237 DISC

Veterinary Dermatology 2001, 12, 129137

TNF-a dependent NF-kB activation in cultured canine

keratinocytes is partly mediated by reactive oxygen species
H. B. K. KOHLER,* B. HUCHZERMEYER,{ M. MARTIN,{ A. DE BRUIN,} B. MEIER} and I. NOLTE*
*Klinik fur kleine Haustiere, Tierarztliche Hochschule Hannover, Bischofsholer Damm 15, 30173
Hannover, Germany
{Institut fur Tierokologie und Zellbiologie, Tierarztliche Hochschule Hannover, Bunteweg 17d, 30559
Hannover, Germany, {Abteilung Molekularpharmakologie, Medizinische Hochschule Hannover, CarlNeuberg-Str. 1, 30625 Hannover, Germany, }Institut fur Tierpathologie, Universitat Bern, Langgassstr. 122,
3012 Bern, Switzerland, }Fakultat fur Physik E17, Ludwig Maximilian Universitat Munchen, Germany
(Received 16 August 1999; accepted 15 June 2000)

Abstract The cytokine TNF-a plays a major role in inammatory and immunological reactions of canine
skin. With respect to a possible therapeutic modulation, we investigated the role of the transcription factor
NF-kB and the involvement of reactive oxygen species (ROS) in the TNF-a signalling pathway in cultured
canine keratinocytes. TNF-a treatment resulted in activation of NF-kB which was partially inhibited by the
antioxidant a-lipoic acid. Using the cytochrome c reduction test no superoxide production could be detected
in the supernatant of TNF-a stimulated keratinocytes. However, TNF-a dependent intracellular hydrogen
peroxide production was demonstrated spectroscopically. With electron energy loss spectroscopy (EELS)
signicant hydrogen peroxide formation was detected in the mitochondria, the endoplasmic reticulum, the
cytosol and partially on the plasma membrane of the keratinocytes. Hence, ROS possibly play an important
role in the TNF-a signalling pathway leading to NF-kB activation in canine skin. An adjunctive therapy with
natural potent antioxidants modulating NF-kB overactivation in canine cutaneous inammation may be of
therapeutic benet.
Keywords: canine, keratinocytes, nuclear factor-kB, reactive oxygen species (ROS), redoxregulation.

INTRODUCTION
The importance of resident skin cells in initiating
inammatory and immunological skin diseases has
been an increasing eld of investigation in the past
years. Keratinocytes respond to a variety of stimuli
with secretion of inammatory products such as
cytokines and expression of adhesion molecules.1
The pro-inammatory cytokine TNF-a, synthesized
by activated macrophages and lymphocytes inltrating the skin as well as epidermal Langerhans
cells, plays a key part in inammatory and allergic
skin reactions.2 By inducing the expression of
cytokines such as IL-1, IL-6, IL-8 and TNF-a as
well as adhesion molecules, it amplies the initial
cutaneous response.2,3
TNF-a induced expression of these inammatory
products is largely regulated by the transcription factor
nuclear factor-kB (NF-kB) in man and rodents.4
Increased intracellular levels of reactive oxygen
species (ROS) possibly function as intracellular secondary messengers in NF-kB dependent gene expression.46

This study was supported by a research grant of the Deutsche

Forschungsgemeinschaft (DFG).
Correspondence: H.B.K. Kohler. Tel.: + 49511856 7251;
Fax: + 49 511856 7686; E-mail: hkoehler@stud.tiho-hannover.de
# 2001 Blackwell Science Ltd

Biologically important ROS, including the superoxide radical O27, hydrogen peroxide H2O2, the
hydroxyl radical OH and hydroperoxides ROOH,
originate from diverse redox systems such as mitochondrial respiratory chain enzymes, cytochrome P450 systems, xanthine oxidase, NADPH-oxidase,
lipoxygenases and cyclooxygenase.7
NF-kB is an inducible transcription factor composed
of a 50 and 65 kDa subunit and an inhibitory subunit
I-kB. Upon activation by various stimuli (viral and
bacterial products, inammatory cytokines, reactive
oxygen species [ROS], UV irradiation and mitogens)
the inhibitory subunit is degraded from the inactive
heteromeric complex after phosphorylation and ubiquitination.8 Upon the release of I-kB from the
complex, the remaining heterodimer p50/65 translocates into the nucleus and binds to dened DNA
sequences in the promoter regions of predominantly
stress-related target genes such as those encoding
Interleukin-1, IL-6, IL-8, TNF-a, growth factors (e.g.
Granulocyte-Colony stimulating factor), cell adhesion
molecules, immunoreceptors or acute phase proteins.6,9
After examining the involvement of ROS in TNF-a
mediated NF-kB activation in canine dermal broblasts10 we investigated these mechanisms in canine
keratinocytes of healthy dogs in order to gain more
insight into ROS-related signalling in inammatory
and allergic diseases of the canine skin.
129

