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Endocrinology Vol. 148, No. 3 1313-1322
Copyright © 2007 by The Endocrine Society

The Role of 12/15-Lipoxygenase in the Expression of Interleukin-6 and Tumor Necrosis Factor-{alpha} in Macrophages

Yeshao Wen, Jiali Gu, Swarup K. Chakrabarti, Kevin Aylor, John Marshall, Yoshitaka Takahashi, Tanihiro Yoshimoto and Jerry L. Nadler

Diabetes and Hormone Center (Y.W., J.G., S.K.C., K.A., J.M., J.L.N.), University of Virginia, Charlottesville, Virginia 22908; Department of Nutritional Science (Y.T.), Okayama Prefectural University, Okayama 700-8558, Japan; and Department of Pharmacology and Internal Medicine (II) (T.Y.), Kanazawa University School of Medicine, Ishikawa 920-8641, Japan

Address all correspondence and requests for reprints to: Jerry L. Nadler, Diabetes and Hormone Center, University of Virginia, Charlottesville, Virginia 22908. E-mail: jln2n{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
12/15-lipoxygenase (12/15-LO) enzyme and products have been associated with inflammation and atherosclerosis. However, the mechanism of effects of the 12/15-LO products has not been fully clarified. To study the role of 12/15-LO in cytokine expression, experiments with direct additions of the12/15-LO products, 12(S)-hydroxyeicosa tetraenoic acid or 12(S)-hydroperoxyeicosa-5Z, 8Z, 10E, or 14Z-tetraenoic acid to macrophages were first carried out, and results showed that the 12/15-LO products stimulated mRNA and protein expression of IL-6 and TNF-{alpha} in a dose-dependent manner. In contrast, an inactive analogue of 12(S)-hydroxyeicosa tetraenoic acid had no effect. To further explore the role of endogenous 12/15-LO in cytokine expression, we used an in vitro and in vivo model to test the effect of 12/15-LO overexpression. The models included Plox-86 cells, a J774A.1 cell line that stably overexpresses leukocyte-type 12/15-LO and primary mouse peritoneal macrophages (MPMs) from 12/15-LO transgenic mice. The results showed a clear increase in IL-6 and TNF-{alpha} expression in Plox-86 cells and MPMs from 12/15-LO transgenic mice, compared with mock-transfected J774A.1 cells and MPMs from control C57BL6 mice. IL-1ß, IL-12, and monocyte chemoattractant protein (MCP)-1 mRNA were also increased in Plox-86 cells. These data clearly suggest a clear role of 12/15-LO pathway in cytokine production. We also demonstrated that signaling pathways including protein kinase C, p38 MAPK (p38), c-jun NH2-terminal kinase as well as nicotinamide adenine dinucleotide phosphate oxidase are important for 12-(S)-hydroxyeicosatetraenoic acid-induced increases in IL-6 and TNF-{alpha} gene expression. These results suggest a potentially important mechanism linking 12/15-LO activation to chronic inflammation and atherosclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MACROPHAGES ARE MONONUCLEAR phagocytes that reside within almost all tissues including adipose tissue, where they are identifiable as distinct populations with tissue-specific morphology, localization, and function (1). During the process of atherosclerosis, monocytes adhere to the endothelium and migrate into the intima, express scavenger receptors, and bind internalized lipoprotein particles resulting in the formation of foam cells (2). In obesity, adipose tissue contains an increased number of resident macrophages (3, 4). Macrophage accumulation in proportion to adipocyte size may increase the adipose tissue production of proinflammatory and acute-phase molecules and thereby contribute to the pathophysiological consequences of obesity (1, 3). These facts indicate that macrophages play an important role in a variety of diseases. When activated, macrophages release stereotypical profiles of cytokines and biological molecules such as nitric oxide TNF-{alpha}, IL-6, and IL-1 (5). TNF-{alpha} is a potent chemoattractant (6) and originates predominantly from residing mouse peritoneal macrophages (MPM) and mast cells (7). TNF-{alpha} induces leukocyte adhesion and degranulation, stimulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and enhances expression of IL-2 receptors and expression of E-selectin and intercellular adhesion molecules on the endothelium (8). TNF-{alpha} also stimulates expression of IL-1, IL-2, IL-6, and platelet-activating factor receptor (9). In addition, TNF-{alpha} decreases insulin sensitivity and increases lipolysis in adipocytes (10, 11). IL-6 also increase lipolysis and has been implicated in the hypertriglyceridemia and increased serum free fatty acid levels associated with obesity (12). Increased IL-6 signaling induces the expression of C-reactive protein and haptoglubin in liver (13). Recombinant IL-6 treatment increases atherosclerotic lesion size 5-fold (14). IL-6 also dose-dependently increases macrophage oxidative low-density lipoprotein (LDL) degradation and CD36 mRNA expression in vitro (15). These data clearly indicate that IL-6 and TNF-{alpha} are important pathogenetic factors associated with obesity, insulin resistance, and atherosclerosis. However, the factors regulating gene expression of these cytokines in macrophages have not been fully clarified.

