in 
contact hypersensitivity expression 
M. Kataranovski, G. Milojevic, L. Kandolf and V. Milosevic 
ABSTRACT 
Objective. Contact hypersensitivity (CHS) is a local inflammatory response of the skin following chal-
lenge of hapten-sensitized animals. The intensity of inflammation could be quantified by ear swelling, a 
classical manifestation of contact hypersensitivity. In this study, skin-organ culture system was em-
ployed to evaluate involvement of nitric oxide (NO) in the expression of CHS in rats. 
Methods. Nitrite accumulation in conditioned medium (CM) of rat ear skin following challenge with 
DNCB was determined by Griess assay. The experiment was conducted on two genetically different rat 
strains (AO and DA), which differ in the magnitude of ear swelling response. 
Results. Dose-dependent increase in nitrite levels was noted in CM following application of 0.65% and 
1.3% DNCB to the ears of sensitized rats of both strains. The response was higher in DA rats, display-
ing a more vigorous ear swelling. Correlation between ear swelling and the levels of nitrites in CM of 
both strains was found. Decrease in nitrite levels was noted in CM in the presence of aminoguanidine, 
a known inhibitor with marked specificity tor the inducible isoform of the NO synthase in rodents. 
Conclusion. Presented data demonstrated the utility of skin organ-culture system in detection of NO 
production during the expression of CHS. 
Introduction 
Contact hypersensitivity (CHS) is a T cell-mediated 
inflammatory response of the epidermis following skin 
challenge of hapten-sensitized animals. The afferent 
phase of contact hypersensitivity response, sensitiza-
tion phase, is initiated by epicutaneous application of 
the hapten to clorsal or abdominal tmnk skin, and is 
characterisecl by the activation ancl clivision of hapten-
specific T lymphocytes in the regional lymph nodes 
ancl the appearance of effector cells in the lymph nodes 
Acta Dermatoven APA Vol 11, 2002, No 1 
and spleen (1). In the efferent pluse, which follows a 
subsequent challenge with hapten, e.g. epicutaneous 
application of the sensitizer to the skin of the ear, primed 
T lymphocytes are recmited to the sife of challenge 
where they produce a variety of inflammatory media-
tors, amplifying a background inflammato1y response 
into a more vigorous process. This is the classical mani-
festation of contact hypersensitivity that is measured as 
"ear swelling". Ear swelling is the early-recognized skin 
_J 
Nitric oxide in contact hypersensitivity 
response to hapten application , characterized histologi-
cally by dermal celi infiltration (2). The magnitude of 
ear swelling reflects the intensity of local inflammatory 
response determined largely by the presence and ac-
tivity of cytokines (3,4) and adhesion molecules (5,6), 
w hich govern leukocyte extravasation and effector 
functions in situ. Among cytokines, tumor necrosis fac-
tor-a. (TNF-a.) seems to play a major role in elicitation 
of CHS, as demonstrated in the mouse model of CHS to 
trinitrochlorobenzene (TNCB) (3). 
Recent data demonstrated the involvement of ni-
trie oxide (NO) in t:he expression of CHS to dinitrofluoro-
benzene (DNFB) in mice (7). Contribution of NO syn-
thesis was inclicated in this model of CHS and the activ-
ity of inducible nitric oxide synthase (iNOS) has been 
suggested as largely responsible for NO production 
during CI-IS (7). These data are in line w ith the reported 
expression of inducible nitric oxicle synthase (iNOS) in 
various types of skin cells , and with the postulatecl role 
of synthesis ofthis biological mediator in inflammat01y / 
immune-mecliated processes in skin (8) . 
Short-term cul ture of skin explants is a new experi-
mental in vitro system designed to emulate in vivo 
conditions. Development of skin organ culture tech-
niques bas provided investigation of various aspects of 
normal and pathological skin biology inclucling mainte-
nance of homeostasis, inflanm1ation, eftects ofchemical and 
physical agent~, as well as celi migration (9). Cutaneous in-
flammation has been investigated frequently by monitoring 
fue presence of various soluble biochemical and inflanm1a-
t01y/ irnmunoregulato1y mediators released from skin explants 
in culture fluids . In this way, vadous mediators are being col-
lec.ted and defined as relevant for cutaneous inflammation 
including biologically active amines, proteolytic enzymes, 
various serum p roteins ancl cytokines (10,11 ,12). 
