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(The FASEB Journal. 1999;13:143-154.)
© 1999 FASEB

Research Communications

Inverse regulation of lipid-peroxidizing and hydroperoxyl lipid-reducing enzymes by interleukins 4 and 13

Kerstin Schnurra, Astrid Borcherta and Hartmut Kuhna,1

a Institute of Biochemistry, University Clinics Charité, Humboldt University, 10115 Berlin, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
12/15-lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are opposite enzymes balancing the intracellular concentration of hydroperoxy lipids. We studied the regulation of both enzymes by interleukins 4 and 13 and found an inverse response. When human lung carcinoma cells A549 were cultured in vitro in the presence of these cytokines, an up-regulation of the 12/15-lipoxygenase and a down-regulation of the phospholipid hydroperoxide glutathione peroxidase were observed. A similar inverse regulation was found in human peripheral monocytes. Interleukin 4-treated A549 cells exhibited an impaired capability of reducing exogenous hydroperoxyl lipids and their levels of endogenous lipid hydroperoxides were markedly increased. To find out whether these regulatory processes also occur in vivo, arachidonic acid oxygenase and phospholipid hydroperoxide glutathione peroxidase activity was assayed in various tissues of transgenic mice that systemically overexpress interleukin 4. In lung, spleen, kidney, and heart, an increased arachidonic acid oxygenase activity was detected when transgenic mice were compared with inbred controls. The phospholipid hydroperoxide glutathione peroxidase activity was impaired in lung, liver, and spleen of the transgenic animals. These data indicate that lipid-peroxidizing and lipid peroxide-reducing enzymes are inversely regulated in various mammalian cells. Up-regulation of the 12/15-lipoxygenase and simultaneous down-regulation of the phospholipid hydroperoxide glutathione peroxidase may lead to an increased oxidizing potential, which is reflected by an augmented intracellular peroxide tone.—Schnurr, K., Borchert, A., Kuhn, H. Inverse regulation of lipid-peroxidizing and hydroperoxyl lipid-reducing enzymes by interleukins 4 and 13. FASEB J. 13, 143–154 (1999)


Key Words: cytokines • eicosanoids • carcinogenesis • atherosclerosis • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GLUTATHIONE PEROXIDASES constitute a family of anti-oxidative enzymes which are capable of reducing organic and inorganic hydroperoxides to the corresponding hydroxy compounds utilizing glutathione as reducing equivalent (1). They contain selenocysteine at their active site (1). Several types selenoperoxidases have been described (2) as translation products of different genes (3). Among glutathione peroxidases, only phospholipid hydroperoxide glutathione peroxidase (PH-GPx)2 is capable of reducing hydroperoxy fatty acids, which are esterified to biomembranes (4) or lipoproteins (5). Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that oxygenate polyenoic fatty acids to their corresponding hydroperoxy derivatives. According to the currently used nomenclature, mammalian LOXs are classified with respect to their positional specificity of arachidonic acid oxygenation in 5-LOXs, 12-LOXs, and 15-LOXs (6). 15-LOX may be subclassified into reticulocyte-type and epidermis-type enzymes (7) and there are three types of 12-LOXs; platelet-type, leukocyte-type, and epidermis-type enzymes (8). The murine leukocyte-type 12-LOX appears to be the functional equivalent of the human reticulocyte-type 15-LOX (9), and thus this enzyme subclass may be called 12/15-LOX. Several LOX subtypes such as 5-LOXs and the platelet-type 12-LOXs only accept free polyenoic fatty acids as substrate. In contrast, 12/15-LOX are capable of oxidizing phospholipids (10, 11), cholesterol esters (12), and even more complex lipid–protein assemblies such as biomembranes (13) and lipoproteins (12). These enzymatic characteristics indicate that 12/15-LOXs and PH-GPx constitute opposite enzymes in the metabolism of complex lipid hydroperoxides.

The regulation of 12/15-LOXs has been studied in various cellular systems (14). When human peripheral monocytes (15, 16), human alveolar macrophages (17), human lung carcinoma cells A549 (18), and murine peritoneal macrophages (19) are cultured in the presence of interleukins 4 or 13 (IL-4 and -13), the 12/15-LOX is up-regulated on a pretranslational level. Unfortunately, no data are currently available as to the regulation of PH-GPx by interleukins. This lack of information and the functional relatedness of both enzymes prompted as to study the effect of IL-4 and IL-13 on the expression of PH-GPx in vitro and in vivo. The data presented here indicate that 12/15-LOXs and PH-GPx are inversely regulated by these cytokines and that the regulatory processes create an oxidizing environment inside the cells. These findings may be of pathophysiological importance for disorders associated with an up-regulation of oxidative metabolic events such as atherosclerosis, cancer, and inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The chemicals used were from the following sources: human AB serum, bovine serum albumin, NADPH, glutathione, glutathione reductase, microperoxidase (MP-11), linoleic acid, dilinoleoyl phosphatidylcholine, sodium selenite, Trypan blue solution, EDTA solution, penicillin-streptomycin solution, RPMI 1640 medium, peroxidase conjugated anti-rabbit immunoglobulin G (IgG), and peroxidase conjugated anti-guinea pig IgG from Sigma (Deisenhofen, Germany); phosphate-buffered saline (PBS), and trypsin/versene from BioWhittaker (Heidelberg, Germany); Dulbeccos modified Eagle's medium (DMEM) and geneticin were from Gibco BRL (Eggenstein, Germany); recombinant human IL-4 and IL-13 from PBH (Hannover, Germany); Taq DNA polymerase from Promega (Heidelberg, Germany); AMV reverse transcriptase from Boehringer (Mannheim, Germany). All solvents used were of high-performance liquid chromatography (HPLC) grade and purchased from J. T. Baker (Deventer, The Netherlands).

