From the
Human eosinophils produce upon treatment with
5-oxo-eicosatetraenoic acid or
(5S,15S)-dihydroxyeicosatetraenoic acid a potent
eosinophil-chemotactic eicosanoid,
5-oxo-15-hydroxy-(6E,8Z,11Z,13E)-eicosatetraenoic
acid (5-oxo-15-HETE). 5-Oxo-15-HETE induces human eosinophil (Eo)
chemotaxis at nanomolar concentrations with an efficacy in vitro comparable to that seen for platelet activating factor. Comparison
of Eo chemotactic activities of several structurally related
eicosanoids with different substituents and/or double bound geometry
led to the conclusion that maximal potency and efficacy of
eosinophil-chemotactic and chemokinetic activity is present in
5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic
acid (5-oxo-ETE). The presence of a hydroxyl group at position C-15 is
not necessary for potent chemotactic activity, whereas a geometric
isomer having trans instead of cis double bond at
C-atom 8, as well as esterified 5-oxo-ETE usually show a
5-10-fold lower potency.
5-Oxo-eicosanoids elicit a
dose-dependent transient rise of intracellular Ca
Eosinophilic granulocytes (Eos)
A number of eosinophil attractants have been described in the past
including C5a (3) and platelet activating factor
(PAF)(4) , which were shown to be the most potent and efficient
(percentage of migrating cells) Eo chemotaxins in vitro.
Recently, a number of cytokines such as RANTES(5) , macrophage
inflammatory protein-1
Apart from these
polypeptide-like chemotaxins eicosanoids were also found to be Eo
attractants. Mono-HETEs were seen to possess chemotactic and
chemokinetic activity toward neutrophils and Eos at micromolar
concentration(11, 12, 13) . The diHETE
leukotriene B
Since it is well documented that human neutrophils produce
their own chemotactic lipid, which is LTB
Its chemical structure could not be solved
in these initial experiments. With the working hypothesis that
eosinophil chemotactic lipid is possibly formed in eosinophils via its
arachidonate-15-lipoxygenase, which is present in large amounts in
human Eos, arachidonate was incubated with the plant 15-lipoxygenase
soybean-lipoxygenase I. The most potent Eo chemotactic lipid present in
these incubates was
5-oxo-(15S)-hydroxy-(6E,8Z,11Z,13E)-eicosatetraenoic
acid (5-oxo-15-HETE)(19) .
Recently Powell et al.(20) have shown that 5-oxo-eicosanoids will be generated
via a selective (5S)-hydroxyeicosanoid-specific dehydrogenase
in human neutrophils. In addition, both, 5-oxo-15-HETE and 5-oxo-ETE
were found to be chemotactic factors for human neutrophils as well as
inducers of intracellular Ca
In the present study we further investigated the
cellular activation profile of 5-oxo-15-HETE in human eosinophils and
report about structural requirements for potent biologic activity in
these cells by comparison of several structurally related
oxo-eicosanoids using Eo chemotaxis assays in vitro and
[Ca
All compounds were
more than 98% pure as revealed by HPLC.
5-Oxo-15-hydroxy-(6E,8Z,11Z,13E)-ETE
was enzymatically synthesized from
5-oxo-(6E,8Z,11Z,14Z)-ETE.
Briefly 10 µg of 5-oxo-ETE, dissolved in 1 ml of 0.05 M Tris-HCl buffer, pH 7.4, were incubated for 60 min with
soybean-lipoxygenase type I-S, (600 units/ml) Sigma, Munich, F.R.G. at
room temperature. Thereafter, the solution was acidified to pH 6.0, and
the lipids were extracted using a Sep-Pak (C
Storage of the 8Z,11Z form of
5-oxo-15-HETE in methanol for several days at -30 °C led to
the formation of two additional peaks with characteristic UV maxima at
229/278 nm (eluting as the first peak from the RP-HPLC column) and an
UV maximum at 225/278 nm (eluting as major peak between peak 229/278
and the 8Z,11Z form).
The hypsochromic shift of 3
nm of the UV maximum at 281 nm together with the hypsochromic shift of
4 nm of the UV maximum at 229 nm in
5-oxo-15-(6E,8Z,11Z,13E)-HETE can
only be explained by the fact that peak 225/278 represents the
8E,11E form of 5-oxo-15-HETE(26) , whereas
peak 229/278 is identical with authentic
8E,11Z-isomer of 5-oxo-15-HETE.
Therefore, only
freshly purified material with defined UV spectra was used for
biological studies.
5-Oxo-15-hydroxy-(6E,8E,11Z,13E)-ETE
was synthesized with identical procedures except that as educt
synthetic 5-oxo-(6E,8E,11Z,14Z)-ETE
was used and incubated with soybean lipoxygenase. The resulting
5-oxo-15-hydroxy-(6E,8E,11Z,13E)-ETE
revealed a single peak upon RP-HPLC, eluting earlier from the column
than the 8-cis-isomer. Its UV spectrum showed absorbance
maxima at 229 and 278 nm (in ethanol).
Storage of this 5-oxo-15-HETE
for several days in methanol resulted in formation of exclusively the
8E,11E-isomer. Therefore, only freshly prepared
8E,11Z-isomer with the correct UV spectrum was used
for biological studies.
15-Oxo-5-hydroxy-(6E,8Z,11Z,13E)-ETE
was isolated from supernatants of activated neutrophils incubated with
synthetic
15-oxo-(5Z,8Z,11Z,13E)-ETE.
Briefly, purified human neutrophils (10
This peak was
purified to homogeneity by a second RP-HPLC step with an increasing
gradient of acetonitrile.