Using a rat model of experimentally inducecl con-
tact hypersensitivity reaction to dinitrochlorobenzene 
(DNCB) (13,30) we have demonstrated a rise in levels 
ofTNF-a. in conditioned medium of o rgan-cultured ear 
skin following challenge w ith increased doses ofDNCB 
(9). Since correlation was noted between the magni-
tude of ear swelling response and an increase in levels 
ofTNF-a. in conditioned medium of organ-cultured ear 
skin following challenge , the utility of this experimen-
tal system in eva luation of inflammato1y mecliators in-
volvernen t in CHS express ion was suggested (9). In 
this study, organ culture of rat skin was used as an ap-
proach in evaluation of nitric oxide involvement in ex-
pression of contact hypersensitivity to DNCB in rats. To 
this aim, accumulation of nitrites was determined in 
culture fluids of organ-cultured ear skin following chal-
lenge , concomitantly w ith changes in ear thickness asa 
classical measure of CHS expression. As there are strain 
differences in reactivity to contact sensitization in rats 
(14) , experiments w ere conducted in tw o genetically 
different rat strains Albino Oxford (AO) and Dark Au-
gust (DA), w hich differ in the magnitude of ear swelling 
response to DNCB (15). 
Basic investigation 
Materials and methods 
Chemicals and reagents 
1-chloro-2,4-dinitrochlorobenzene, DNCB (BDI-I 
Chemicals LTD, England) was dissolved in vehicle (ac-
etone ancl olive oil, 4: 1) . AminoguAnidine (Sigma, USA) 
was dissolved in culture medium RPMI-1640 (ICN Flow, 
USA) supplemented w ith foetal calf serum, FCS 5% (v/ 
v) ancl gentamycin (1ml / 1). 
Animals 
All experiments were done in aclherence to the NII-I 
gu idelines for the use of experimental animals, w ith 
permission of the Ethical Committee of our Institute . 
Inbred male Albino Oxford (AO) and Dark August (DA) 
rats (Farm for Experimental Animals, Militaiy Medica! 
Academy, Belgrade , Yugoslavia), 3 months of age, were 
housed in an air-conditioned rooms at 25°C ona 12-h 
light/ dark cycle . Animals were provided pelleted food 
and water acl lihitum. During the experiment, animals 
were cagecl incl ividually. 
Contact sensitization and ear swelling 
response 
Groups of six to eight animals receivecl 100 µl of 
DNCB (2% and 4%, w/ v in vehicle) oran equal volume 
of vehicle ( 4: 1 acetone: olive oil) on the shavecl area for 
two consecutive days. Three days later, rats were chal-
Fig 1. Mean percent increase in earthickness 
following challenge ot AO and DA rats. Results 
are expressed as means ± SD tor each animal 
group (7 animals). Significance at * p<0.05, 
**p<0.01 or ***p<0.001 vs vehicle (0% DNCB) 
treatment or p<0.001 vs respective values tor 
AO rats. 
Increase in 
ear thickness 
( %) 100 ,--- ---- --- ------- ---, 
80 
60 
40 
20 
o 
igj AO. DA 
o 0.65 
DNCB concentration (%w/v) 
*** ••• ' 
1.3 
4 -------- - --- --- - - ------ --- --- --- - - - Acta Dermatoven APA Vol 11, 2002, No 1 
Basic investigation Nitric oxide in contact hypersensitivity 
Fig 2. Histology of ear skin from AO (A-C) or DA (D-F) rats following application of 0.66% (B, E) or 
1.3% (C, F) of DNCB or following application of vehicle solely (A, D). H&E, x 32. 
lenged by application of 50 µl of three times lower con-
centration of DNCB than that used in sensitizations 
phase (i.e. 0.66% and 1.3%) on the dorsal skin of the 
left ear. The contact hypersensitivity reaction was as-
sessed by measuring the ear thickness with an engi-
neer' s micrometer 24 hours after challenge. The re-
sponse was quantitated as the difference in the thick-
ness between challenged and non-treated ears of the 
same animal , according to the formula (C-N)/NxlOO, 
where C is thickness of the challenged ear, N is thick-
ness of non-treated ear. The response was expressed 
as percent of increase in ear thickness. 
