Veterinary Dermatology 2002, 13, 301 305

Blackwell Science, Ltd

Influence of vitamin E on mast cell mediator release

THOMAS GUECK,* JRG RUDOLPH ASCHENBACH and HERBERT FUHR MANN*
*Institute of Physiological Chemistry, and Institute of Physiology, Faculty of Veterinary Medicine, University
of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany
(Received 26 Febraury 2002; accepted 13 June 2002)

Abstract We investigated the influence of vitamin E on mediator activity and release in a canine mastocytoma
cell line (C2) as a model for canine atopic dermatitis. Cells were incubated without and with vitamin E (100 )
for 24 h. The histamine and prostaglandin D2 (PGD2) release as well as the chymase and tryptase activity were
measured. To stimulate the PGD2 and histamine release, cells were incubated with the wasp venom peptide mastoparan (50 ) for 30 or 45 min. Nonstimulated as well as mastoparan-stimulated histamine and PGD2 release
was reduced significantly in vitamin E-treated cells. The activity of chymase tended to decrease, but the tryptase
activity of C2 cells was not influenced by vitamin E. These results indicate that vitamin E decreased the production and release of inflammatory mediators in C2 cells, suggesting that vitamin E might have a possible beneficial
effect in inflammatory diseases.
Keywords: atopic dermatitis, chymase, histamine, mastoparan, prostaglandin, RRR--tocopherol, tryptase

INTRODUCTION
Dietary vitamin E supplementation is reported to alleviate the symptoms of inflammatory diseases.1 The
pathogenesis of atopic dermatitis (AD), an immunoglobulin (Ig)E-mediated hypersensitivity reaction, is
characterized by the release of inflammatory mediators
from mast cells. Effects of the preformed mast cell
mediator histamine are mediated through histamine
receptors (H1, H2). Stimulation of H1 on nerve cells
causes pruritus. However, stimulation of H 2 on
neutrophils, lymphocytes, and monocytes inhibits the
release of inflammatory mediators. The effect of histamine on blood vessels seems to be mediated via H1 and
H2, whereby it influences the vascular tone and permeability.2 It was hypothesized that in AD mast cells
have an enhanced releasability for histamine.3 Proteases
such as chymase and tryptase are also preformed mast
cell mediators. In AD they are involved in the generation of bioactive peptides and they take part in the
destruction of the extracellular matrix. The expression
of mast cell proteases seems to be dependent on the
microenvironment. Ricarelli et al.4 showed an inhibition of protease expression by vitamin E. Other mast
cell mediators, such as prostaglandins, are produced
and released immediately after stimulation. These de novo
synthesized prostaglandins, especially prostaglandin
D2 (PGD2), are known as pro-inflammatory mediators. PGD2 leads to peripheral vasodilatation,

Correspondence: Thomas Gueck, Institute of Physiological

Chemistry, Faculty of Veterinary Medicine, University of Leipzig, An
den Tierkliniken 1, 04103 Leipzig, Germany. Tel.: +49-341-9738107;
Fax: +49-341-9738119; E-mail: gueck@vetmed.uni-leipzig.de
2002 Blackwell Science Ltd

inhibits platelet aggregation and functions as a chemoattractant for neutrophils. The rate-limiting enzyme
in prostaglandin production (prostaglandin H synthase, PGHS) is regulated by reactive oxygen species
(ROS).5 The production and release of these mast cell
mediators therefore seems to be dependent on membrane composition and redox state.6 Vitamin E accumulates in biological membranes and stabilizes them.
Furthermore, mast cells are able to generate ROS in
response to different stimulants. It has been shown that
ROS generation induced during mast cell activation is
prevented by the antioxidant diphenyleneiodinium
leading to reduced release of histamine.7 Here we demonstrate that vitamin E as a natural antioxidant also is
involved in modulation of histamine release, prostaglandin D production and possibly chymase activity in
the well-characterized canine mastocytoma cell line
(C2).8

MATERIALS AND METHODS

All chemicals and reagents were obtained from SigmaAldrich (Taufkirchen, Germany) unless noted otherwise.
Cell culture
Canine mastocytoma cells (cell line C2) were seeded at
a density of 106 cells per mL in Iscoves medium (Biochrome KG, Berlin, Germany). To measure PGD2,
Dulbeccos modified Eagles medium/HAMs F12
50:50 (DEH; Biochrome KG) containing linoleic acid
(0.15 ) was used. Both media were supplemented
with 2 m glutamine, 25 m HEPES (pH 7.4), 1.6 m
histidine, 100 U mL1 penicillin, 100 g mL1 streptomycin and 10% FCS. C2 were serially passaged to
301

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T. Gueck et al.