237 DISC
130

H. B. K. Kohler et al.

In this study we demonstrate that NF-kB is

activated by TNF-a and that this activation is
partially inhibited by the antioxidant a-lipoic acid.
TNF-a dependent intracellular superoxide and/or
hydroperoxide formation could be demonstrated at
nonspecic sites with a spectrographical method.
MATERIALS AND METHODS
Cell culture
Canine keratinocytes were isolated and cultured by a
modication of the protocol of Wilkinson et al.11
Briey, healthy skin from the linea alba was obtained
from anaesthesized dogs undergoing surgery, cut into
small pieces and incubated in William's Medium E
(Sigma, Munich, Germany) with 10 mg mL71 dispase
(Boehringer, Mannheim, Germany) overnight at
4 8C. The epidermis was stripped o the dermis and
incubated with trypsin/EDTA (0.25%/0.1%) at room
temperature for 30 min. The cell suspension was then
centrifuged for 5 min at 700 g and the keratinocytes
were seeded into tissue culture asks containing
William's Medium E supplemented with 2 mM
glutamine, 20% foetal calf serum (Gibco, Karlsruhe,
Germany), 0.1 nM choleratoxin (Sigma), 10 ng mL71
Epidermal Growth Factor (EGF), 100 mg mL71
neomycin, 50 mg mL71 gentamicin and 2.5 mg mL71
amphotericin B (Biochrom, Berlin, Germany).
For passages 25, low calcium medium containing
0.3 mM calcium and 10% foetal calf serum was used.
Decalcication was achieved by incubation of unsupplemented William's Medium E with 19 g 500
mL71 Chelex 100 Resin (Biorad, Munich, Germany)
for one hour.
Electrophoretic mobility shift assay (EMSA)
Keratinocyte cultures from passage 35 were plated
in 25 cm2 asks and grown to conuency. Cells were
stimulated by incubation with 10 ng mL71 human
recombinant TNF-a only for one hour (in conditioned medium) or incubated with 18 mM a-lipoic
acid (dissolved in HBSS, pH 7.4) two hours before
TNF-a stimulation with 10 ng mL71. The Trypan
blue exclusion test was used to ensure that none of
the concentrations and solvents employed aected
cell viability (95% cell viability tolerated). Two asks
were used for each experiment containing a total cell
number of approximately 3 million cells. After
stimulation, asks were repeatedly rinsed with
phosphate-buered saline (PBS, pH 7,4, Sigma) and
cells scraped into centrifugation tubes. The cell
suspension was pelleted at 500 g for 10 min and
nuclear extracts harvested according to a modication of the method of Dignam et al.12 Briey, the cells
were lysed in hypotonic buer containing 10 mM
HEPES, 10 mM KCl, 200 mM EDTA, 1 mM
dithiothreitol (DTT), 1 mM phenylmethyl-sulphonyl
uoride (PMSF), 1 mg mL71 pepstatin, 200 mM
leupeptin, 10 mM transepoxy-succinyl-leucylamid (E
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 129137

64) and 0.3 mM Na3VO4 (Sigma) for 15 min on ice.