The purified product showed an UV spectrum
with maxima at 229 and 279 nm (in ethanol). GC-MS analyses of the
hydrogenated and derivatized lipid revealed the presence of double
oxidized arachidonic acid derivative (at C-atoms 5 and 15). Its
structure is
15-oxo-5-(6E,8Z,11Z,13E)-ETE.
5-Oxo-(6E,8Z,11Z,13E)-ETE-methylester
was prepared from the free acid by treatment with etherical
diazomethane for 60 s. The single product was purified by RP-HPLC and
was found to show the same UV spectrum as the free acid.
GC-MS
analyses of a hydrogenated and silylated derivative revealed that the
methylester is an arachidonic acid derivative and not an elongated
homoarachidonic acid product, which easily is formed by treatment of
double conjugated oxo compounds for longer time periods with
diazomethane.
The viability of the cells as measured
by trypan blue dye exclusion was always greater than 94%.
Briefly, blind well Boyden
chambers (Nuclepore GmbH, Tübingen, Germany) were filled with
samples at appropriate dilutions and covered with
polyvinyl-pyrrolidone-containing polycarbonate filters (pore size, 3
µm) (Nuclepore GmbH).
Human eosinophils were suspended in PBS
containing 0.9 mM CaCl
Boyden chambers were incubated
in a humidified atmosphere at 37 °C for 1.5 h. Thereafter, cells
remaining in the upper part of the chambers were removed, and migrated
cells present in the lower part of the Boyden chambers were lysed by
adding Triton X-100 (final concentration 0.1% (v/v).
For calculation of the number of migrated cells, known
amounts of eosinophils were lysed with Triton X-100 and incubated with
the
Chemotactic activity was
expressed as the number of cell equivalents, which were detected in the
lower part of the Boyden chambers.
The chemotactic index was
calculated by the following formula: chemotactic index (CI) =
stimulated migration/random migration.
For control in some
experiments migrated cells adhering to the lower part of a cellulose
nitrate filter (used instead of a polycarbonate filter) were fixed,
stained, and directly counted under a microscope as
described(24) .
In some cases eosinophil peroxidase
activity was determined. 100 µl of supernatants were incubated with
100 µl of 0.01 o-phenylendiamine in 0.05 M Tris-HCl buffer, pH 8.0, containing 0.001% (v/v) hydrogen peroxide
and 0.1% (v/v) Triton X-100 for 10 min at room temperature. The
enzymatic reaction was stopped by adding 100 µl of 2 M
H
Eosinophil cationic protein was measured with an
enzyme-linked immunosorbent assay (Pharmacia CAP System, ECP FEIA, Kabi
Pharmacia Diagnostica AB, Uppsala, Sweden).
Eosinophils (1.5-3
Experiments were performed at 37
°C using a Perkin Elmer fluorescence spectrophotometer equipped
with a magnetic stirrer. The excitation wavelengths were set at 333 and
373 nm and the emission wavelength at 500 nm.
Prior to the addition
of agonists, CaCl
The responses to the agonists were measured after
stabilization of the base-line fluorescence. F
A dissociation constant of 224 nM for the
Fura-2
Furthermore, the product was
found to be as active as authentic material in an eosinophil chemotaxis
assay system (data not shown).
When sonicated eosinophils were
incubated with (5S,15S)-DiHETE (1.6 µM)
in the presence of 1 mM NADP
Up
to 20% of the DiHETE were converted to 5-oxo-15-HETE.
As shown in Fig. 2,
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
was found to be the most potent Eo chemotactic 5-oxo-15-HETE.
A change
of the positions of the oxo group and the hydroxyl group, as seen in
15-oxo-5-(6E,8Z,11Z,13E)-HETE (Fig. 2), led to a shift of the dose-response curve to micromolar
concentrations.
In order to investigate whether the hydroxyl group
at position C-15 is important for Eo chemotactic activity, we tested
two 5-oxo-eicosanoids, which lack this hydroxyl group. 5-Oxo-ETE was
found to be as active as
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
in eliciting eosinophil chemotactic responses (Fig. 3). In
addition its 8-trans-isomer
5-oxo-(6E,8E,11Z,14Z)-ETE appeared
to be 4-fold less potent than
5-oxo-(6E,8Z,11Z,14Z)-ETE. The
15-oxo-(5Z,8Z,11Z,13E)-ETE,
however, lacked Eo chemotactic activity up to 4 µM concentration (Fig. 3), which is in accordance with the
finding that 15-HETE is also a poor Eo attractant (18).
Interestingly, esterification of the carboxylic group in
5-oxo-eicosanoids, as in the methylester of
5-oxo-(6E,8Z,11Z,14Z)-ETE, gave a
dose-response curve of Eo chemotaxis with an ED
In order to test the hypothesis that
the methylester of 5-oxo-ETE is hydrolyzed during the chemotaxis
experiment, human Eos (10
As shown in Fig. 5,
neither 5-oxo-eicosanoids,
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE,
or 5-oxo-(6E,8Z,11Z,14Z)-ETE showed
any significant release of granule constituents in cytochalasin
B-treated human eosinophils at concentrations up to 10 µM,
whereas both control agents fMLP and LTB
Desensitization was measured by adding the first stimulus
to the suspension of Fura-2-loaded Eos and subsequently adding the
second stimulus after reaching the blank value.
As shown in Fig. 7, 5-oxo-15-HETE is capable of desensitizing responses
toward 5-oxo-ETE and vice versa.
Interestingly the LTB
In the effect of different
stimuli as first and second challenge upon
[Ca
It is interesting to note, that 5-oxo-15-HETE as well
as 5-oxo-ETE as primary stimuli desensitize, apart from responses to
itself, the [Ca
There is no significant cross-desensitization between
5-oxo-eicosanoids and unrelated Eo chemotaxins such as LTB
Similar findings were observed
when the influence of a preincubation of Eos with different stimuli
upon subsequent chemotactic activation was investigated.