For immunohistochemical staining, a polyclonal anti-rabbit-15-LOX antibody (FPLC-purified IgG fraction of a guinea pig) was used. This antibody was tested to cross-react with the human 15-LOX, the murine leukocyte-type 12-LOX, and the porcine leukocyte-type 12-LOX but not with the human platelet-type 12-LOX or human 5-LOX. For PH-GPx staining, a polyclonal antibody was used that was raised in rabbits against PH-GPx prepared to apparent homogeneity from pig hearts. This antibody, which does not cross-react with the classical glutathione peroxidases, was kindly provided by F. Ursini (Pavia, Italy).

Cell lines and culture conditions
The cell lines used were obtained from the following vendors: U937 (human promyelomonocytic cells) from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany); A549 (human lung carcinoma cells) from A.T.C.C. (Rockville, Md.). The 15-LOX transfected U937 cells (B8 cells) were a kind gift from A. Habenicht (Heidelberg, Germany).

All cell lines were cultured according to the recommendations of the vendors unless stated otherwise. Human lung carcinoma cells (A549) were maintained in DMEM containing 10% human AB serum, antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), and sodium selenite (50 nM). Human promyelomonocytic cell line (U937 and B8) were cultured in RPMI 1640 medium supplemented with 10% human AB serum, antibiotics, and sodium selenite. The 15-LOX transfectants (B8) were selected from untransfected cells by addition of geneticin (0.6 mg/ml). Human peripheral monocytes were isolated from buffy coats of healthy volunteers by density gradient centrifugation and adherence to plastic dishes (16), and were cultured in RPMI 1640 medium supplemented with 10% human AB serum, antibiotics, and sodium selenite for 5 days. Cell viability (usually greater than 98%) was determined by Trypan blue exclusion. After plating the cells at a density of about 2 x 106 cells/Petri dish, they were cultured for 24 h in the absence of exogenous cytokines and then IL-4 or IL-13 (670 pM) was added.

Immunohistochemistry
The immunoperoxidase method was used for immunohistochemical staining. The cells were spun down to microscopic slides (cytospin preparations) and fixed with ice-cold acetone. Afterward, the slides were incubated serially with the following solutions: 1) 10 min at room temperature with 0.3% hydrogen peroxide to inactivate endogenous peroxidase activity; 2) 10 min at room temperature with 3% bovine serum albumin to minimize nonspecific background staining; 3) 2 h at room temperature with the primary anti-15-LOX or anti-PH-GPx antibodies, which were diluted 1:100 for 15-LOX staining and 1:25 for PH-GPx staining; 4) 1 h at room temperature with the secondary antibody. For 12/15-LOX staining, a peroxidase conjugated goat anti-guinea pig IgG (diluted 1:500) was used, and for PH-GPx staining, a goat anti-rabbit IgG (diluted 1:500); 5) development with 4 mg diaminobenzidine in 10 ml 0.1% H2O2. Antibody solutions were prepared in PBS containing 0.2% Tween and 0.5% bovine serum albumin. After each step, the cytospin preparations were washed with PBS containing 0.2% Tween and 0.5% bovine serum albumin. All slides were counterstained with hematoxylin, embedded in artificial resin, and inspected under a light microscope (Olympus BH2-RFCA, Japan).

Enzyme activity assays
For measurements of cellular PH-GPx activity, the cells were trypsinized from the culture dishes, washed, and resuspended in sodium chloride/phosphate solution (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4), pH7.4. The cells were sonicated on ice (15 times for 0.5 s at 40 W with a microtip sonifier; Braun; Melsungen, Germany). The homogenate was centrifuged for 5 min at 20,000 x g and the supernatant was assayed for PH-GPx activity. The assay mixture constituted a 0.1 M Tris-HCl (pH7.4) containing 5 mM EDTA, 0.1% Triton X-100, 0.2 mM NADPH, 3 mM glutathione, and 1 U of glutathione reductase. As a source of PH-GPx, the 20,000 x g supernatant of the cell homogenate (1 mg protein/ml) was added. This mixture was preincubated at 37°C for 5 min and then the reaction was started by addition of 25 µM dilinoleoyl phosphatidylcholine hydroperoxide as substrate. The decrease in absorbance at 340 nm reflecting the consumptions of NADPH was followed at 37°C with a Shimadzu UV-2100 spectrophotometer. After 10 min, the reaction was stopped by the addition of 1 ml of ice-cold methanol and the lipids were extracted (20). The organic phase was recovered, the solvent was removed under vacuum, and the remaining lipids were reconstituted in 0.5 ml of methanol. Aliquots of this solution were analyzed by HPLC with postcolumn chemiluminescence detection to quantify the remaining substrate. Dilinoleoyl phosphatidylcholine hydroperoxide was prepared with the soybean LOX, as described earlier (21).