After addition of Nonidet P-40 to a nal concentration of 0.6%, the keratinocyte suspension was passed
through a 20-gauge and subsequently 26-gauge needle
to achieve complete lysis of cells. Nuclear extracts
were pelleted at 10,000 g for 30 s and resuspended in
hypertonic buer containing 20 mM HEPES, 400 mM
NaCl, 1 mM EDTA, 10% Glycerin, 1 mM DTT, 1 mM
PMSF, 1 mg mL71 pepstatin, 200 mM leupeptin, 10 mM
E 64 and 0.3 mM Na3VO4 and stored on ice for 30
min. The suspension was centrifuged at 13 000 g for
10 min and the supernatant collected as the nuclear
extract. Protein concentration was determined according to Bradford.13
The binding assay was performed using a 32Plabelled, double-stranded oligonucleotide (Pharmacia, Freiburg, Germany) containing the consensus
binding sequence for NF-kB:14 5'-TGA CAG AGG
GGA CTT TCC AGA GA-3'. The oligonucleotides
were labelled using g-(32P)ATP and T4-polynucleotidekinase (Pharmacia) and the labelled probes puried
from free nucleotides by gelltration using S-200 H
columns (Stratagene, Heidelberg, Germany).
For the binding reaction 10 mL of nuclear extracts
containing 8 mg protein were incubated with a
reaction buer containing 3 mL 5 6 binding buer
(20 mM HEPES, 60 mM KCl, 1 mM EDTA, 5%
glycerin and 1 mM DTT), 1 mL poly(dI-dC) (Pharmacia) as nonspecic competitor DNA and 1 mL of
the 32P-labelled oligonucleotide. After a 30-min
binding reaction at room temperature, the samples
were loaded on a native 6% polyacrylamide gel and
run in 0.25 6 TBE buer, pH 8.3 (10 6 TBE buer
containing 0.9 M TRIS-borate and 0.025 M EDTA) at
200 V for 2.5 h at room temperature. The gels were
dried under vacuum at 80 8C and exposed overnight
to a Kodak Biomax X-ray lm. Specicity of the
binding to the oligonucleotide containing the NF-kBDNA consensus site was shown by competition
studies using unlabelled NF-kB and an unlabelled
irrelevant motif (containing the binding site for the
transcription factor Activator Protein-1, AP-1). The
illustrated autoradiograms are representative results
of three independent experiments.
Superoxide dismutase-inhibitable cytochrome c reduction
Keratinocytes were seeded into glass cuvettes at
0.8 6 106 cells/cuvette and cultured for 2472 h until
conuent. Cuvettes were washed in Hank's balanced
salt solution (HBSS; Sigma) and pre-incubated with
100 mM oxidized cytochrome c (ferricytochrome c) in
HBSS for 10 min. The amount of superoxide released
into the medium of the cuvettes after TNF-a or
lipopolysaccharide (positive control) addition (LPS
from E. Coli; Sigma) was immediately monitored by
measuring the amount of reduced cytochrome c
(ferrocytochrome c) at 550 nm (37 8C) every 2 min
(averaging time = 5 s) for 2 h against a reference
cuvette containing HBSS and 100 mM ferricytochrome
c without cells in a Uvikon 930 spectrophotometer

237 DISC
TNF-a dependent NF-kB activation in cultured canine keratinocytes
(Kontron, Hannover, Germany).15 TNF-a and LPS
alone had no eect on the oxidation status of
cytochrome c. At the end of the experiments cell
adherence was veried by microscopic evaluation, cell
viability was assessed with trypan blue exclusion and
cells counted in a Neubauer chamber after trypsination (0.025% trypsin in HBSS).
The raw data were converted into ASCI format
and transferred to Excel 8.0 software. The mean
superoxide production rate was calculated using the
extinction coecient for reduced cytochrome c
De550nm = 21 000 M71 cm71 15 and expressed in
nmol O27/106 cells/120 min. The specicity of
cytochrome c reduction was veried by simultaneous
addition of 2 mM superoxide dismutase (SOD)
leading to a degradation of superoxide radicals and
thus inhibition of cytochrome c reduction. Superoxide-independent cytochrome c reduction as well as
basal superoxide generating activity of unstimulated
cells was taken into consideration by subtraction
from the results.
The experiments were each carried out in quadruple and the mean superoxide production rate and
standard deviation calculated.
Spectroscopical detection of intracellular hydrogen
peroxide with cerium chloride
For the localization of intracellular hydrogen peroxide production a modied technique by Briggs et
al. was applied:16 Canine keratinocytes were seeded
into transwell inserts of 12 well plates (Corning
Costar, Bodenheim, Germany) at a density of 105
cells/well and grown to conuency. The monolayer
was washed in buer A consisting of 0.1 M TRISmaleate buer, 10 mM glucose and 160 mM sucrose
(pH 7.3) and incubated with 5 mM cerium chloride
(CeCl3) alone or in combination with 10 mM
aminotriazole for 30 min at 37 8C. Subsequently, 25
ng mL71 TNF-a was added and cells were incubated
for another 30 min. The monolayers were washed in
buer A before prexing the adherent cells with 2%
glutaraldehyde in buer B consisting of 0.1 M
cacodylate buer (pH 7.3) and 150 mM sucrose at 4
8C for 45 min. In order to eliminate cerhydroxides
which precipitate in alkaline milieu, cells were washed
in buer B at pH 6 for another 60 min before postxation with 2% osmium tetroxide included in buer
B. The transwell membrane containing the cell
monolayer was excised and the cells processed for
electron microscopy as described by Lehmann et al.17
Briey, cells were dehydrated with acetone and
progressively embedded in the epoxy resin Durcupan
ACM (Fluka, Buchs, Switzerland). Cells were subjected to 24 h pure resin at 40 8C followed by
polymerization for 48 h at 60 8C. Blocks were
sectioned (30 nm), mounted on uncoated copper
grids without staining and examined for analysis of
elements by electron energy loss spectroscopy (EELS)
using a Zeiss EM 902 transmission electron microscope (Zeiss, Jena, Germany).