In
preliminary experiments optimal concentrations of various stimuli were
determined and used for the preincubation of the cells. After
subsequent washing pretreated cells were investigated in the Boyden
chamber chemotaxis system for responsiveness toward different stimuli.
Results of these experiments are shown in .
There is
more than 50% inhibition of the Eo chemotaxis toward 5-oxo-eicosanoids
as well as (5S)-HETE when cells were pretreated with
5-oxo-eicosanoids or (5S)-HETE. In addition pretreatment with
5-oxo-ETE seems to desensitize to a less degree responses toward PAF,
which might be nonspecific. In addition these experiments clearly show
that none of the chemotaxins used as primary stimuli (LTB
Human Eos are able to produce their own lipid-like
chemotaxins, when they are incubated with exogenous arachidonic acid.
The quantitatively dominating attractants are
(8S,15S)-DiHETE and
(5S,15S)-DiHETE(18) .
These eicosanoids,
however, elicit chemotactic responses in human eosinophils only at
micromolar concentrations(18) .
Apart from these eosinophils
produce a 100-fold more potent Eo chemotactic lipid, which structurally
shows similarities to 5-oxo-15-HETE (18, 19) and seems
to represent the 6E,8Z,
11Z,13E-isomer, when compared with authentic
geometric isomers.
Recently, it has been
shown that 5-oxo-eicosanoids are produced by human neutrophils upon
incubation with exogenous 5-HETE(20) . This reaction is
catalyzed by a (5S)-hydroxyeicosanoid-specific
dehydrogenase(21) . As yet it is speculative by which molecular
mechanism Eos produce 5-oxo-15-HETE. One possibility is the conversion
of 5-oxo-ETE by the Eo-derived 15-lipoxygenase. As shown in Fig. 1B formation of 5-oxo-15-HETE indeed does occur,
when human Eos are incubated with 5-oxo-ETE. Similar results were
obtained when 5-oxo-ETE was incubated with soybean-lipoxygenase I.
These findings are consistent with former results that
5-oxo-eicosanoids are used as substrates for
15-lipoxygenases(29) . In these investigations, however, the
products were not analyzed.
Preliminary studies in our laboratory
revealed that eosinophils, similar to neutrophils(30) , are
capable of producing 5-oxo-ETE, the precursor of 5-oxo-15-HETE, when
incubated with Ca-Ionophore and a phorbol ester.
In addition
Eos could use (5S,15S)-DiHETE, which is one of the
quantitatively dominating eicosanoids produced by Eos (31), as a
substrate for production of 5-oxo-15-HETE. Sonicated human eosinophils
indeed convert (5S,15S)-DiHETE but not
(5R,15S)-DiHETE (data not shown), to 5-oxo-15-HETE (Fig. 1B) indicating also that these cells contain a
(5S)-hydroxyeicosanoid-specific dehydrogenase activity, which
originally was found in neutrophils(20) .
5-Oxo-15-(6E,8Z,11Z,13E)-HETE is
unstable due to the presence of a conjugated oxo-diene system
containing cis-double bounds. Such compounds are known to
easily form enols with reforming oxo-dienes containing an all-trans structure, which represents the energetically most stable
geometric isomer(32) .
Therefore, this most likely
(nonenzymatic) conversion of 5-oxo-15-HETE into its geometric isomers
has to be taken into account when this family of substances will be
studied in in vitro investigations.
When synthetic
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
was analyzed for chemotactic activity for human Eos an ED
In order to determine which structure elements are
necessary for maximal Eo chemotactic activity, we tested several
structurally related molecules for Eo chemotactic activity.
The data
from Fig. 2and Fig. 3allowed us to conclude that the
structural basis for being a potent Eo chemotactic lipid seems to be
the presence of an oxo group at C-atom 5 together with conjugated trans, cis double bonds at the positions C-6
and C-8. Both,
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
and 5-oxo-(6E,8Z,11Z,14Z)-ETE are
equipotent and efficient Eo chemotaxins, whereas a change of the double
bound geometry at C-8 leads to a 5-fold increase of the ED
All-trans-5-oxo-15-HETE
(which can originate from the natural
5-oxo-(15S)-hydroxy-(6E,8Z,11Z,13E)-ETE
during storage or alkali treatment) elicited a similar dose-response
curve as found for
5-oxo-(15S)-(6E,8E,11Z,13E)-HETE
indicating that the change of the double bond configuration at C-11
from cis to trans is of minor importance for the
expression of Eo chemotactic activity.
An exchange of the positions
of the hydroxyl-group at C-15 and the oxo-group at C-5 in 5-oxo-15-HETE
toward 15-oxo-5-HETE led to a nearly complete loss of Eo chemotactic
activity (Fig. 3). Moreover, 15-oxo-ETE lacks Eo chemotactic
activity (Fig. 3).
5-Oxo-(6E,8Z,11Z,14Z)-ETE was
found to be as potent and efficient in Eo chemotaxis as 5-oxo-15-HETE (Fig. 3). Powell et al.(21) observed that
5-oxo-ETE was more potent than 5-oxo-15-HETE when neutrophil chemotaxis
was investigated. In our hands when neutrophils were tested instead of
Eos, both 5-oxo-15-HETE and 5-oxo-ETE were also equipotent in eliciting
neutrophil chemotaxis (data not shown). Technical reasons, i.e. possibly isomerization of 5-oxo-15-HETE, could be responsible for
these discrepancies.
These findings indicate that the presence of a
hydroxyl group at C-15 seems to be less important for biologic
activity.
In support with data obtained from geometric isomers of
5-oxo-15-HETE, the 8-trans form of 5-oxo-ETE showed 5-fold
lower specific activity as the 8-cis form (Fig. 3).