For measurement of LOX activity, cells were washed and resuspended in PBS at a concentration of 1 x 106 to 1 x 107 cells/ml. After addition of linoleic acid as substrate (0.1 mM final concentration), the cell suspension was sonicated on ice for 15 s. The homogenates were incubated for 20 min at 37°C and the reaction was stopped by the addition of 2.5 ml methanol. The hydroperoxyl linoleic acid derivatives formed were reduced to their corresponding alcohols by addition of sodium borohydride. After acidification to pH 3, the lipids were extracted (20) and aliquots of the extracts were injected to reverse phase-HPLC (RP-HPLC) for quantification of the LOX products. RP-HPLC was carried out on a Shimadzu instrument coupled with a Hewlett Packard diode array detector 1040A. Compounds were separated on a Nucleosil C-18 column (Macherey/Nagel, Düren, Germany; KS-system, 250 mm x 4 mm; 5 µm particle size) with a solvent system of methanol/water/acetic acid (85:15:0.1; vol/vol) and a flow rate of 1 ml/min. The absorbance at 235 nm (detection of conjugated dienes) was recorded and the chromatograms were quantified by peak areas. A calibration curve (5 point measurements) for 13-hydro(pero)xy linoleic acid [13-H(P)ODE] was established.

Quantification of endogenous hydroperoxyl lipids was carried out by HPLC with postcolumn chemiluminescence detection. Cells were washed in sodium chloride/phosphate solution and the lipids were extracted (20). The phospholipid classes were separated by HPLC on a Zorbax-NH2 column (250 mm x 4.6 mm; 5 µm particle size) with simultaneous detection of the absorbance at 235 nm and of the chemiluminescence. For HPLC separation of the phospholipid classes, a solvent system consisting of methanol/40 mM monobasic sodium phosphate solution (95:5; vol/vol) and a flow rate of 1 ml/min were used (22). The chemiluminescence was measured on-line with a Berthold HPLC radioactivity monitor LB 506 C-1 (EG&G Berthold, Bad Wildbad, Germany) after mixing the column effluent with microperoxidase/isoluminol solution (10 µg/ml microperoxidase-11 and 0.5 mM isoluminol in 30% methanol and 70% 100 mM sodium borate, pH 10). Chemiluminescence data were evaluated with the Winflow program (WinFlow-1.21) on a Compaq DeskPro computer.

Reverse transcriptase polymerase chain reaction
Total RNA was prepared by guanidinium thiocyanate-phenol-chloroform extraction (23) and stored as ethanol precipitates at -20°C. The cDNAs were obtained by reverse transcription using the avian myeloblastosis virus reverse transcriptase (AMV-RT) and oligo(dT) as primer. Polymerase chain reaction (PCR) was performed on a Biometra TRIO Thermoblock 2.51BB (Biometra, Göttingen, Germany). Total RNA (3 µg) was reverse transcribed at 37°C for 90 min in 45 µl of 50 mM Tris/HCl buffer, pH 8.2, containing 8 mM MgCl2, 30 mM KCl, 1 mM DTT, 100 µg/ml bovine serum albumin, 30 U of RNase inhibitor, 0.166 mM of each dNTP, 150 pmol of oligo(dT) primer, and 15 U of reverse transcriptase. To stop the reaction, samples were heated to 95°C for 10 min. For quantification of 15-LOX and PH-GPx mRNAs, we used two different methods. 1) The expression of the mRNAs of both enzymes was related to the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. As `housekeeping' enzyme, the GAPDH mRNA was not supposed to be regulated by these cytokines. For amplification of PH-GPx cDNA, the PCR primers 5'-TGTGCGCGCTCCATGCACGAGT-3' and 5'-AAATAGTGGGGCAGGTCCTTCTCT-3' were selected from a cDNA region displaying minimal sequence homology to the classical human glutathione peroxidase. The primers for the human 15-LOX and GAPDH were the following: for 15-LOX, 5'-GGGGCTGGCCGACCTCGCTATC-3' and 5'-TCCTGTGCGGGGCAGCTGGAGC-3'; for GAPDH, 5'-TCGGAGTCAACGGATTTGGTCGTA-3' and 5'-ATGGACTGTGGTCATGAGTCCTTC-3'. Two microliters of the reverse transcriptase reaction were used for amplification and the PCR mixture consisted of a 10 mM Tris/HCl buffer, pH 8.3, containing 50 mM KCl, 1.5 mM MgCl2 (for PH-GPx), or 2 mM MgCl2 (for 15-LOX and GAPDH), 6 pmol of primer sets, 0.1 mM of each dNTP, and 2.5 U of Taq DNA polymerase. After initial denaturation for 4 min at 94°C, 30 cycles of PCR were performed. Each cycle consisted of a denaturing period (40 s at 94°C), an annealing phase (60 s at 67°C for PH-GPx, 71°C for 15-LOX, or 66°C for GAPDH), and an extension period (120 s at 72°C). After the last cycle, all samples were incubated for additional 10 min at 72°C. PCR products were separated by 2% agarose gel electrophoresis, the DNA was stained with ethidium bromide, and the electropherograms were quantified densitometrically. 2) Competitive PCR using truncated cDNAs of the 15-LOX, PH-GPx, and GAPDH as external standards (24). These standards were prepared by deletion of some 100 bp in the region, which was amplified with our primer combinations. To 2 µl of the reverse transcriptase reaction were added known amounts of the standards (human GAPDH: cDNA with a 105 bp deletion, human 15-LOX: cDNA with a 117 bp deletion, human PH-GPx: cDNA with a 115 bp deletion in the PCR). The PCR conditions, primer sets, and densitometric evaluation were the same as described above. The densitometric data were corrected for molar equivalence and plotted on a log/log scale as the formation of standard-derived PCR products.