131

Mitochondria, the endoplasmic reticulum, the

plasma membrane and cytosol were analysed in
uncontrasted ultrathin sections of TNF-a stimulated
cells for the existence of cerium precipitates by
electron energy loss spectroscopy, a technique which
is briey described. Conventional transmission electron microscopy does not dierentiate between
elastically and inelastically scattered electrons from
the primary electron beam. Inelastically scattered
electrons detected with spectrometric techniques
interact with atoms of the specimen and lose a
dened amount of energy (DE) characteristic for
each element and within each element for each shell
electron (DEK, DEL, etc.).18 Electrons with an
element specic energy loss can be separated by a
magnetic prism producing a line spectrum of
dierent electron wavelengths.19
The characteristic electron energy loss for the
element cerium is 883 eV for the M5 shell and 905 eV
for the M4 shell resulting in a double peak. The
uncontrasted sections were scanned qualitatively for
cerium using a circular blend of 100 mm diameter
corresponding to an area of 368 nm in diameter at
the standard magnication of 30 000. In two
independent experiments, six measurements in each
of the above mentioned specic sites were performed
and the cerium-positive ndings for each sample
documented (qualitative analysis). Representative
data is shown.
RESULTS
Electrophoretic mobility shift assay
Nuclear extracts of cultured canine keratinocytes
treated with 10 ng mL71 TNF-a for 1 h exhibited
two inducible bands in the EMSA (Fig. 1, lane 2).
Specicity of the binding to the oligonucleotide
containing the NF-kB-DNA consensus site was
shown by competition studies using unlabelled
NF-kB and an unlabelled irrelevant motif (containing the binding site for the transcription factor
Activator Protein-1, AP-1). Complete blocking
was achieved with both 10 and a 100-fold excess
of the cold NF-kB-competitor whereas 10 and a
100-fold excess of the AP-1 oligonucleotide had no
eect on TNF-a induced NF-kB activation in these
cells. Saturation of band intensity in the EMSA
was achieved at concentrations of 10 ng mL 71
TNF-a (Fig. 2).
The appearance of two inducible bands upon TNF
activation can be attributed to dierent hetero- and
homodimer formations as assessed in supershift
assays using antibodies against p50 and p65 in
canine broblasts.10
TNF-a induced NF-kB activation was partially
abrogated by 2-hour pre-incubation with the antioxidant a-lipoic acid in a dose-dependent manner
(Fig. 3). Higher antioxidant concentrations were not
feasible due to the strong acidity of the reagent.
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 129137

237 DISC

10

25

50

Control

(1:10)

TNF- (1h)
[ng / m l -1]

+ AP-1

+ AP-1 (1:100)

+ NF- B (1:10)

+ NF- B (1:100)

H. B. K. Kohler et al.
TNF- [10 ng / m l -1]

Control

132

Figure 2. Autoradiogram after treatment of canine keratinocytes

with various TNF-a concentrations as indicated for one hour. The
lled arrows indicate the inducible bands, the open arrow
designates the unbound 32P-labelled NF-kB oligonucleotide.