Acta Dermatoven APA Vol 11, 2002, No 1 
Histology 
Ear samples taken 24 h after challenge were placed 
into 4% bufferecl formalin, pH 6.9, embeclded in paraf-
fin wax for sectioning at 5 µm and stained with hema-
toxylin/ eosin (H&E). 
Skin Organ Culture 
Exposed ears were tak en 24 h after elicitation of the 
CHS, split into ventral and clorsal halves and submergecl 
in 1.5 ml of culture medium in wells of 24-well culture 
J 
Nitric oxide in contact hypersensitivity 
plates. After 24h and 48 hours of incubation in humid 
atmosphere with 5% CO
2 
at 37°C (Heraus, Germany), 
the conditioned medium (CM) was collected. In some 
experiments, ear halves were cultured in the presence 
of 25 and 50 µg / ml aminoguanidine . 
Nitrite measurement 
Nitrite accumulation, as indica tor of NO production, 
was measured in 24 h and 48 h conditioned medium 
using the Griess reagent (16) . Briefly, 50 µl aliquots of 
CM were mixed with an equal volume of Griess re-
agent (a mix ture of 0.1% naphtylethylenediamine 
dihydrochloride in water and 1 % sulphanilamide in 5% 
phosphoric acicl) and incubated for 10 min at room tem-
perature. The absorbance was measured at clual wave-
length 570nm/ 650nm by an ELISA 96-well plate reader 
(Behring, FR Germany). The nitrite concentration was 
calculated from a sodium nitrite standard curve. 
Statistical analysis 
Results are expressed as mean value ±SD for each 
experimental animal group (6-8 animals) . Statistical sig-
nificance was determinecl by the Mann-Whitney test. P 
values less than 0.05 were considerecl significant. The 
relationship between the levels of nitrites in CM and ear 
swelling was examinecl by regression analysis. 
Results 
Ear swelling response and skin histology following 
challenge of ears of sensitizecl animals 
The mean percentage increase in ear thickness is 
presented in Fig. 1. A dose-dependent increase in ear 
swelling response was eviclent for both AO and DA rats, 
being more pronouncecl in animals ofDA strain. Appli-
cation of DNCB to the ears of sensitized animals pro-
duced both epidermal and dermal alterations charac-
teristic for contact dermatitis. Histological picture of ear 
skin 24 hours after challenge of AO and DA rats w ith 
DNCB is presentecl in Fig. 2 illustrating the increase in 
ear o edema following application of ha pten , compared 
to vehicle. The higher the dose of the hapten, more 
intense skin oedema was notecl comparecl to vehicle 
application. In the dermis, oeclema was associatecl with 
leukocyte infiltration : 14±2 and 29±8 cells/ microscopic 
fielcl in skin of AO rats and 19±4 ancl 34±4 cells/ micro-
scopic field in skin of DA rats following challenge with 
0.65% and 1.3% DNCB, respectively, comparecl to 9-
10 cells/ fielcl in the dermis of vehicle treatecl rats. 
Nitrite levels in culturejluids oj ear skin 
explants 
Nitrite accumulation was measured in conditioned 
Basic inv e sti ga tion 
Nitrite 
acumulation 
(10·6 M) 
30 
mAO-DA 
25 
20 24h 
15 
10 
5 
o 
o 0.65 1.3 O 0.65 
DNCB concentration (% w/v) 
Fig 3. Nitrite levels in conditioned medium of 
organ-cultured ear skin following challenge with 
DNCB. Values are expressed as means of mM of 
nitrites.± SO from seven animals. Significance at 
* 0.05, **0.01 or ***0.001 vs vehicle treatment or 
=I: p<0.05, *** p<0.001 vs respective values tor 
AO rats. 
meclium (CM) of ear skin from challenged AO and DA 
rats (Fig 3) . Increasecl levels of nitrites were eviclent in 
24-hour conclitioned medium (CM) following applica-
tion of0.65% and 1.3% DNCB to the ears ofboth sensi-
tized AO and DA rats comparecl to CM of respective 
control, vehicle treatecl ears. Rise in nitrite concentra-
tions was greater in CM of organ-cultured ear skin from 
DA rats compared to these measured in CM of AO rats . 