Transgenic animals
Transgenic mice that overexpress IL-4 under the control of MHC I regulatory elements (25) were kindly provided by Dr. A. Schimpl (Würzburg, Germany). The animals were fertile but showed signs of autoimmune disorders (26) and a marked hepato/splenomegaly. In contrast, the inbred nontransgenic controls with identical genetic background develop normally. Animals were killed by diethyl ether inhalation, the organs were removed and were shock-frozen in liquid nitrogen. After thawing, the organs (about 100–500 mg wet weight) were homogenized on ice in 6 ml of sodium chloride/phosphate solution, pH 7.4, using an Ultraturax microhomogenizer. The homogenates were centrifuged at 20,000 x g and aliquots of the 20,000 x g supernatant were used for PH-GPx activity assays in the standard spectrophotometric assay (see above). To measure arachidonic acid oxygenase activity, frozen pieces of the tissues (about 100–300 mg) were added to 1–1.5 ml of PBS containing 0.1 mM of arachidonic acid. After homogenization on ice, the mixture was incubated for 20 min at room temperature and the lipids were extracted with ethyl acetate under reducing conditions. The organic phase was recovered, the solvent was evaporated under vacuum, the remaining lipids were reconstituted in 200 µl of methanol, and aliquots were injected to RP-HPLC for quantification of the arachidonic acid oxygenase products. For selected organs (spleen, lung), the oxygenated arachidonic acid derivatives were prepared by RP-HPLC and further analyzed by normal phase and/or chiral phase HPLC for more detailed structural information. Normal phase HPLC was carried out on a Zorbax SIL column (DuPont, Wilmington, Del.; 250 x 4.6 mm, 5 µm particle size) with the solvent system n-hexane/2-propanol/acetic acid (100:2:0.1, vol/vol) and a flow rate of 1 ml/min. The enantiomer composition of the hydroxylated fatty acids was analyzed on a Chiracel OD column (Diacel Chem. Industries, Tokyo, Japan; 250 x 4.6 mm, 5 µm particle size) with the solvent system n-hexane/isopropanol/acetic acid (100:5:0.1, vol/vol). The absorbance at 235 nm was recorded in both cases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytokine-dependent regulation of 15-LOX and PH-GPx in human cells
Stimulation of A549 human lung carcinoma cells with IL-4 and IL-13 leads to a strong increase in linoleic acid oxygenase activity. From Fig. 1A, it can be seen that the cells kept in culture for 6 days in the absence of any cytokines were unable to convert exogenous linoleic acid to 13-H(P)ODE, the major product of the 15-LOX reaction. In contrast, when the cells were cultured in the presence of IL-4 or IL-13, large amounts of 13S-H(P)ODE were formed, indicating the expression of a 15-LOX. When cellular 15-LOX activity was assayed at different time points of IL-4 treatment (inset to Fig. 1A), a time-dependent increase was observed. It should be stressed, however, that short-term incubations (up to 5 h) of the cells with IL-4 or IL-13 did not lead to a significant induction of the 15-LOX as indicated by activity assays and quantitative RT-PCR (data not shown). These data suggest that the 15-LOX gene may not belong to the family of immediate early genes turned on by IL-4 and IL-13. In contrast to 15-LOX activity, a time-dependent decrease in the cellular PH-GPx activity was observed during IL-4 treatment ( Fig. 1B). After 48 h of incubation, PH-GPx activity was reduced to about 50%; after 144 h, a residual activity of 14% was measured in this experiment.



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  		Figure 1. Up-regulation of 15-LOX and concomitant down-regulation of PH-GPx activities in A549 cells. A549 cells were cultured for 6 days in the absence (control) or presence of 670 pM IL-4 or 670 pM IL-13. A) After harvesting, the cells were resuspended in 1 ml PBS, disintegrated by sonication, and incubated for 20 min at 37°C with 100 µM linoleic acid. RP-HPLC analysis was performed as described in Materials and Methods. Inset: Time dependence of 15-LOX activity. In this experiment the cells were incubated with IL-4 for the times indicated and the formation of 13-H(P)ODE was assayed by RP-HPLC. The chromatograms were quantified by peak areas. B) Spectrophotometric assay of cellular PH-GPx activity. A549 cells were cultured for 6 days in the absence (control) or presence of 670 pM IL-4 or 670 pM IL-13 and PH-GPx activity was assayed spectrophotometrically as described in Materials and Methods. Inset: Time course of PH-GPx activity during IL-4 treatment. The effect of inverse regulation of both enzymes has been confirmed on the level of enzyme activity in 15 independent experiments under various conditions.

To find out whether the increase in 15-LOX activity and the decrease of PH-GPx activity can also be detected on the level of enzyme protein, immunohistochemical staining was performed. Figure 2 illustrates that A549 cells cultured for 6 days in the absence of cytokines were stained 15-LOX negative. However, when the cells were treated with IL-4, most of the cells were stained 15-LOX positive. The expression level of the enzyme in some cells (about 10% of the entire population) was extremely high whereas other cells were stained weakly. Similar results were obtained when the cells were cultured in the presence of IL-13 (data not shown). When A549 cells were stained with our polyclonal anti-PH-GPx antibody, all cells were stained positive ( Fig. 2). In contrast, nonimmune control stainings (not shown) turned out to be negative. After treatment with IL-4 for 6 days, the level of PH-GPx expression appeared to be markedly reduced as suggested by the lower intensity of the immunohistochemical staining. Some cells (about 20%) even appeared PH-GPx negative under these conditions.



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  		Figure 2. Expression of 15-LOX and PH-GPx in A549 cells. A549 cells were cultured for 6 days in the absence or presence of 670 pM IL-4. Cells were harvested by trypsinization and cytospin preparations were immunostained using polyclonal anti-15-LOX and anti-PH-GPx antibodies, as described in Materials and Methods. Immunohistochemical stainings were carried out in 14 independent experiments. In addition, in six experiments immunoblots were performed that also confirmed the down-regulation of PH-GPx.