Figure 1. Electrophoretic mobility shift assay (EMSA) detecting

activated NF-kB in the nucleus after TNF-a treatment of canine
keratinocytes for 1 h. To show the specicity of the TNF-a induced
complexes a 10-and 100-fold excess of unlabelled NF-kB or
Activator Protein-1 (AP-1) oligonucleotide was added as a
competitor. The lled arrows indicate the inducible NF-kB
bands, the open arrow designates the unbound 32P-labelled NFkB oligonucleotide.

TNF-a induced superoxide production

In order to evaluate whether cellular superoxide
production was involved in TNF-a mediated signalling, we determined superoxide formation in the
supernatant after addition of TNF-a to adherent
keratinocytes cultured in cuvettes: No superoxide
production could be detected using TNF-a concentrations of 10150 ng mL71. Positive controls were
implemented with 10 and 80 mg mL71 LPS rendering
a superoxide generation of 2.82 + 1.15 nmol O27/106
cells/120 min and 8.75 + 0.29 nmol O27/106 cells/120
min, respectively (results not shown). TNF-a dependent cytochrome c reduction was inhibited 100% by
addition of 2 mM SOD.
Spectroscopic detection of TNF-a induced intracellular
hydrogen peroxide formation
A second, more sensitive approach to determine the
involvement of intracellular superoxides in TNF-a
signalling was based on the reaction of hydrogen
peroxide with cerium chloride (CeCl3) to form
electron-dense, insoluble precipitates of cerium perhydroxides, Ce(OH)2OOH and Ce(OH)3OOH.16
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 129137

The presence of cerium deposits, not detectable

with standard transmission electron microscopy,
could be demonstrated with the electron energy loss
spectroscopic technique (EELS): Canine keratinocytes incubated with only cerium chloride (negative
control) showed modest intracellular electron-dense
deposits of cerium perhydroxides in the mitochondria, the endoplasmic reticulum, the cytoplasm and
the plasma membrane. The addition of 3-amino1,2,4-triazole, a catalase inhibitor leading to the
accumulation of cellular hydrogen peroxide (positive
control), induced the formation of hydrogen peroxide-derived reaction products in all 4 sites. Treatment with 25 ng mL71 TNF-a for 30 min after preincubation with 5 mM cerium chloride (TNF-stimulation) lead to a higher percentage of cerium positive
results in all analysed sites in comparison to the
negative controls (Fig. 4a,b).
DISCUSSION
Our in vitro study demonstrated rapid TNF-a
dependent nuclear translocation and DNA binding
of the transcription factor NF-kB in cultured
keratinocytes from healthy dogs as previously shown
in canine dermal broblasts.10
Biological activity of human recombinant TNF-a
in the dog exhibiting 92% structural homology to
canine TNF-a20 has previously been shown by
signicant TNF-a dependent PGE2 synthesis in
canine broblasts.10 NF-kB activating concentrations

237 DISC
TNF-a dependent NF-kB activation in cultured canine keratinocytes
TNF - [10 ng /ml -1]

8 mM

1 mM

5 mM

Control

- lipoi c acid (2h)

Figure 3. NF-kB binding activity in canine cultured keratinocytes

after pre-incubation with the indicated concentrations of a-lipoic
acid for two hours and stimulation with 10 ng mL71 TNF-a for 1 h.
Filled arrows indicate the position of the inducible NF-kB-DNA
complex, the open arrow demonstrates the unbound 32P-labelled
NF-kB oligonucleotide.

of 5 ng mL71 TNF-a in canine keratinocytes are

within the range of the receptor binding constant for
the 55 kDa receptor at 8.5 ng mL71 21 implying
receptor dependent signalling mechanisms.
The fact that canine keratinocytes required higher
TNF-a concentrations (5 ng mL71) for NF-kB
activation than canine dermal broblasts, which were
stimulated by concentrations as low as 0.01 ng mL71
TNF-a,10 is possibly attributable to a higher activating threshold due to decreased TNF-a receptor
expression by keratinocytes. With regard to the
localization of keratinocytes and their permanent
exposure to various physical and chemical stimuli,
this would be a biologically plausible phenomenon.
In order to assess whether ROS are involved in
TNF-a mediated NF-kB mobilization in canine skin
we examined the inhibitory eect of the antioxidant
a-lipoic acid. Pre-incuabtion with a-lipoic acid
partially blocked TNF-a dependent NF-kB mobilization in canine keratinocytes: alpha-lipoic acid is a
natural cofactor of the pyruvate dehydrogenase and
a-ketoglutarate dehydrogenase in the mitochondria.