Further increase in nitrite levels was noted in 48-hour 
skin-conditioned medium of both strains, being higher 
in CM of skin from DA rats ancl eviclent even in CM of 
vehicle-treatecl ears. 
There is a correlation between increase in ear thick-
ness and levels of nitrites in 24-hour and 48-hour CM of 
ear skin from AO rats (Fig 4 A, B) (the respective for-
mula is y=2 .836x - 1.101 , r=0.76, p<0.001 for 24-hour 
CM ancl y=l.6188x+ 1.3466, r=0.785, p<0 .00lfor 48-
hour CM. Significant correlation was notecl between re-
spective parameters in DA rats (Fig 4 C, D). The re-
spective formula is y=4.5884x-6.6693, w ith r =0.828 
and p<0.001 for 24-h CM and y= 2.2527x, r=0 .866, 
p<0,001, for 48-hour CM. 
Ejfect oj aminoguanidine on nitrite 
accumulation. 
In order to examine contribution ofNO production 
to the observed increase in nitrite accumulation in cul-
1.3 
6 --------------- - ----------- ----- ---Acta Dermatoven APA Vol 11, 2002, No 1 
Basic investigation 
ture fluids of ear skin, specimens harvested following 
challenge with DNCB or following vehicle application 
to DA rats were cultured in the presence of amino-
guanidine , inducible isoform of nitric oxide synthase 
(iNOS) inhibitor, with a marked specificity for iNOS in 
rodents (17). As shown in Fig 5, significant and dose-
dependent reductions in nitrite levels were noted both 
in 24-hour and 48-hour CM of ear skin explants cultured 
with 25 µg/ ml and 50 µg/ ml of aminoguanidine. 
Discussion 
Contact hypersensitivity (CHS) is a local inflamma-
tory response of the skin following challenge of sensi-
tized animals. The intensity of inflammation following 
Ear 
swelling 
(%) 
A 
100 -,-----------------, 
y = 2.836x - 1.101 
80 - r=0.7596 
o 5 10 
Ear C 
swelling 
(%) 
100 
y = 4.5884x - 6.6693 
80 r = 0.8277 
• 
60 
40 • • 
•l • 
20 
o 
o 5 10 
15 20 25 
Nitrites (10-GM) 
• • • .. 
15 20 25 
Nitrites (10-GM) 
Nitric oxide in contact hypersensitivity 
hapten application could be quantified by ear swelling, 
since dose-dependent increase in ear thickness was 
noted. Ear swelling is the early recognized manifesta-
tion of contact hypersensitivity characterised by der-
mal celi infiltration (2) , identified as T cells, granulo-
cytes, monocytes / macrophages, and Langerhans cells 
(18). It is based largely on activity of skin cells, which 
through release of cytokines modulate skin microenvi-
ronment facilitating extravasation of inflammatory leu-
kocytes. In the dermis , leukocytes produce a variety of 
inflammatory mediators, amplifying the local response 
into vigorous inflammatory process expressed as ear 
swelling (19). This local inflammatory response is de-
termined by protein-type factors (cytokines) , (3,4) ad-
hesion molecules (5,6) and, as recently demonstrated, 
nitric oxide (7). 
Ear 
swelling 
(%) 
B 
100 ~-------------~ 
80 
60 
40 
20 
y =1.6188x + 1.3466 
r =0.785 
o -i=----....i'----''!L--3'-'--,---------,--------,----! 
o 5 10 15 20 25 
Ntrites (106M) 
Ear D 
swelling 
(%) 
100 
y = 2.2527x 
80 r =0.8664 
60 
40 
20 
o 
o 5 10 15 20 25 
Nilrites (10-GM) 
Fig 4. The regression lines for changes in ear swelling and nitrite levels in conditioned medium of ear 
skin following challenge of AO (A, B) or DA (C, D) rats. 