obtain the required number of cells. To investigate the

influence of vitamin E, the growth medium was supplemented with 100 RRR--tocopherol (Serva, Heidelberg, Germany) using ethanol as a vehicle (0.1% v/v
final ethanol concentration). The growth medium of
controls was supplemented with the vehicle only. Cells
were incubated at 37 C in a humidified atmosphere
with 5% CO2. After 24 h, replicate aliquots were harvested to measure RRR--tocopherol, histamine,
PGD2, chymase and tryptase. RRR--tocopherol was
determined by HPLC.9
Histamine release
For studies of histamine content and release, cells were
washed three times in calciummagnesium-free
(CMF) Tyrodes buffer and then suspended in complete Tyrodes buffer containing 25 m HEPES and
0.1% BSA. We incubated 2 106 cells per mL with
the mast cell-degranulating peptide mastoparan (50 ;
Bachem, Heidelberg, Germany) at 37 C in polypropylene tubes. Spontaneous histamine release was
measured in samples incubated in the absence of
mastoparan. After 45 min, the reaction was stopped
on ice and the cells centrifuged at 450 g for 10 min.
Supernatants were separated and the pellets were
resuspended in PBS (pH 7.4). Both were kept on ice for
20 min with 3 m trichloroacetic acid and centrifuged
at 2000 g for 15 min. Histamine was extracted from
pellets and supernatants with a 50 m 2-ethylhexylhydrogenphosphate-heptane solution and converted
to ortho-phtaldialdehyde and measured by HPLC.10
Histamine release was calculated as the amount of
histamine in the supernatant expressed as percentage
of total histamine present in both supernatant and
pellet.
PGD2 release
To measure PGD2 release, cells were washed twice in
CMF Tyrodes and then suspended in HBSS containing 0.3% -globulin. We incubated 2 106 cells per
mL with mastoparan (50 ) at 37 C in polypropylene
tubes. Spontaneous PGD2 release was measured in
samples incubated in the absence of mastoparan. After
30 min, the reaction was stopped on ice and the cells
were centrifuged at 450 g for 10 min. Supernatants
were separated and held on ice until measured with a
PGD2 methoxime enzyme immunoassay (Cayman

Table 1. RRR--tocopherol content of C2 cells when cultured with

and without RRR--tocopherol
C2 cells

g -tocopherol per 107 cells

Without RRR--tocopherol
RRR--tocopherol (100 )

0.041 0.027a
3.27 1.09b

Data are shown as means SD of six different experiments.

Different letters indicate significant differences P < 0.0001.

Chemical Company, Ann Arbor, MI, USA). The procedure for the measurement was performed according
to the manufacturers instructions.
Chymase and tryptase activity
To measure tryptase and chymase activity, 2 107 cells
lysed with bis-2-hydroxyethylimino-trishydroxymethyl
methane (10 m; pH 6.1) containing NaCl (2 ) were
sonicated for 15 s. Tryptase and chymase activities
were measured spectrophotometrically with the substrate N-benzoyl-Phe-Val-Arg-p-nitroanilide and Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide, respectively.
The amount of nitroaniline released per unit of time,
monitored by the increase in absorbance at 394 nm,
was a measure of the catalytic activities of chymase and
tryptase.11,12
Statistics
Data are shown as means SD of six different experiments. Means were tested for differences between control and tocopherol treatment by the separate variance
t-test with = 0.05 ( statistical software,
version 3.0; 1994). P-values < 0.05 were assumed to
denote significant differences.

RESULTS
We observed effects of vitamin E on the canine
mastocytoma cell line C2. After 24 h of incubation
with 100 RRR--tocopherol, we found a marked
increase in the RRR--tocopherol content in C2 cells
(Table 1).
Pellets of RRR--tocopherol-treated C2 cells contained more histamine than pellets of untreated cells
(Table 2). However, the histamine content of the corresponding supernatants was reduced compared with

Table 2. Histamine content and release of C2 cells

Mastoparan
(50 )

Mastoparan (50 )
+ RRR-tocopherol (100 )

Influence of
mastoparan
(50 )

Influence of
+ RRR-tocopherol (100 )

Histamine

Control

Control
+ RRR-tocopherol (100 )

Pellet
(pg/cell)
Supernatant
(pg/cell)
Release
(%)

0.064 0.016

0.086 0.017

0.047 0.007

0.069 0.006

P = 0.0039

P = 0.0003

0.005 0.0005

0.004 0.0005

0.026 0.005

0.021 0.005

P < 0.0001

P = 0.046

7.9 1.2

5.0 0.3

35.2 3.5

22.5 2.3

P < 0.0001

P < 0.0001

Data are shown as means SD of six different experiments.