133

Upon absorption from the medium it is reduced to

dihydrolipoic acid intracellularly by various oxidoreductases.22 The reduced disulfhydryl compound can
act as a potent antioxidant through a variety of
mechanisms: it can regenerate glutathione-pools,
scavenge superoxide radicals, singlet oxygen, peroxyl-and hydroxyl-radicals and prevent lipid peroxidation by regenerating vitamin E and recycling vitamin
C. In the past years a-lipoic acid has been clinically
employed for the treatment of various ROS-mediated
diseases such as atherosclerosis, neurodegenerative
diseases or diabetic vasculopathies.23,24,25 As a-lipoic
acid also shows transition metal chelating capacities24
it could potentially interfere with DNA binding of
NF-kB in the nucleus thus inhibiting the transcription factor through redox-independent mechanisms.
However, Bierhaus et al.25 as well as Suzuki et al. 26
proved that the inhibitory capacity is not related to a
reaction in the nucleus but to a suppression of the
activating process already in the cytoplasm.
Furthermore, we investigated whether the production of superoxide or the dismutation product
hydroperoxide was specically induced by TNF-a
treatment in canine skin cells. With the photometrical
determination of SOD-inhibitable cytochrome c
reduction, no superoxide production could be demonstrated in canine keratinocytes using concentrations of 10150 ng mL71 TNF-a in contrast to a
signicant superoxide generation after LPS treatment. This method implies a diusion of intracellularly produced superoxides into the supernatant as
the molecular weight of cytochrome c (and SOD as
well) prohibits this reagent to diuse into the cells.
We therefore speculated that the amount of intracellularly generated superoxides was not high enough
for this extracellular detection method.
Thus, a second ultrastructural technique allowing
the precise localization of intracellular sites of
hydrogen peroxide accumulation, the dismutation
product of the superoxide radical, was employed.
This method was rst described to detect membrane
associated hydrogen peroxide production in neutrophilic granulocytes16 and plant tissues.27 Kausch et
al.28 used a combination of hydrogen peroxide
entrapment with cerium chloride and spectroscopic
cerium detection to identify oxidases in peroxisomes
of plant cells.
Taking into consideration that pre-incubation with
cerium chloride allows cerium to diuse freely into
the cell, the cerhydroperoxide precipitates should
form directly at the site of hydrogen peroxide
production. We therefore analysed four cellular
locations with potential superoxide/hydrogen peroxide generating capacities in respect to the presence of
TNF-a induced cerium precipitate formation. As
representative hydrogen peroxide accumulation was
shown in mitochondria, the endoplasmic reticulum,
the cytosol and to a lesser extent on the plasma
membrane in keratinocytes of the dog, one specic
site of TNF-a dependent superoxides or hydrogen
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 129137

237 DISC
134

H. B. K. Kohler et al.

6.0E3

(b)

Ce

M5

Ce

M4

Intensity LIN

4.5E3

3.0E3

1.5E3

0.0E0
850.0

875.0

900.0

925.0

eV

950.0
LIN

Figure 4. (a): Electron micrograph of an ultra thin section from a canine keratinocyte monolayer pre-incubated with 5 mM cerium chloride for
30 min and subsequently stimulated with 25 ng mL71 TNF-a for 30 min (60 000 6). Encircled areas indicate location of electron energy loss
spectroscopic (EELS) analysis for the detection of cerium precipitates in the mitochondria (M), endoplasmic reticulum (ER), plasma
membrane (Pl) and cytoplasm (Z).
(b): Representative electron energy loss spectrogram (EELS) for the element cerium showing characteristic increase of intensity at 883 eV
(CeM5) and 905 eV (CeM4) after subtraction of background activities in cases of positive ndings in the encircled areas shown in Figure 4 (a).
The x-axis delineates energy loss (DE) and the y-axis represents intensities measured as electron impulses per 0.5 s.