Similar to neutrophil chemotactic activity of LTB
The most important structural
requirement for potent Eo chemotactic activity seems to be the presence
of an oxo-group at C-5. When instead of an oxo-group a
(5S)-hydroxy-group was present, as in (5S)-HETE or
(5S,15S)-DiHETE, compared to 5-oxo-ETE nearly 50-fold
higher concentrations were necessary for half-maximal Eo chemotaxis.
Similar to other leukocyte attractants both 5-oxo-eicosanoids,
5-oxo-ETE as well as 5-oxo-15-HETE also induce chemokinesis in Eos,
although its efficacy is lower than under chemotaxis conditions
indicating that these lipids rather act as chemotactic factors than
chemokinetic factors in Eos.
The chemotactic and chemokinetic
effects of 5-oxo-eicosanoids in Eos are accompanied by raises of the
intracytoplasmatic Ca
All
5-oxo-eicosanoids showing Eo chemotactic activity elicited a
dose-dependent raise in [Ca
Both 5-oxo-eicosanoids tested elicited
a significant raise in [Ca
In the case of
neutrophils, highest [Ca
When we used human neutrophils instead of eosinophils we saw
significant and maximum raises of both
[Ca
The
concentration-response data in both, Eo chemotaxis and
[Ca
Since there is no cross-desensitization between 5-oxo-eicosanoids
and other unrelated chemotaxins such as fMLP, PAF, LTB
Due to the finding that
(5S)-HETE is able to desensitize both chemotactic responses
and [Ca
In contrast to other
eosinophil chemotaxins such as fMLP, LTB
It is noteworthy, that in
human monocytes we did not see any chemotactic responses toward either,
5-oxo-15-HETE or 5-oxo-ETE up to 10 µM (data not shown)
indicating that 5-oxo-eicosanoids appear to represent preferential
chemotaxins for eosinophils and neutrophils.
Owing to their effects
on human eosinophils and neutrophils the potent 5-oxo-eicosanoids must
be regarded in addition to some of the other well-characterized
leukocyte chemotaxins as possibly major mediators of effector cell
recruitment in different types of inflammation with eosinophil and
neutrophil tissue accumulation.
The actual involvement of
5-oxo-eicosanoids in eosinophil-dependent inflammatory reactions will
mainly depend upon the release of precursor forms of 5-oxo-eicosanoids,
particularly (5S)-HETE and (5S,15S)-diHETE
and conditions of the expression of the
(5S)-hydroxyeicosanoid-specific dehydrogenase. Additional
studies, now in progress, will show whether some of the potent Eo
chemotactic 5-oxo-eicosanoids are involved in diseases where
eosinophils could play a role, such as allergic asthma, allergic skin
reactions, as well as atopic dermatitis.
Results represent the mean ± S.D. of four experiments. The
change of intracellular Ca
We thank Prof. E. Christophers for support, D. Tiaden
for technical assistance, and G. Tams for editorial help.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
levels in human Eos, however, in contrast to some other Eo
chemotaxins do not induce degranulation. Cross-desensitization of
Ca
mobilization and Eo chemotaxis revealed that the
geometric isomers of 5-oxo-eicosanoids, 5(S)-HETE, and
(5S,15S)-diHETE cross-deactivate Eo responses to each
other, whereas other, unrelated stimuli did not interfere with these
lipids indicating that 5-oxo-eicosanoids activate Eos via a separate
receptor.
(
)are
believed to play an important role apart from parasite infections in
the development of chronic asthma(1) , late phase allergic
reactions, and atopic dermatitis(2) . Immigration of Eos into
inflammatory sites is thought to be mediated by chemotactic factors.
(6) , MCP-2(7) , and MCP-3 (7, 8) have been reported to be Eo chemotactic factors.
These cytokines have rather leukocyte-selective chemotactic properties
and belong to the chemokine
-family. In addition a polypeptide
termed LCF (9) was reported to be the most potent and efficient
Eo chemotaxin known so far(10) .
(14) is known to be 100-1000 times
more potent in eliciting chemotaxis in human neutrophils (15) and guinea pig Eos (16) when compared to the
Mono-HETEs. In human Eos, however, LTB
appears to have less
efficacy in activating Eo chemotaxis(17) , in contrast to guinea
pig Eos.
(14) , we
originally addressed the question whether human Eos similarly are
capable of producing their own chemoattractants. In order to test this
working hypothesis, a highly potent eicosanoid we tentatively termed
eosinophil chemotactic lipid could be isolated from Eo
supernatants(18) .
mobilization in these
cells(21) .
]
mobilization
experiments.
Eicosanoids
Synthetic (5S)-HETE,
(5S,15S)-diHETE,
5-oxo-(6E,8Z,11Z,14Z)-ETE,
5-oxo-(6E,8E,11Z,14Z)-ETE and
15-oxo-(5Z,8Z,11Z,13E)-ETE were
purchased from Cascade, London, United Kingdom.
) cartridge
(Waters, Milford, MA). The lipids, which have bound to the cartridge,
were stripped from the cartridge by the use of methanol. Thereafter, a
10-fold molar excess of triphenylphosphine was added, the mixture
incubated for 20 min at room temperature, and then separated by the use
of a RP-18-HPLC column and a gradient of increasing concentrations of
methanol. A single product was isolated which revealed a single peak in
different RP-HPLC systems and an UV-Spectrum with maxima at 229 and 281
nm (in ethanol). This product is identical with
5-oxo-15-hydroxy-(6E,8Z,11Z,13E)-ETE
by direct comparison of its retention time upon RP-HPLC and straight
phase-HPLC, its UV spectrum and its GC-MS properties (after
derivatization).
cells/ml),
prewarmed to 37 °C, were incubated with 100 µM
15-oxo-(5Z,8Z,11Z,13E)-ETE in the
presence of 10 µM Ca-Ionophore A 23187 (Sigma), 0.9 mM CaCl
, and 0.5 mM MgCl
for 10 min.