2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 301 305

nkatal/mg protein

Vitamin E and mediator activity

303

16
14
12
10
8
6
4
2
0

Chymase
Tryptase

0
Figure 1. Influence of RRR--tocopherol on spontaneous and
mastoparan-stimulated prostaglandin D 2 release of C2 cells
(measured as PGD2 methoxime). Data are shown as means SD
of six different experiments. Different letters indicate significant
differences at P < 0.05.

untreated controls (Table 2). Percentage of spontaneous

and mastoparan-stimulated histamine release was
reduced significantly in RRR--tocopherol-treated
cells (Table 2). The PGD2 production was measurable
only when C2 were cultured in DEH medium containing linoleic acid. In this case, supplementation with
RRR--tocopherol decreased mastoparan-stimulated
PGD2 release significantly (Fig. 1). Statistically, no
difference in the chymase (P = 0.07) and tryptase
activities (P = 0.11) were found between RRR-tocopherol-treated and untreated cells (Fig. 2).

DISCUSSION
Our results show that mastoparan induces histamine
release from C2 cells. The release of this mediator was
diminished by treatment with RRR--tocopherol,
both in unstimulated cells and cells stimulated with
mastoparan. Similar effects of vitamin E on histamine
release have been shown on rat mast cells.13 It is well
known that ROS are involved in the secretory process
of histamine release.14 Recently, it has been shown
that it is possible to diminish the histamine release of
stimulated mast cells by blocking ROS generation with
diphenyleneiodonium (DPI).7 The authors concluded
that ROS control the tyrosine phosphorylation of
adapter proteins like the pp125FAK and the FAKassociated protein (FAP, 77 kDa) which appear to be
essential for the degranulation process of mast cells.15
A reduction of ROS by DPI or, as in our experiments, by the main nonenzymatic antioxidant RRR-tocopherol could be the reason for the reduced
histamine release. In our investigations RRR-tocopherol increased the histamine content of C2 pellets
and decreased it in the supernatants in controls and in
mastoparan-stimulated cells. This result underlines
the effective chain-breaking antioxidant properties of
vitamin E in biological membranes, where it contributes to membrane stability.1

100
0
100
M RRR-alpha-tocopherol

Figure 2. Influence of RRR--tocopherol on chymase and tryptase

activity of C2 cells. Data are shown as means SD of six different
experiments. nkatal = nanokatal. One katal corresponds to the
transformation of one mole of substrate per second.

Mastoparan also increased PGD2 production in

C2 cells. The response was diminished by treatment
with RRR--tocopherol. Prostaglandin production
depends on the activity of the isoforms of the enzyme
prostaglandin H synthase (PGHS), the constitutively
expressed PGHS-1 and the inducible PGHS-2. The
PGHS activities are influenced by changes of cellular
hydroperoxide levels.16 Vitamin E decreases PGHS
activity directly by inhibiting the free-radical-induced
formation of hydroperoxy lipids and therefore the prostaglandin production. Vitamin E probably reduces
PGHS activity by decreasing nitric oxide (NO) production,17 which leads to lower production of peroxinitrite.
Peroxinitrite has been shown to increase the activity
of PGHS. However, vitamin C, which regenerates
vitamin E, did not enhance the inhibition of PGHS-2
induction by vitamin E.18 Therefore the effect of
vitamin E on PGHS-2 mRNA expression and protein
seems to be independent of the redox state. Meydani19
showed a reduction of prostaglandin E2 in mice after
feeding vitamin E. Our results underline the inhibitory
effect of vitamin E on prostaglandin production.
C2 cells express both tryptase and chymase similar
to connective tissue mast cells.20 However, in our experiments only the chymase activity tended to be
decreased by vitamin E. This effect could be the result
of a direct interaction of vitamin E with the enzyme.
The inhibitory effect of nonpeptidic ketones on chymase but not on tryptase at the substrate binding site of
chymase was described previously.21 Such interactions
are also conceivable with RRR--tocopherol and
chymase. Apart from this, RRR--tocopherol could
have an inhibitory effect on chymase expression. Azzi
et al.22 reviewed specific cellular responses to tocopherols and postulated a regulation of several genes via a
receptor protein for tocopherol, a tocopherol-sensitive
transcription factor or promoter element. In addition,
it cannot be excluded that the chymase activity
depends on the antioxidative cell status. Further investigations on the effects of RRR--tocopherol on the
activity of proteases in mast cells are necessary.
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T. Gueck et al.