24 hour-conditioned medi um (A, C) or 48 hour-conditioned medium (B, D). 
Acta Dermatoven APA Vol 11, 2002, No 1 7 
Nitric oxide in contact hypersensitivity 
In this study, the expression of contact hypersensi-
tivity was evaluated by traditional measurements of 
changes in ear thickness following challenge with DNCB 
and by organ-cultured skin approach. Nitrite concen-
trations were determined using organ-culture in culture 
fluids of ears following hapten challenge. The measure 
of NO production was estimated with the Griess reac-
tion, since it is only possible to evaluate the concentra-
tion ofNO indirectly, measuring its metabolites - nitrites/ 
nitrates (20). In concordance with data, which demon-
strated measurement of nitrite accumulation in culture 
supernatants of macrophages as an indicator ofNO pro-
duction in conditions of inflammation (21, 22), our data 
demonstrated the utility of the ear skin organ culture 
system in nitrite leve! measurements in conditioned 
medium of tissue (skin) explants. Our results that dem-
onstrated dose-dependent increase in ear thickness fol-
lowing administration of DNCB (noted both in AO and 
DA rats), which was accompanied by increase in levels 
of nitrites in challenged ear-skin conditioned meclium, 
suggest the utility of nitrite accumulation measurements 
in conditioned meclia as an approach in evaluation of 
NO contribution to CHS expression. Data demonstrat-
ing higher levels ofNo?- in CM of ear skin from DA rats, 
which respond to DCNB challenge with more vigorous 
inflammatory response (greater magnitude of ear swell-
ing), support this assumption. 
Significant correlation between levels of nitrites in 
CM of skin following DNCB challenge and the magni-
tude of ear swelling response suggested relationship 
between nitrite accumulation in skin culture fluid and 
the intensity of local skin inflammatory response dur-
ing the elicitation of CHS. These data are further in line 
with that obtained in mouse model of CHS to DNFB 
which demonstrated involvement of nitric oxide in ear 
swelling response and contribution of NO production 
to this response (7). 
The skin organ culture system has been exploited 
in detection of various inflammatory mediators pro-
duced and released by skin explants in culture fluid 
(10,11 ,12,23,9). It appeared to be valuable in studying 
local skin cytokine response following topical applica-
tion/exposure to various chemical and physical agents 
including heat, mechanical inju1y, some drugs and toxi-
cants (12,23). Using this experimental system a rise in 
levels of TNF-a in conditioned medium of organ-cul-
turecl ear skin explants was notecl following application 
of increased doses of DNCB to the ears of sensitizecl 
rats (9). As correlation was established between the in-
tensity of ear swelling response to DNCB ancl an in-
crease in levels of TNF-a in conclitioned medium of 
organ-cultured ear skin following challenge, the utility 
of this experimental system in evaluation of inflamma-
toty mecliators involvement in CHS expression was sug-
gested (9). Presentecl clata suggest nitrite accumulation 
measurements in challengecl ear skin conclitionecl me-
clia as an approach in evaluation of NO contribution to 
CHS expression. 
Basic investigation 
Nitrite 
acumulation 
(10-6 M) 
30 
25 
20 24h 
15 
10 
5 
o 
o 25 50 O 
Aminoguanidine (10.,;g/ml) 
Fig. 5. Nitrite levels in conditioned medium of ear 
skin from DA rats, cultured in the presence of 
aminoguanidine. Values expressed as means of 
mM of nitrites.± SD from seven animals. 
Significance at * p<0.05, **p<0.01 or ***p<0.001 
vs nitrites in CM from cultures without 
aminoguanidine. 