Thereafter, cells were spun down and supernatants were applied to a Sep
Pak RP-18 cartridge to separate lipids after acidification to pH 6.0.
After stripping the lipids from the cartridge with methanol and
reduction of the hydroperoxides with triphenylphosphine, these were
separated upon RP-HPLC using a methanol gradient. A single peak showing
the typical UV spectrum of an oxo-diene-hydroxy-diene-ETE with two
absorbance maxima near 230 and 280 nm was collected.
Isolation of Eosinophils and
Neutrophils
Eosinophils were isolated from human peripheral
blood as described previously(18, 19) . Briefly 100 ml
of freshly isolated veneous blood from healthy persons with mild
eosinophilia (3-10% Eos) was mixed with 20 ml of citrate-dextran
solution containing 65 mM citric acid, 85 mM sodium
citrate in 2% (w/v) Dextran T 70 (Pharmacia, Freiburg, Germany) and
centrifuged for 20 min at 500 g and room temperature.
Thereafter, the plasma and the buffy coat containing mononuclear cells
and platelets were sucked away and the remaining sediment was mixed
with the same volume of a gelatine solution (2, 5% (w/v) in 0.9%
saline) and incubated for 30 min at 37 °C. The granulocytes
containing supernatant were centrifuged, and the cell sediment was
washed three times with PBS containing 0.1% (w/v) bovine serum albumin.
Thereafter, the cell sediment was further separated by centrifugation
in a gradient of Percoll (Sigma) similarly formed according to the
method of Gärtner(22) . A discontinuous density gradient
consisting of six Percoll solutions of different density (1.080, 1.085,
1.088, 1.090, 1.092, and 1.095 g/ml) was prepared, and the granulocyte
preparation was layered onto the top of the gradient and centrifuged
(400
g, 20 min, 4 °C). Cells were collected from
each interface and contaminating erythrocytes were lysed by hypotonic
shock. Fractions containing more than 90% Eos were used as Eo
preparations. These were usually identical with
``normodense''-Eos(23) . Neutrophils with a purity of
more than 95% were collected from the interphase between 1.080 and
1.085 g/ml Percoll solution.
Conversion of 5-Oxo-ETE by Activated
Eosinophils
Eosinophil preparations (4 10
cells/ml), suspended in PBS, were treated with 5-oxo-ETE (final
concentration: 0.5-10 µM) for 5-60 min in the
presence of 10
M calcium ionophore A 23187
(Sigma). After centrifugation (500
g 10 min, 4 °C)
supernatants were isolated, and lipids were separated by RP-HPLC using
a methanol gradient.
Conversion of (5S,15S)-DiHETE by
Eosinophils
Eosinophil preparations (4-7 10
cells/ml, purity >95%) suspended in PBS, were disrupted by
ultrasound (150 watts, 6
5 s; ice-water chilling) and
subsequently centrifuged (800
g) for 15 min at 4
°C. After adding 5 mM CaCl
, 2.5 mM MgCl
and 1 mM NADP
ethanolic
(5S,15S)-DiHETE (5 µg in 10 µl) was injected
into the solution and incubated for 5-60 min. Thereafter, the
mixture was applied to a RP-HPLC column, and the lipids were separated
by a gradient of methanol.
Measurement of In Vitro Eosinophil Chemotaxis
Eo
chemotactic activity of the putative attractants was determined with an
established method as described previously (18, 24) and
successfully used for the purification of novel Eo
attractants(5, 19) .
, 0.5 mM MgCl
, and 1% (w/v) bovine serum albumin at a density
of 1
10
cells/ml, and 100 µl of the cell
suspension was added to each chamber.
-glucuronidase activity in the lysates was determined using p-nitro-phenyl-
-D-glucuronide (Sigma) as a
substrate.
-glucuronidase substrate.
Measurement of In Vitro Eosinophil
Chemokinesis
Chemokinesis experiments were performed as
described for chemotaxis experiments except that to the upper part of
the Boyden chamber (cell containing compartment) the same final
concentration of a stimulus was added, which was present in the lower
part of the Boyden chamber. Therefore, no gradients of chemotactic
factors were present in the Boyden chamber.
Degranulation
Measurement of degranulation of Eos
was performed similarly to a method we used for enzyme release in
neutrophils(24, 25) . Briefly Eos (5 10
cells/ml in PBS) were preincubated with cytochalasin B (5
µg/ml, Sigma) for 5 min. Thereafter, 100 µl of samples at
appropriate dilutions were added and incubated for an additional 30
min. After centrifugation, 100 µl of supernatants were incubated
for 18 h with 100 µl of 0.01 Mp-nitrophenyl-
-D-glucuronide in 0.1 M sodium acetate, pH 4. The enzymatic reaction was stopped by adding
200 µl of 0.4 M glycine buffer, pH 10.
-Glucuronidase release was expressed in percentage of a total
control, where Triton X-100 (final concentration, 0.1% (v/v)) was added
instead of the stimulus.
SO
, and the brownisch color was examined in
microtiter plates at 486 nm using a multichannel photometer (SLT 210,
Kontron).
Measurement of Intracellular Calcium
Levels
Cytosolic calcium levels were determined by measuring the
fluorescence of Fura-2 (Sigma) according to the methods of Grynkiewicz et al.(27) and Sozzani et al.(28) .
10
cells/ml) were
preincubated for 15 min at 37 °C in Ca
- and
Mg
-free PBS and thereafter loaded with Fura-2 by
incubation with its acetoxymethylester (1 µM) for a
further 30 min. The Fura-2-loaded cells were then washed twice in
Ca
- and Mg
-free PBS and resuspended
in the same medium to obtain a final cell density of 0.5-2
10
cells/ml.
and MgCl
were added to an
aliquot of the eosinophil suspension to give final concentrations of 1
mM each.