peroxide production is unlikely. Our ndings rather

suggest multiple production sites in the cell.
Irregular intercellular cerium precipitates, as noticeable in Fig. 4(a), were seen in all samples and
controls. These proved to be residual pure cerium
precipitates from the incubation medium as experiments with 100 mg mL71 catalase co-incubation in all
samples could not eliminate this precipitate.
The origin of superoxides, potentially involved in
TNF-a signalling pathways, has been a matter of
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 129137

dispute in the last decade: TNF-a mediated superoxide formation in the mitochondria and subsequent
dismutation to H2O2 by the mangan superoxide
dismutase has been implicated both in apoptosis and
NF-kB activation.29 In contrast, Meier et al.30,31
could attribute a TNF-a dependent superoxide
generation to a specic membrane bound NADPHoxidase in stimulated human broblasts. The existence of this specically regulated superoxide-producing enzyme could also be conrmed in chondrocytes

237 DISC
TNF-a dependent NF-kB activation in cultured canine keratinocytes
and cells of the glomus caroticum by Cross et al.32
Our results, however, clearly show that a sole
membrane-associated or mitochondrium-derived
superoxide production cannot be discerned in canine
skin cells.
The biomembrane may represent an unspecic
target for TNF-a associated signal transduction. A
TNF-a induced modulation of membrane integrity
could lead to unspecic activation of various
membrane-associated
redox-systems
producing
superoxides as a by-product. These could comprise
cytochromes, avines, ubiquinones, oxygenases and
oxido-reductases. The membrane coated peroxisomes
located in the cytoplasma possibly account for the
cerium precipitates found in this localization as an
identication of peroxisomes was not possible with
the applied spectroscopic technique.
These in vitro studies demonstrate a partial
involvement of superoxides and/or hydroperoxides
in TNF-a mediated NF-kB activation in canine
keratinocytes. The existence of a redox-sensitive
kinase leading to phosphorylation of the inhibitory
subunit of NF-kB preceeding its degradation has
been described.8
However, the fact that only partial reduction of
NF-kB activity was achieved with the antioxidant
a-lipoic acid, demonstrates the coexistence of
redox-independent TNF-a signalling pathways in
keratinocytes. Thommesen et al.33 have described
the involvement of the arachidonic cascade in the
TNF signal transduction pathway leading to the
activation of NF-kB in the human keratinocyte cell
line HaCaT.
As a total inactivation of NF-kB, blocking the
immune system in general, is not the intended aim of
therapy, potent natural antioxidants such as a-lipoic
acid may prove benecial to balancing an overactivation of NF-kB, possibly associated with inammatory and allergic cutaneous disease in the dog.
Moreover, in contrast to glucocorticoids, which not
only completely inhibit NF-kB34 but also show other
numerous side-eects, a-lipoic acid is a safe therapeutic agent. Owing to the multiple antioxidative
actions of a-lipoic acid, it should prove superior to
other natural antioxidants such as vitamin E, which
showed little to no eect in canine allergic disease.35
Thus, further investigations should be encouraged
to assess in vitro and clinical ecacy of potent
antioxidants in canine dermatology.
ACKNOWLEDGEMENTS
This study was supported by a research grant from
the Deutsche Forschungsgemeinschaft (DFG). We
also thank the H.W. Schaumann Stiftung, Hamburg,
for the generous support.
The assistence of H. Lehmann and U. Kunz in the
electronmicroscopical studies are gratefully acknowledged. Furthermore, we thank J. Knoop for the