In summary, it seems that the balance of oxidative and

antioxidative metabolism plays an important role in
mast cell mediator production and release. However, a
redox-independent effect of vitamin E on these processes cannot be excluded.
It should been noted that we worked with a canine
neoplastic cell line as a model for canine atopic dermatitis. The biochemical and functional variations
of mast cells from different species requires the use of
mast cells from the target species for investigations on
mast cell mediators.23 DeVinney & Gold8 have shown
that C2 cells resemble normal dog mast cells morphologically and functionally. Nevertheless, further examinations in mast cells from atopic dogs are necessary to
prove the effects of vitamin E in a dose-dependent
manner on mast cell mediator release.

ACKNOWLEDGEMENTS
We thank Professor W.M. Gold, University of California, San Francisco, for providing us with C2 cells. The
study was supported by the Gesellschaft zur Frderung
Kynologischer Forschung e.V. (GKF).
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3. Hill, P.B., Olivry, T. The ACVD task force on canine
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9. Fuhrmann, H., Sallmann, H.P. Phospholipid fatty acids

of brain and liver are modified by -tocopherol and dietary fat in growing chicks. British Journal of Nutrition
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1988; 952: 1429.
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mastocytoma tryptase. Affinity purification, characterization, and amino-terminal sequence. Archives of Biochemistry and Biophysics 1987; 258: 55563.
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mast cells: modulation by nitric oxide synthase inhibition. Journal of Pharmacology 1999; 128: 58590.
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Protein tyrosine phosphorylation as a mechanism of
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305

Rsum Nous avons tudi linfluence de la vitamine E sur lactivit et la liberation des mdiateurs par une
ligne cellulaire de mastocytome (C2) comme modle de dermatite atopique canine. Les cellules ont t incubes
avec ou sans vitamine E (100 M) pendant 24 heures. Les mesures concernaient la libration dhistamine et de
prostaglandine D2 (PGD2) ainsi que lactivit de la chymase et de la tryptase. Pour stimuler la libration de PGD2
et dhistamine, les cellules taient incubes avec le mastoparan du venin de gupe (50 M) pendant 30 ou 45 min.
Les librations dhistamine et de PGD2 taient significativement diminues aprs traitement par la vitamine E,
avec ou sans stimulation. Lactivit de la chymase avait tendance diminuer, mais lactivit de la tryptase ntait
pas influence par la vitamine E. Ces rsultats indiquent que la vitamine E diminue la production et la libration
des mdiateurs de linflammation par les cellules C2, ce qui suggre que la vitamine E puisse avoir un rle
bnfique dans les maladies inflammatoires.
Resumen Investigamos la influencia de la vitamina E en la actividad mediadora y liberadora en una lnea celular
de mastocitoma canino (C2), como modelo para la dermatitis atpica canina. Las clulas fueron incubadas sin
y con vitamina E (100 M) durante 24 horas. Se midieron la liberacin de histamina y prostaglandina D2 (PGD2)
as como la actividad de quimasa y triptasa. Para estimular la liberacin de PGD2 e histamina, las clulas fueron
incubadas con el pptido de veneno de avispa mastoparan (50 M) durante 30 o 45 min. La liberacin de
histamina y PGD2 no-estimulada as como estimulada por mastoparan fue significativamente reducida en
clulas tratadas con vitamina E. La actividad de la quimasa tendi a disminuir, pero la actividad de la triptasa
de clulas C2 no se vio influida por la vitamina E. Estos resultados indican que la vitamina E disminuy la
produccin y liberacin de mediadores de la inflamacin en clulas C2, sugiriendo que la vitamina E puede haber
tener un papel beneficioso en las enfermedades inflamatorias.
Zusammenfassung Wir untersuchten den Einfluss von Vitamin E auf Aktivitt und Freisetzung von Mediatoren
in einer Hundemastozytomzellinie (C2) als Modell fr atopische Dermatitis des Hundes. Zellen wurden mit und
ohne Vitamin E (100 M) fr 24 Stunden intubiert. Die Freisetzung von Histamin- und Prostaglandin D2 (PGD2)
sowie die Aktivitt von Chymase und Tryptase wurden gemessen. Um die Freisetzung von PGD2 und Histamin
zu stimulieren, wurden Zellen mit dem Wespenvenompeptid Mastoparan (50 M) fr 30 oder 45 Minuten inkubiert. Sowohl nicht-stimulierte wie auch Mastoparan-stimulierte Freisetzung von Histamin und PGD2 waren
in mit Vitamin E behandelten Zellen signifikant reduziert. Die Chymaseaktivitt tendierte zur Verminderung,
die Trypytaseaktivitt der C2 Zellen wurde durch Vitamin E nicht beeinflusst. Diese Resultate deuten darauf hin,
dass Vitamin E die Produktion und Freisetzung von Entzndungsmediatoren in C2 Zellen vermindert, was
darauf schliessen lsst, dass Vitamin E mglicherweise eine positive Wirkung auf mit Entzndung einhergehende
Erkrankungen haben knnte.

2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 301305