(fluorescence intensities of Ca
-saturated
Fura-2) was obtained by lysing the cells with reduced Triton X-100 (50
µM, Sigma) in the presence of Ca
(1
mM); F
(fluorescence intensities of
Ca
-free Fura-2) was determined by exposing the lysed
cells to EGTA (40 mM) after adjusting the pH to 7.2 with
sodium hydroxide.
Ca
complex was used to calculate
[Ca
]
(26) .
Eosinophils Convert Both (5S,15S)-DiHETE and 5-Oxo-ETE
to 5-Oxo-15-HETE
In order to test the working hypothesis whether
eosinophils are capable of synthesizing
5-oxo-15-(6E,8Z,11Z,13E)-HETE
directly from physiological substrates, these cells were incubated with
authenic synthetic
5-oxo-(6E,8Z,11Z,14Z)-ETE in the
presence of a Ca-Ionophore. RP-HPLC analyses of supernatants revealed
among others a major peak eluting at similar retention time as
authentic 5-oxo-15-HETE. Purification of this peak by straight phase
and reversed phase HPLC using different solvent systems revealed a
single peak showing the same characteristic UV spectrum (Fig. 1A) as seen for 5-oxo-15-HETE.
Figure 1:
A, RP-HPLC of an incubate of 5-oxo-ETE
with human eosinophils. Human Eos (4 10
cells/ml)
were incubated with
5-oxo-(6E,8Z,11Z,14Z)-ETE (3
µM) and Ca-Ionophore A 23187 (10 µM) for 15
min, and thereafter the mixture was separated by RP-18-HPLC as
described under ``Materials and Methods.'' The inset shows the UV spectrum of the product
5-oxo-(15S)-hydroxy-(6E,8Z,11Z,13E)-ETE,
which eluted at 21 min. B, RP-HPLC of an incubate of
(5S,15S)-DiHETE with Eo lysates. 10000
g supernatants of 7
10
human eosinophils were
incubated with 1.6 µM (5S,15S)-DiHETE in
the presence of 1 mM NADP
for 20 min, and
thereafter the mixtures were analyzed by RP-HPLC. A single product
eluting later than the educt was obtained, which shows the same
chromatographic properties and UV spectrum (inset) as
authentic
5-oxo-15-(6E,8Z,11Z,13E)-ETE.
GC-MS analyses
of its hydrogenated derivative revealed the presence of a
5,15-dioxygenated derivative of arachidic acid (data not shown)
supporting the idea that the product represents 5-oxo-15-HETE.
HPLC-analyses and coinjection experiments using different columns and
solvents revealed identity with synthetic
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE,
but not with its 8E- or
8E,11E-stereoisomers.
(Fig. 1B) a single product was formed, which by
its UV spectrum and its RP- as well as straight phase-HPLC retention
time is identical with authentic
5-oxo-15-(6E,8Z,11Z,13E)-ETE.
Structural Requirements for Potent Eosinophil Chemotactic
Activity of Oxo-eicosanoids
In order to determine whether
changes in the double bound geometry of 5-oxo-15-HETEs affect
chemotaxis, we were interested in determining specific chemotactic
activities of the geometric 5-oxo-15-HETE-isomers. All preparations
were tested for stability and identity upon HPLC after dissolving these
lipids in physiologic buffers and were immediately used for chemotaxis
assays. Under these conditions these lipids were found to be stable.
Figure 2:
Comparison of the eosinophil chemotactic
properties of different oxo-hydroxyeicosanoids.
5-Oxo-(15S)-(6E,8Z,11Z,13E)-HETE
([cirof]),
5-oxo-(15S)-(6E,8E,11Z,13E)-HETE
(),
5-oxo-(15S)-(6E,8E,11E,13E)-HETE
(
), and
15-oxo-(5S)-(6E,8Z,11Z,13E)-HETE
(
) were tested for eosinophil chemotactic activity at different
concentrations using the Boyden chamber assay system. 300 nM PAF (C
) was used as positive control. Data shown
represent the mean ± S.D. of eight separate experiments each
performed in duplicate.
5-Oxo-(15S)-(6E,8E,11Z,13E)-HETE
representing the 8-trans-isomer of the 5-oxo-15-HETE mentioned
above, gives a dose-response curve in Eo chemotaxis, which is shifted
to 5-fold higher doses, expressing similar efficacy (percentage of
input migrating cells) of eosinophil chemotaxis. The conversion product
of
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
after alkaline treatment (a peak absorbing at 225 and 278 nm, data not
shown), which represents the all-trans form of 5-oxo-15-HETE,
reveals a dose-response curve, which is similar to that of
5-oxo-(15S)-(6E,8E,11Z,13E)-HETE
indicating that a change of the double bound configuration at C-atom 11
appears to be less important for Eo chemotactic activity.
Figure 3:
Comparison of the eosinophil chemotactic
properties of different oxo lipids.
5-Oxo-(6E,8Z,11Z,14Z)-ETE (),
5-oxo-(6E,8Z,11Z,14Z)-ETE-methylester
(
), 5-oxo-(6E,8E,11Z,14Z)-ETE
(
), 15-oxo-(5Z,8Z,11Z,13E)-ETE
(
), and 5-oxo-hexanoic acid (
) were tested for eosinophil
chemotactic activity at different concentrations. 300 nM PAF
(C
) was used as positive control. The data represent the
mean ± S.D. for 12, 3, 10, 4, and 3 experiments each performed
in duplicate, respectively.