Quantitative RT-PCR was performed in order to find out whether the up- and down-regulation of 15-LOX and PH-GPx, respectively, can also be detected on the mRNA level. A549 cells cultured in the absence of cytokines did not show any 15-LOX signal after 30 PCR cycles ( Fig. 3, inset). After treatment with IL-4 or IL-13 strong bands at the expected size of 432 bp were observed, indicating expression of the 15-LOX mRNA. In contrast, PH-GPx mRNA was detected in A549 cells cultured for 6 days in the absence of cytokines, but the level of mRNA expression was reduced when IL-4 or IL-13 was present. For more precise quantification, the ratio of 15-LOX and GAPDH mRNAs on one hand and PH-GPx/GAPDH mRNAs ratio on the other hand were quantified ( Fig. 3, main part). Here, a time-dependent increase in the 15-LOX/GAPDH mRNA ratio and a decrease in PH-GPx/GAPDH mRNA ratio were observed during the treatment with IL-4. In this particular experiment the expression of PH-GPx mRNA was impaired by about 50%. A similar up-regulation of 15-LOX mRNA expression and down-regulation of PH-GPx mRNA expression were observed by competitive RT-PCR using truncated internal standards for 15-LOX, PH-GPx, and GAPDH (data not shown).



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  		Figure 3. Time courses of 15-LOX and PH-GPx mRNA expression. A549 cells were cultured in the presence of 670 pM IL-4 for the times indicated and reverse transcriptase PCR was performed as described in Materials and Methods. Cellular 15-LOX mRNA content (black columns) and PH-GPx mRNA content (gray columns) are expressed as percentage of the 15-LOX mRNA/GAPDH mRNA ratio (100% represents 1514 copies of 15-lipoxygenase mRNA/106 GAPDH transcripts) or PH-GPx mRNA/GAPDH mRNA ratio (100% represents 9450 copies of PH-GPx mRNA/106 GAPDH transcripts). Inset: Agarose gel electrophoresis (2%) of the 15-LOX and PH-GPx PCR products before and after treatment with cytokines. n.d., not detectable. RT-PCR of 15-LOX and PH-GPx was performed in 12 independent experiments (9 experiments with IL-4 stimulation, 3 experiments with IL-13 stimulation).

It has been reported before that the 15-LOX of human peripheral monocytes is also induced when the cells are cultured in the presence of IL-4 (16) or IL-13 (15). We repeated these experiments and assayed simultaneously the cellular activities of both 15-LOX and PH-GPx at different time points in IL-4 treatment. When human monocytes were cultured in the absence of IL-4 for 96 h, we were unable to detect 15-LOX activity (formation 0 nmol LOX products/mg protein x 15 min from exogenous linoleic acid). Under these conditions, a PH-GPx activity of 0.283 {Delta}A340nm/min x mg cell lysat protein was measured using the coupled optical test. However, when the cells were cultured in the presence of IL-4 for the same time period, a 15-LOX activity of 17.9 nmol LOX products/mg protein x 15 min was assayed, whereas PH-GPx activity was attenuated to 0.114 {Delta}A340nm/min x mg cell lysate protein. A similar up-regulation of 15-LOX and a concomitant down-regulation of PH-GPx was also observed after 72 and 120 h of IL-4 treatment and with monocyte preparations of three different donors.

15-LOX expression is not sufficient for down-regulation of PH-GPx
To answer the question of whether the down-regulation of PH-GPx expression may be a regulatory consequence of an augmented intracellular 15-LOX concentration, experiments with 15-LOX transfectants were performed. For the transfection studies, U937 cells were selected because they are unable to respond to IL-4 stimulation with an induction of the 15-LOX, although they express a functional IL-4 cell surface receptor (27). To characterize the 15-LOX pathway of the transfected cells, oxygenase activity with exogenous linoleic acid as substrate was measured. Whereas the nontransfected and mock-transfected cells were unable to oxygenate exogenous fatty acids (no formation of 13-H(P)ODE as indicated by HPLC analysis), the transfected cells formed between 5 and 10 nmol 13-H(P)ODE/mg cellular protein x min (data not shown). Figure 4 shows that nontransfected controls and 15-LOX transfected U937 cells do not differ with respect to their cellular PH-GPx activity, suggesting that expression of 15-LOX is not sufficient for down-regulation of PH-GPx. When both the transfected and nontransfected U937 cells were cultured in the presence of IL-4, an impaired PH-GPx activity was detected ( Table 1). Here again, there was no significant difference in the cellular PH-GPx activity when transfected and nontransfected U937 cells were compared. As observed before, 15-LOX activity with exogenous linoleic acid as substrate remained unchanged after IL-4 stimulation of the transfected U937 cells.



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  		Figure 4. PH-GPx activity of 15-LOX transfected and untransfected U937 cells. For measurements of the cellular PH-GPx activity, the cells were washed and resuspended in sodium chloride/phosphate solution. Cells were sonicated and the 20,000 x g supernatant was used for the assay. Two independent experiments were carried out under identical conditions; PH-GPx activity is expressed as mean ±SD. Similar results were obtained in two additional experiments that were performed under different conditions.


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  		Table 1. Cellular activity of 15-LOX and PH-GPx in 15-LOX transfected U937 cells and untransfected controls1tfa

These data suggest that down-regulation of the intracellular PH-GPx may not be a direct consequence of an augmented 15-LOX expression. Thus, PH-GPx down-regulation appears to be associated with the IL-4-induced intracellular signal transduction cascade.