Our data that clemonstratecl a clrop in nitrite levels in 
CM in the presence of aminoguanicline, which has a 
marked specificity for iNOS in roclents (17), suggestecl 
that NO procluction coulcl be moclulatecl in skin-organ 
culture. The results ofthese inhibition experiments cor-
roborate clata obtainecl in mice in vivo in which incluc-
ible NOS is suggested as largely responsible for NO pro-
duction during CHS to DNFB in mouse. In that model of 
CHS, keratinocytes and Langerhans cells were suggested 
to be NO producing cells (7). NO produced by these 
cells may be involved in CHS reaction by formation of 
oedema ancl inflammatory celi infiltrate during effector 
phase. We suggest that leukocyte (cells in dermal infil-
trate) NO procluction, also contribute to skin NO pro-
duction. The clynamics of nitrite accumulation, with high-
est values obtained by 48 hours in culture, are in con-
cordance with that of rat leukocytes (macrophages and 
granulocytes) in culture (22 and unpublished data). In-
flammatory cells stimula teci by epiclermal celi cytokines 
as well as their own cytokines produce NO and thereby 
amplification could occur. From numerous studies it is 
known that inflammatory cytokines (IFN-y, IL-8, TNF-
a, IL-1 ancl others) could stimulate keratinocytes to pro-
cluce NO (8) and that some (e.g. IFN-y) have a capacity 
of stimulating iNOS expression in Langerhans cells (24). 
In addition , TNF-a (21) and IFN-y (25) coulcl stimulate 
macrophage NO production. Following hapten appli-
cation, skin cell-derived NO coulcl incluce vasodilata-
48h 
* 
25 50 
s ------- - ---------------------------Acta Dermatoven APA Vol 11, 2002, No 1 
Basic investigation Nitric oxide in contact hypersensitivity 
tion, causing an increase in the volume of blood and 
delive1y of leukocytes at the site of inflammation. The 
role ofNO in formation of inflammato1y celi infiltrate in 
dermis was supported by recent data which demon-
strated the relevance of nitric oxide in neutrophil re-
cruitment to inflammatory focus in various models of 
inflammation (26) . Inhibitors ofNO synthesis employed 
in these models of inflammation have caused reduction 
in the accumulation of neutrophils at the site of inflam-
mation (26). 
29) , determination of changes in nitrite levels, as indi-
cators of NO production in skin-organ culture system 
might be used as an accompanying test. Thus valuable 
information regarding agents causing skin toxicity and 
inflammatory celi accumulation/ function in contact hy-
persensitivity could be obtained. 
Conclusion 
One aspect regarding nitrite measurements in cul-
ture fluids of ear skin should be mentioned. Skin organ 
culture has been recommended recently as a new ex-
perimental system suitable for studying and testing the 
effects of various physical, chemical and biological 
agents on skin (27). As CHS is frequently employed in 
investigations of various agents of immunotoxicity (28, 
In conclusion, data presented in this paper demon-
strate the evaluation of CHS response by in vitro mea-
surement of nitrite accumulation in cul ture fluids of or-
gan-cultured skin. The relevance of measurement of 
nitrites as an indicator of NO production by inflamed 
skin in CHS is indicated by a correlation between ear 
swelling and levels of nitrite accumulation. 
l
., 
l FE.R.ENC 
Acta Dermatoven APA Vol 11, 2002, No 1 
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Nitric oxide in contact hypersensitivity Basic investigation 
AUTHORS' 
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Milena J(ataranovski, PhD, Projessor ojimmunobiology, Faculty oj 
Biology, University ojBelgrade, Studentska 4 and Senior research associate, 
Institutejor MedicalResearch, JV!ilitary MedicalAcademy, Crnotravska 17, 
11000 Belgrade, Yugos lavia, 
e-mail: vmaimi@eunet.yu- corresponding author 
Gordana Milojevic, BSc, Institutefor Medical Research, Military M edical 
Academy, Crnotravska 17, 11000 Belgrade, Yugos lavia, 
e-mail: vmaimi@eunet.yu 
Lidija Kandoif Sekulovic, MD, Department oj Dermatology, Military 
MedicalAcademy, Crnotravska 17, 11000 Belgrade, Yugoslavia, e-mail: 
bdd_sld@yubc.net 
VericaMiloševic, PhD, Senior research associate, Institutefor Biological 
Research Siniša Stankovic, 29 Novembra 142, 11000 Belgrade, Yugoslavia 
_10 ------- - --------------------- -----Acta Dermatoven APA Vol 11, 2002, No 1