To determine
whether an oxo group at C-5 of a short chain carbonic acid is
sufficient for expression of Eo chemotactic activity, we tested whether
the saturated 5-oxo-carbonic acid 5-oxo-hexanoic acid is an active Eo
attractant in a Boyden chamber system. As shown in Fig. 3, no
significant Eo chemotactic activity up to 10M concentration of 5-oxo-hexanoic acid could be seen.
10-fold
higher than the free acid (Fig. 3) indicating also that the
presence of a free carboxylic group is necessary for expression of
potent Eo chemotactic activity.
cells/ml) were incubated for 1.5
h at 37 °C with 5-oxo-ETE-methylester (200 nM) and the
supernatants were analyzed for free 5-oxo-ETE. Neither 5-oxo-ETE nor
5-oxo-15-HETE was seen under these conditions (data not shown).
Therefore, it is unlikely that 5-oxo-ETE methylester is chemotactic for
human Eos due to in situ hydrolysis toward highly active
5-oxo-eicosatetraenoic acids.
5-Oxo-eicosanoids Elicit Chemokinetic Responses in Human
Eosinophils
When the most potent Eo chemotactic lipids
5-oxo-15-(6E,8Z,11Z,13E)-HETE as
well as 5-oxo-(6E,8Z,11Z,14Z)-ETE
were investigated for chemokinetic activity (absence of any chemotaxin
gradient in the Boyden chamber), it became clear that both
5-oxo-eicosanoids are chemokinetically active showing a dose-response
behavior (Fig. 4) with an ED near 8 and 20
nM, respectively. The efficacy of the chemokinetic stimulation
is less than that of chemotaxis (Fig. 4).
Figure 4:
Induction of eosinophil chemokinesis by
5-oxo-eicosanoids. A,
5-Oxo-(15S)-(6E,8Z,11Z,13E)-HETE
was tested for eosinophil chemokinetic activity at various doses (A, ). For comparison experiments were performed,
where this lipid was added to the upper part of the chambers (
)
(negative chemotaxin gradient) as well as to the lower part of the
chambers (
) (positive chemotaxin gradient). B,
5-oxo-(6E,8Z,11Z,14Z)-ETE was
tested for chemokinetic activity (
). In other experiments this
lipid was added to the upper part of the Boyden chambers (
) as
well as to the lower part (
). As positive control 300 nM PAF (C
) was used in the lower well of the chambers.
The mean ± S.D. of six experiments performed in duplicate is
shown.
5-Oxo-eicosanoids Do Not Degranulate Human
Eosinophils
Human Eos, pretreated with cytochalasin B, were
investigated for the induction of the liberation of granule components
by 5-oxo-eicosanoids. As control agents well known secretagogues such
as fMLP and LTB were used.
induced a
significant and dose-dependent liberation of the marker enzymes
-glucuronidase (Fig. 5) and eosinophil peroxidase (data not
shown).
Figure 5:
Eosinophil degranulation induced by
different stimuli. Human eosinophils were pretreated with cytochalasin
B and thereafter stimulated with various doses of fMLP (▾),
LTB (
),
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
(
), or
5-oxo-(6E,8Z,11Z,14Z)-ETE
(
). Data shown represent the percentage of
-glucuronidase
release (of a total control). Background release revealed to be 7% of
the total control. Note the absence of any significant release of
-glucuronidase by both 5-oxo-eicosanoids up to 10 µM concentration. Results shown represent a representative out of
five experiments with different donors.
The eosinophil cationic protein was determined by
radioimmunoassay in the supernatants of cytochalasin B-pretreated Eos:
no significant release could be observed with
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
and 5-oxo-(6E,8Z,11Z,14Z)-ETE (data
not shown), whereas 10M C5a and
10
M fMLP released 80 and 50% of a total
control, respectively.
5-Oxo-eicosanoids Raise Intracellular Ca
In order to investigate whether
5-oxo-eicosanoids raise [Ca in Human Eosinophils
]
in eosinophils, we added to a suspension of normodense human
eosinophils that had been loaded with the fluorescent dye Fura-2
increasing concentrations of the 5-oxo-eicosanoids 5-oxo-15-HETE (Fig. 6A) and 5-oxo-ETE (Fig. 6B),
respectively. Changes in the fluorescence were monitored using a
spectrofluorimeter. Addition of 5-oxo-eicosanoids corresponding to
final concentrations of 0.1 nM did not induce significant
changes, whereas in both cases 1.1 nM concentrations lead to a
significant raise in
[Ca
]
. Half-maximal
[Ca
]
raise was seen at
3 and 6 nM of the 5-oxo-eicosanoids, respectively.
Figure 6:
Effects of 5-oxo-eicosanoids on
intracellular calcium levels in human eosinophils. Increasing
concentrations of
5-oxo-(15S)-(6E,8Z,11Z,13E)-ETE (A) and
5-oxo-(6E,8Z,11Z,14Z)-ETE (B) were added successively to Fura-2-loaded human
eosinophils. Changes in the fluorescence were monitored as described
under ``Materials and Methods.'' The arrows indicate
the time point of addition of the stimulus as well as the final
stimulus concentration (in nM) in the cuvette. A
representative out of five experiments is
shown.
Interestingly, after addition of the eicosanoids at 400 nM final concentrations responses were nearly absent indicating
desensitization.
Desensitization of 5-Oxo-eicosanoid-dependent
[Ca
In
preliminary investigations concentrations of the stimuli were
determined, which were optimal for
[Ca]
Raises and Chemotactic
Responses in Human Eosinophils by Different Agonists
]
measurement and
usually were identical with doses exhibiting maximal Eo chemotactic
activity.