IL-4 treatment of A549 cells leads to an augmented intracellular lipid peroxide level
An increased 15-LOX expression and a concomitant impaired cellular PH-GPx activity should lead to a higher level of endogenous hydroperoxyl lipids. To test this hypothesis, A549 cells were cultured in the presence of IL-4 for different periods and the endogenous hydroperoxyl lipids were quantified by HPLC with postcolumn chemiluminescence detection. For direct comparison, we calculated the relative hydroperoxyl lipid content of the IL-4-treated cells, which normalizes the amount of hydroperoxyl lipids detected after IL-4 treatment to that of the control incubations (no IL-4). After 72 h of IL-4 treatment ( Table 2), the endogenous level of hydroperoxyl lipids was twice as high as in the corresponding controls (72 h culture in the absence of IL-4). After 6 days, a 10 fold-increase was observed.


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  		Table 2. Levels of endogenous hydroperoxy lipids in IL-4-treated and untreated A549 cells2tfa

It has been suggested that LOXs may be catalytically silent under resting conditions, even though the enzyme is expressed at relatively high levels (28). This `cryptic enzyme' may be activated by stimulation of the cells with calcium ionophore or by disrupting cell integrity prior to activity assay. We found that the level of endogenous hydroperoxyl lipids after 72 h of IL-4 treatment was 6.7-fold higher than that of control cells (no IL-4) when the cells were treated with calcium ionophore 5 min before lipid extraction. After 144 h of IL-4 treatment a 20-fold increase was observed. These data indicated activation of the 15-LOX, which was induced by IL-4. Comparison of PH-GPx activity before and after ionophore treatment did not reveal major differences. Disruption of the cell integrity by sonication did not lead to increased levels of lipid hydroperoxides when compared with intact cells. However, under these conditions, too, the content of endogenous hydroperoxyl lipids was much higher after 144 h of IL-4 treatment as compared with a 72 h incubation period.

In additional experiments we addressed the question of whether IL-4 may also act pro-oxidative in cells that are not capable of expressing the 15-LOX. For this purpose, U937 cells were cultured for 5 days in the presence and absence of IL-4 and the lipid extracts were analyzed for hydroperoxides. We found that the extracts of both IL-4-treated and untreated cells contained only very small amounts (0.05 nmol/106 cells) of hydroperoxyl phospholipids. In contrast, IL-4-treated A549 cells contained 0.68 nmol hydroperoxide/106 cells. Thus, one may conclude that IL-4 treatment may not augment the oxidizing potential of U937 cells. Although PH-GPx is down-regulated, we found no evidence for the up-regulation of lipid-peroxidizing enzymes in these cells. Similar results were obtained when 15-LOX transfected U937 cells were analyzed. Only small amounts of hydroperoxides were detected in the membrane phospholipids of IL-4-treated (0.04 nmol/106 cells) and untreated cells (0.05 nmol/106 cells). These data suggset that although the cells contain 15-LOX (immunohistochemistry), the enzyme may not be capable of oxidizing the membrane lipids. This finding is in line with earlier observations suggesting that the transfected enzyme requires additional activation in order to act on endogenous substrates (unpublished data).

Summarizing these data, one may conclude that the regulation of the activity of the 15-LOX induced by IL-4 in A549 cells, on the one hand, and of the enzyme transfected into U937 cells appears to be different. It is well known that the intracellular 15-LOX activity can be regulated on posttranlational levels. Calcium-dependent membrane association, the intracellular peroxide tone, the presence of alternative LOX activators (29, 30), and the susceptibility of the membrane phospholipid have been identified as critical parameters. For the time being it remains to be investigated which of these factor may be responsible for the functional silence of the transfected LOX on endogenous substrates in U937 cells.

Studies of transgenic mice that overexpress IL-4
To find out whether these regulatory effects may also occur in vivo, we assayed arachidonate oxygenase activity and PH-GPx activity in various tissues of mice that systemically overexpress IL-4 under the control of MHC I regulatory elements. Several metabolic processes such as the LOX pathway, the cytochrome P-450 pathway, and possibly yet unidentified metabolic routes may contribute to the cellular arachidonic acid oxygenase activity. Table 3 reveals that in lung, spleen, heart, and perhaps the kidney, arachidonate oxygenase activity was higher in the transgenic mice when these animals were compared with inbred controls. When PH-GPx activity was assayed, an impaired activity was detected in spleen, lung, and perhaps liver, whereas heart and kidney did not show significant differences. These data suggest that an increased arachidonate oxygenase activity is not necessarily accompanied by impaired PH-GPx expression, suggesting tissue specific regulatory differences. We observed increased arachidonate oxygenase activity in kidney and heart, but PH-GPx was not significantly altered. On the other hand, we found strongly decreased PH-GPx activity in the liver of the transgenic animals, whereas arachidonate oxygenase activity was not significantly different. From Table 3it can also be seen that a particular striking increase in arachidonic acid oxygenase activity and a concomitant strong decrease in PH-GPx activity were observed in the spleen. To estimate which share the 12/15-LOX contributes to arachidonic acid oxygenation, more detailed structural analysis of the oxygenation products was performed. For this purpose, arachidonic acid oxygenation products were prepared by RP-HPLC with a solvent system allowing the separation of hydroxy arachidonic acid (HETE) positional isomers ( Fig. 5). It can be seen that the major arachidonic acid metabolite, which contained a conjugated diene chromophore (left inset), comigrated with an authentic standard of 12-HETE. This compound was prepared and further analyzed by chiral phase HPLC in order to quantify its enantiomer composition. As indicated by the right inset, the majority of the 12-HETE formed was in the S configuration; only trace amounts of 12R-HETE were detected. In contrast to the transgenic mice, only small amounts of 12-HETE were formed by the spleen of inbred controls. These data strongly suggest that the majority of the arachidonic acid oxygenation products formed by the spleen of IL-4-overexpressing mice are generated via the 12/15-LOX pathway. A similar pattern of specific 12-LOX products was detected in the lung (data not shown), suggesting that in this organ, too, the 12-LOX pathway is up-regulated in IL-4-overexpressing mice.