135

assistance with the EMSA and S. Aydogdu for

helping with the tissue cultures.
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Resume La cytokine TNF-a joue un role important dans les reactions inammatoires et immunologiques
dans la peau chez le chien. Dans l'espoir d'une possibilite therapeutique, nous avons etudie le role du facteur
de transcription NF-kB et l'implication des derives oxygenes (ROS) dans l'activation par le TNF-a des
cultures de keratinocyte. Le traitement par le TNF-a a provoque une activation du NF-kB qui etait
partiellement inhibee par l'acide a-lipoique (un antioxydant). A l'aide d'un test de reduction par le cytochrome
c, nous n'avons pas detecte de production de superoxyde dans le surnageant des keratinocytes stimules par le
TNF-a. Cependant, une production intracellulaire de peroxide d'hydrogene dependant du TNF-a a ete
demontree par spectroscopie. Par spectroscopie, une formation signicative de peroxide d'hydrogene a ete
detectee dans les mitochondries, le reticulum endoplasmique, le cytoplasme et partiellement sur la membrane
plasmique des keratinocytes. Les ROS pourraient donc jouer un role important dans l'activation par le TNF-a
des NF-kB dans la peau du chien. Un traitement avec des antioxydants naturels modulant l'activation
exageree des NF-kB dans les inammations cutanees pourrait etre interessant. [Kohler, B. K., Huchzermeyer,
B., Martin, M., de Bruin, A., Meier B., Nolte I. TNF-a dependent NF-kB activation in cultured canine
keratinocytes is partly mediated by reactive oxygen species. (L'activation NF-kB dependante du TNF-a dans
les cultures de keratinocyte est partiellement modulee par les derives oxygenes.) Veterinary Dermatology 12:
129137.]
Resumen La citoquina TNF-a juega un papel principal en las reacciones inamatorias e inmunologicas de la
piel canina. Con respecto a una posible modulacion terapeutica, investigamos el papel de la transcripcion del
factor NF-k B y la implicacion de especies reactivas de ox geno (ROS) en senalizar las v as del TNF-a en
cultivos de queratinocitos caninos. El tratamiento con TNF-a resulto en la activacion de NF-kB, que se
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237 DISC
TNF-a dependent NF-kB activation in cultured canine keratinocytes
encontraba parcialmente inhibido por el antioxidante acido alipoico. Utilizando el test de reduccion de
citocromo c no se pudo detectar la produccion de superoxido en el sobrenadante de queratinocitos
estimulados con TNF-a. Sin embargo, la produccion intracelular de peroxido de hidrogeno intracelular
dependiente de TNF-a fue demostrada espectroscopicamente. Con espectroscopia de perdida de energ a de
electrones (EELS) se detecto una formacion signicativa de peroxido de hidrogeno en las mitocondrias, en el
ret culo endoplasmico, el citosol y parcialmente en la membrana plasmatica de queratinocitos. As , los ROS
posiblemente juegan un papel importante en la senalizacion de las v as del TNF-a llevando a la activacion por
NF-kB en la piel canina. Una terapia adyuvante con antioxidantes naturales potentes que module la sobreactivacion de NF-kB en la inamacion cutanea canina puede ser terapeuticamente beneciosa. [Kohler, B. K.,
Huchzermeyer, B., Martin, M., de Bruin, A., Meier B., Nolte I. TNF-a dependent NF-kB activation in cultured
canine keratinocytes is partly mediated by reactive oxygen species. (La activacion de NF-kB dependiente de
TNF-a en queratinocitos caninos cultivados se encuentra parcialmente mediada por especies reactivas de
ox geno.) Veterinary Dermatology 12: 129137.]
Zusammenfassung Das Zytokin TNF-a spielt eine massgebliche Rolle in Entzundungs-und
immunologischen Reaktionen der Hundehaut. Wir untersuchten die Rolle des Transkriptionsfaktors NFkB und die Beteiligung reaktiver Sauerstospezies (RSS) in TNF-a vermittelten Signalbahnen von caninen
kultivierten Keratinozyten bezuglich moglicher therapeutischer Beeinussung. Behandlung mit TNF-a
resultierte in Aktivierung von NF-kB und war teilweise durch das Antioxidans alipoische Saure gehemmt.
Mit dem Zytochrom C Reduktionstest konnte keine Superoxidproduktion im Supernatant der mit TNF-a
stimulierten Keratinozyten festgestellt werden. Allerdings wurde TNF-a abhangige intrazellulare
Hydrogenperoxidproduktion spektroskopisch demonstriert. Mittels Elektronverlustspektroskopie wurde
bedeutende Hydrogenperoxidbildung in den Mitochondrien, dem endoplasmatischen Retikulum, dem
Zytosol und teilweise auf der Plasmamembran der Keratinozyten festgestellt. Daher spielt RSS
moglicherweise eine wichtige Rolle in zur NF-kB Aktivierung fuhrenden TNF-a vermittelten Signalbahnen
in der Hundehaut. Unterstutzende Therapie mit naturlichen, starken, die exzessive NF-B Aktivierung in der
Entzundung der Hundehaut modulierenden Antioxidantien ist moglicherweise von therapeutischem Vorteil.
[Kohler, B. K., Huchzermeyer, B., Martin, M., de Bruin, A., Meier B., Nolte I. TNF-a dependent NF-kB
activation in cultured canine keratinocytes is partly mediated by reactive oxygen species. (TNF-a abhangige NFkB Aktivierung caniner kultivierter Keratinozyten ist teilweise durch reaktive Sauerstospezies vermittelt.)
Veterinary Dermatology 12: 129137.]

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