Figure 7:
Effects of different eicosanoids on the
changes in eosinophil cytosolic calcium levels induced by
5-oxo-15-HETE, 5-oxo-ETE, and LTB. Top left, human
eosinophils (10
cells/ml) were treated with
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
(100 nM) followed by
5-oxo-(6E,8Z,11Z,14Z)-ETE (100
nM). Top right, eosinophils were first treated with
5-oxo-(6E,8Z,11Z,14Z)-ETE and
thereafter with
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE. Bottom, human eosinophils were first stimulated with
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
(100 nM), followed by LTB
(40 nM) (left) or first stimulated with 40 nM LTB
followed by
5-oxo-(15S)-(6E,8Z,11Z,13E)-HETE
(100 nM) (right).
In both cases autologous
responses to the respective stimuli were also desensitized (data not
shown).
-dependent
[Ca
]
raise was not
affected by the use of 5-oxo-15-HETE as primary stimulus and vice versa (Fig. 7).
]
raises by human
Eos is shown.
]
raises in Eos elicited by 5(S)-HETE, whereas
LTB
does not desensitize 5-HETE-dependent
[Ca
]
mobilization in
Eos.
,
PAF, fMLP, and C5a ().
,
PAF, fMLP, and C5a) led to a more than 50% inhibition of chemotaxis
toward 5-oxo-eicosanoids and (5S)-HETE ().
(
)
(
)Therefore, one mechanism of 5-oxo-15-HETE formation
by Eos could be that via cellular 5-oxo-ETE synthesis.
of 10 nM as well as high efficacy (Fig. 2) was
found, which is comparable to that seen for PAF, the so far most active
and potent lipid-like Eo chemotaxin(4) . These findings indicate
that 5-oxo-15-HETE represents beside PAF one of the most potent and
effective Eo chemotactic lipids. In our hands only the complement split
product C5a showed a similar efficacy in eliciting Eo chemotactic
responses.
of Eo chemotactic activity.
, which is
drastically reduced in its 6-trans-stereoisomers(33) ,
potent Eo chemotactic activity of 5-oxo-eicosanoids seems to depend
upon the geometry of the double bound at C-8, although such a drastic
drop of activity as in the trans-isomers of LTB
is
not seen in 5-oxo-eicosanoids.
levels.
]
in human Eos (Fig. 6, ). Therefore,
5-oxo-eicosanoids have similar properties in activating Eos as found
for other chemotactic agonists such as PAF, C5a, RANTES, and
MCP-3(7, 8) .
]
at concentrations between 1 and 4 nM and highest
release at 100 nM, which are in the same order of the
concentrations necessary for eliciting significant and maximum Eo
chemotaxis responses ( Fig. 2and Fig. 3).
]
were reported to be at 4-5 nM, whereas 90
nM 5-oxo-ETE were necessary for maximum neutrophil
chemotaxis(21) . It has been suggested that the difference seen
in neutrophils may come from 5-oxo-ETE bound to the chemotaxis filters.
]
and chemotaxis at
doses near 2 and 100 nM, respectively (data not shown), which
are comparable to those found for human eosinophils.
]
mobilization and
the possibility of homologous desensitization in chemotaxis and
[Ca
]
would suggest
that a putative 5-oxo-ETE receptor could exist in human eosinophils.
, and
C5a (Tables I and II) and the chemokines RANTES and MCP-3 (data not
shown) in intracellular [Ca
]
mobilization as well as in Eo chemotaxis this putative
receptor seems to be distinct from receptors known for RANTES, fMLP,
PAF, C5a, and especially LTB
.
]
raises
elicited by 5-oxo-eicosanoids, not, however, LTB
(Fig. 7, Tables I and II), it seems not to act via the
LTB
receptor. These data could be interpreted by action of
5-HETE via the same receptor as 5-oxo-eicosanoids used for Eo
activation. This conclusion is supported by studies from Powell et
al.(21) in neutrophils, who found 5-oxo-ETE as an
efficient chemotaxin for these cells. In addition desensitization of
5-oxo-ETE stimulated [Ca
]
mobilization in neutrophils by (5S)-HETE, but not
LTB
, suggested that a putative 5-oxo-ETE receptor does
exist on human neutrophils(27) .
or C5a 5-oxo-ETE
as well as 5-oxo-15-HETE surprisingly did not elicit any significant
release of lysosomal enzymes or eosinophil cationic protein, when these
cells were pretreated with cytochalasin B. This may point toward
different signal transduction pathways in eosinophils, when these cells
are stimulated with 5-oxo-eicosanoids.
Table: Effects of different stimuli upon changes in
eosinophil cytosolic calcium levels induced by different agonists
after a second challenge
is shown as the percentage of the stimulus dependent mean (n = 3) of [Ca
]
rise. Purity of eosinophils was higher than 95%.
Table: 0p4in
ND, not determined.(119)
], intracellular free
calcium concentration; diHETE, dihydroxyeicosatetraenoic acid; ETE,
eicosatetraenoic acid; GC-MS, gas chromatography-mass spectrometry;
HETE, hydroxyeicosatetraenoic acid; LTB
, leukotriene
B
; MCP, monocyte chemotactic protein; PAF, platelet
activating factor; RANTES, regulated upon activation in normal T cells
expressed and secreted; RP-HPLC, reversed-phase high performance liquid
chromatography; 5-HETE,
(5S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic
acid; 5-oxo-ETE,
5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic
acid; 15-HETE,
(15S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic
acid; 15-oxo-ETE,
15-oxo-(5Z,8Z,11Z,13E)-eicosatetraenoic
acid; (5S,15S)-diHETE,
(5S,15S)-(6E,8Z,11Z,13E)-dihydroxyeicosatetraenoic
acid; (8S,15S)-diHETE,
(8S,15S)-(5Z,9E,11Z,13E)-dihydroxyeicosatetraenoic
acid; HPLC, high performance liquid chromatography; PBS,
phosphate-buffered saline; fMLP, formylmethionylleucylphenylalanine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.