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  		Table 3. Arachidonate acid oxygenase activity and PH-GPx activities of various tissues of IL-4 overexpressing mice and of inbred controls3tfa



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  		Figure 5. Formation of specific 12/15-LOX products by the spleen of IL-4-overexpressing mice. For measurements of arachidonic acid oxygenase activity, 120 mg of spleen was homogenized in 1 ml of PBS containing 100 µM of arachidonic acid. After homogenization on ice the mixture was incubated for 20 min at room temperature. Lipid extraction and RP-HPLC analysis of the 15-LOX products were performed as described in Materials and Methods except that a solvent system consisted of methanol/water/acetic acid (75/24/0.1; by vol.). Left inset: UV spectrum of the major oxygenation product (12-HETE). Right Inset: Enantiomer composition of 12-HETE formed. The retention times of authentic standards are indicated by arrows. Similar chromatograms were obtained with two different mice.

In additional experiments, we attempted to compare the levels of endogenous hydroperoxyl lipids in various tissues of transgenic mice with the corresponding controls using HPLC with chemiluminescence detection. Unfortunately, large luminescence quenching effects were observed with the lipid extracts of the most interesting tissues (lung, spleen) so that the chromatograms could not be evaluated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Lipid peroxidation is considered to be a deleterious process because it may lead to a decomposition of biomembranes and, thus, to a loss of cellular and subcellular integrity (31). However, when lipid peroxidation proceeds in a controlled manner according to a biological program and when it is restricted to certain subcellular components, it may have beneficial effects for the cell and the entire organism. As an example of the beneficial effects of locally restricted and biologically controlled enzymatic lipid peroxidation, the breakdown of mitochondria during the maturation process of rabbit reticulocytes may be discussed (32, 33). It has been suggested that enzymatic oxidation of the membrane lipids may initiate the breakdown cascade of mitochondria, which proceeds according to a genetic program (33). Up- and down-regulation of 15-LOX expression appears to be a part of this program since the enzyme is expressed only within a narrow time window during erythropoiesis. Several elements of pretranslational (16), translational (34, 35), and posttranslational regulations (29, 30) of the cellular 15-LOX activity have been described in various cellular systems, but it remains to be investigated how these elements are connected to each other to form the regulatory network of 15-LOX expression. In contrast to the 12/15-LOXs, little information is available at this point on cytokine-mediated regulation of PH-GPx. The data presented here show for the first time that the expression of PH-GPx can be down regulated in vitro when human cells are cultured in the presence of IL-4 and IL-13. Since the expression of both PH-GPx protein and PH-GPx mRNA is impaired, the regulatory processes may proceed on pretranslational levels. For the time being it remains unclear whether IL-4 may also affect the specific activity of PH-GPx. Our experimental data cannot rule out that the cellular selenium metabolism may be influenced by this cytokine or that processes of posttranslational modification may be altered.

PH-GPx may be considered an enzymatic counterpart of the 12/15-LOX within the regulation of the intracellular concentration of hydroperoxyl lipids. Up-regulation of 15-LOX expression and a concomitant down-regulation of PH-GPx activity lead to an augmented cellular level of hydroperoxyl ester lipids; thus, these cytokines may increase the oxidizing potential of certain cells. Moreover, such changes may lead to alterations of the cellular redox status and, thus, to a differential expression of redox sensitive genes.

IL-4 and IL-13 are pleiotropic cytokines that have a broad range of biological activities (36). IL-4 plays a critical role in the pathogenesis of allergic disease since it is necessary for the isotype switching of B cells to produce IgE (37). In addition, IL-4 stimulates the growth and the function of lymphocytes, macrophages, and mast cells (38). It is difficult to speculate whether the up-regulation of the 15-LOX and the concomitant down-regulation of PH-GPx may be related to any of the known effects of IL-4 or IL-13. Experiments with specific LOX inhibitors and/or studies with transgenic animals that lack or overexpress either 12/15-LOXs and/or PH-GPx are required in order to sort out which role both enzymes may play in the cytokine-induced metabolic network. 12/15-LOX-deficient mice have already been created (39) and IL-4 treatment of peritoneal macrophages of these animals did not lead to a significant up-regulation of the cellular LOX activity. If these animals would be crossbred with IL-4-overexpressing mice (25, 26), more detailed information on the role of 15-LOX in the IL-4-induced signal transduction may be obtained.

12/15-LOXs have been well characterized with respect to their protein chemical and enzymatic properties (40) and the molecular biology of these enzymes has also been studied in detail (41–43). The enzymatic properties of PH-GPx have also been investigated (44, 45), but our knowledge of the molecular biology of this enzyme is rather limited. PH-GPx cDNAs have been cloned from pig blastocyst, pig hearts, human testis, rat brain, and murine testis (46–50), and there appears to be a high degree of amino acid identity among these species. In addition, the structure of the porcine PH-GPx gene, including the 5'-flanking region, was identified (47). However, little is known as to the tissue-specific expression of PH-GPx isoforms in various mammalian species.

