©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
5-Oxo-eicosanoids Are Potent Eosinophil Chemotactic Factors
FUNCTIONAL CHARACTERIZATION AND STRUCTURAL REQUIREMENTS (*)

Uwe Schwenk , Jens-Michael Schröder (§)

From the (1)Department of Dermatology, Klinische Forschergruppe, University of Kiel, D-24105 Kiel, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Eosinophilic granulocytes (Eos)()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.

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(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) .

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(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.

Since it is well documented that human neutrophils produce their own chemotactic lipid, which is LTB(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) .

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 mobilization in these cells(21) .

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]mobilization experiments.


MATERIALS AND METHODS

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.

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) 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).

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 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.

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.

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.

The viability of the cells as measured by trypan blue dye exclusion was always greater than 94%.

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 10M 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) .

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, 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.

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). -glucuronidase activity in the lysates was determined using p-nitro-phenyl--D-glucuronide (Sigma) as a substrate.

For calculation of the number of migrated cells, known amounts of eosinophils were lysed with Triton X-100 and incubated with the -glucuronidase substrate.

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) .

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.

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 HSO, and the brownisch color was examined in microtiter plates at 486 nm using a multichannel photometer (SLT 210, Kontron).

Eosinophil cationic protein was measured with an enzyme-linked immunosorbent assay (Pharmacia CAP System, ECP FEIA, Kabi Pharmacia Diagnostica AB, Uppsala, Sweden).

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) .

Eosinophils (1.5-3 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.

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 and MgCl were added to an aliquot of the eosinophil suspension to give final concentrations of 1 mM each.

The responses to the agonists were measured after stabilization of the base-line fluorescence. F (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.

A dissociation constant of 224 nM for the Fura-2Ca complex was used to calculate [Ca](26) .


RESULTS

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.

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 (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.

Up to 20% of the DiHETE were converted to 5-oxo-15-HETE.

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.

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.


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.

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).


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.

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 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.

In order to test the hypothesis that the methylester of 5-oxo-ETE is hydrolyzed during the chemotaxis experiment, human Eos (10 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.

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 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 10M fMLP released 80 and 50% of a total control, respectively.

5-Oxo-eicosanoids Raise Intracellular Ca in Human Eosinophils

In order to investigate whether 5-oxo-eicosanoids raise [Ca] 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] Raises and Chemotactic Responses in Human Eosinophils by Different Agonists

In preliminary investigations concentrations of the stimuli were determined, which were optimal for [Ca] measurement and usually were identical with doses exhibiting maximal Eo chemotactic activity.

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.


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).

Interestingly the LTB-dependent [Ca] raise was not affected by the use of 5-oxo-15-HETE as primary stimulus and vice versa (Fig. 7).

In the effect of different stimuli as first and second challenge upon [Ca] raises by human Eos is shown.

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] raises in Eos elicited by 5(S)-HETE, whereas LTB does not desensitize 5-HETE-dependent [Ca] mobilization in Eos.

There is no significant cross-desensitization between 5-oxo-eicosanoids and unrelated Eo chemotaxins such as LTB, PAF, fMLP, and C5a ().

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, PAF, fMLP, and C5a) led to a more than 50% inhibition of chemotaxis toward 5-oxo-eicosanoids and (5S)-HETE ().


DISCUSSION

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.()Therefore, one mechanism of 5-oxo-15-HETE formation by Eos could be that via cellular 5-oxo-ETE synthesis.

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 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.

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 of Eo chemotactic activity.

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, 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.

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 levels.

All 5-oxo-eicosanoids showing Eo chemotactic activity elicited a dose-dependent raise in [Ca] 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) .

Both 5-oxo-eicosanoids tested elicited a significant raise in [Ca] 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).

In the case of neutrophils, highest [Ca] 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.

When we used human neutrophils instead of eosinophils we saw significant and maximum raises of both [Ca]and chemotaxis at doses near 2 and 100 nM, respectively (data not shown), which are comparable to those found for human eosinophils.

The concentration-response data in both, Eo chemotaxis and [Ca] 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.

Since there is no cross-desensitization between 5-oxo-eicosanoids and other unrelated chemotaxins such as fMLP, PAF, LTB, 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.

Due to the finding that (5S)-HETE is able to desensitize both chemotactic responses and [Ca] 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) .

In contrast to other eosinophil chemotaxins such as fMLP, LTB 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.

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.

  
Table: Effects of different stimuli upon changes in eosinophil cytosolic calcium levels induced by different agonists

Results represent the mean ± S.D. of four experiments. The change of intracellular Ca 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)


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grant Ch 37/7-1 and in part by Bundesministerium für Forschung und Technologie Grant 01 KC 8907/4. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Dermatology, University of Kiel, Schittenhelmstr. 7, D-24105 Kiel, Germany. Tel.: 431-597-1536; Fax: 431-597-1509.

The abbreviations used are: Eos, eosinophilic granulocytes; [Ca], 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.

U. Schwenk, H. Pareigis, and J.-M. Schröder, unpublished results.

H. Pareigis and J.-M. Schröder, unpublished results.


ACKNOWLEDGEMENTS

We thank Prof. E. Christophers for support, D. Tiaden for technical assistance, and G. Tams for editorial help.


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