The regulatory effects of IL-4 and IL-13 on 12/15-LOX and PH-GPx are not restricted to in vitro incubations, but also occur in vivo. This finding is of particular importance if one considers the fact that up-regulation of 15-LOX in vitro requires cytokine concentrations that are at least two orders of magnitude higher than the IL-4 plasma concentrations measured in humans (51). In a parallel study, we quantified the IL-4 plasma concentrations of 30 healthy volunteers and 70 patients suffering from atopic disorders by immunoassay and found peak levels of IL-4 in the range of 10 pg/ml plasma, which is in fair agreement with previously published data (51). Thus, in humans the IL-4 plasma concentration may not be high enough to induce the regulatory effects reported in vitro. However, the plasma level may not reflect the IL-4 concentrations in other body compartments. IL-4 is produced mainly by TH1 and TH2 subpopulations of CD4+ T-lymphocytes, but is also produced in mast cells (52). Thus, in organs of the lymphatic and hematopoietic systems, the IL-4 concentration could by much higher than in the plasma. In fact, when IL-4 plasma concentrations were assayed in the transgenic mice that overexpress the cytokine (25), no significant differences to the inbred control animals were observed. However, clear-cut IL-4-related effects on T cell subset distribution or on B cell hyperreactivity (25) have been reported in these animals. In addition, we found that 12/15-LOX activity in the spleen and lung was markedly increase in IL-4-overexpressing mice. In the spleen, this increase in 12/15-LOX expression was accompanied by a strong down-regulation of PH-GPx activity. Moreover, a strong decrease in PH-GPx activity was observed in the liver, although the arachidonic acid oxygenase activity was not altered in this organ. These data suggest that the IL-4 plasma concentration may not be of major relevance for IL-4-mediated effects in vivo.

In human peripheral monocytes IL-4 up-regulates the 15-LOXs and simultaneously down-regulates PH-GPx. This effect may be of pathophysiological importance for the pathogenesis of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) has been implicated in atherogenesis, and increasing the oxidative potential of monocytes may lead to enhancement of their LDL oxidizing capability. In fact, unspecific inhibitors of the 15-LOX pathway appear to inhibit LDL oxidation (53), and peritoneal macrophages prepared from mice that lack the expression of 12/15-LOX (12/15-LOX K.O. mice) exhibit impaired LDL oxidizing capabilities (54). We undertook LDL oxidation studies with IL-4-treated and untreated monocytes but did not find significant differences in the LDL oxidizing capabilities.

The intracellular mechanism of IL-4- and/or IL-13-induced up-regulation of 12/15-LOX and of the simultaneous down-regulation of PH-GPx remains to be investigated. Our in vitro and in vivo data suggest that although both enzymes are inversely regulated in A549 cells, in peripheral monocytes and in the spleen of IL-4-overexpressing mice there is no strong coupling of up- and down-regulation of both enzymes. For instance, we observed a down-regulation of PH-GPx in U937 cells and in the liver of the transgenic animals, but there was no concomitant up-regulation of the 12/15-LOX. In liver, PH-GPx was drastically down-regulated, but no up-regulation of 12/15-LOX was seen.

The intracellular signal transduction cascade induced by IL-4 and IL-13 involves activation of the JAK/STAT6 system (55). Recent experiments with STAT6-deficient mice (56) suggested an involvement of the STAT6 transcription factor in the up-regulation of 12/15-LOX expression. In the promoter region of various 12/15-LOXs, putative STAT6-responsive elements with the consensus sequence TTC NNN(N) GAA (57) are present; though their functionality has not been studied so far. On the other hand, the time course of up-regulation of 12/15-LOX expression, particularly the fact that an increase in steady-state concentrations of 15-LOX transcripts can be observed only after 24 h of incubation with IL-4 (18), suggests that the 12/15-LOX gene may not belong to the immediate early genes turned on by this cytokine. More work is needed to characterize the signal transduction cascade leading to the up-regulation of 12/15-LOXs and down-regulation of PH-GPx. For the up-regulation of the 15-LOX in human monocytes, preliminary data may suggest the involvement of a new transcription factor (58). Whether this protein also occurs in other cells and whether it is involved in the down-regulation of PH-GPx remain to be investigated.


   ACKNOWLEDGMENTS
 
The authors are indebted to Dr. F. Ursini (Pavia) and Dr. A. Habenicht (Heidelberg) for providing PH-GPx antibody and the transfected U937 cells, respectively. We would also like to thank Dr. A. Schimpl (Würzburg) for sending the transgenic IL-4-overexpressing mice.


   FOOTNOTES
 
1 Correspondence: Institut für Biochemie, Universitätsklinikum Charité, Humboldt Universität, Hessische Str. 3–4, 10115 Berlin, F.R. Germany. E-mail: hartmut.kuehn{at}rz.hu-berlin.de

2 Abbrevations: DMEM, Dulbecco's modified Eagle's medium; HPLC, high-performance liquid chromatography; 13-H(P)ODE, 13-hydro(pero)xy linoleic acid; Ig, immunoglobulin; PH-GPx, phospholipid hydroperoxide glutathione peroxidase; LOX, lipoxygenase; IL, interleukin; H(P)ODE, hydro(pero)xy octadecadienoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcriptase polymerase chain reaction; LDL, low density lipoprotein, PBS, phosphate-buffered saline.

Received for publication March 31, 1998. Revision received September 4, 1998.
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