There is evidence that MPM and murine macrophage cell lines contain a lipoxygenase (LO) that possesses both 12- and 15-LO activity (12/15-LO) (16). This form of LO is homologous to human 15-LO type 1 (17). 12/15-LO can react with arachidonic acid to form predominantly 12(S)-hydroperoxy-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid [12(S)-HPETE], 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and smaller quantities of 15(S)-HPETE. 12/15-LO can also catalyze stereoselective oxidation at position 13 over position 9 in linoleic acid to preferentially form 13-(S)-hydroperoxyoctadecadienoic acid (13-HPODE), which is the predominant oxidized fatty acid in LDL. 13-HPODE is esterified to cholesterol as cholesteryl-HPODE in LDL particles leading to the oxidative modification of LDL (18, 19, 20). Oxidation of LDL is believed to contribute to foam cell formation and lipid accumulation in lesions as well as the formation of necrotic areas in the core of the plaque (21). These facts suggest that the interaction of 12/15-LO with macrophages may play a role in atherogenesis. The in vivo role of 12/15-LO in atherogenesis has been shown using mouse models (17, 18). However, the role of 12/15-LO in regulation of cytokine expression in macrophages has not been studied.

In a recent publication, Funk and collaborators (22) have shown that macrophages from 12/15-LO–/– mice have a selective defect in lipopolysaccharide-induced IL-12 syntheses, suggesting a novel link of 12/15-LO to inflammatory cytokine production. In this study, we evaluated whether12/15-LO products can directly regulate expression of IL-6 and TNF-{alpha} in macrophages and the signaling mechanisms of 12/15-LO product action. Experiments were conducted with MPM from wild-type and 12/15-LO transgenic (Tg) mice as well as macrophage-like cells, J774A.1 and J774A.1 cells stably expressing leukocyte type 12/15-LO (19, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
J774A.1 cells and DMEM were purchased from the American Tissue Culture Collection (Manassas, VA). C57BL6 mice were from Jackson Laboratory (Bar Harbor, ME). 12/15-LO Tg mice were generated as previously described (24) and are maintained in the mouse core of the Cardiovascular Research Center at the University of Virginia. Thioglycollate was from Sigma (St. Louis, MO). 12(S)-HETE (>98%) and 12(S)-HPETE (>98%) were from Biomol (Plymouth Meeting, PA). RNeasy minikit was from QIAGEN (Valencia, CA). Mouse IL-6 and mouse TNF-{alpha} immunoassay kits were from R & D System (Minneapolis, MN). Culture medium, RPMI 1640, and Hanks’ balanced salt solution were from Invitrogen (Carlsbad, CA).

J774A.1 cell culture and depletion
J774A.1 cells were cultured following instructions from American Tissue Culture Collection. Plox-86 cells and mock-transfected J774A.1 cells were established by Dr. Tanihiro Yoshimoto (Kanazawa University School of Medicine, Kanazawa, Japan) (10). Cells were grown in DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate and 4.5 g/liter glucose, 10% of fetal bovine serum (FBS) and maintained in 5% CO2 incubator at 37 C. Before being treated with various agonists, cells were depleted in DMEM medium containing 0.5% FBS and 0.2% BSA for 18–24 h followed in serum-free DMEM containing 0.2% BSA for 2 h.

MPM isolation and culture
Six to eight male, 8- to 10-wk-old, C57BL6 or 12/15-LO Tg mice were injected ip with 2 ml 4% thioglycollate solution. Three days later, the ascites of these mice was collected and cells were spun down, resuspended in RPMI 1640 medium containing 10% FBS and 100 U/ml of penicillin/streptomycin (P/S), seeded, and cultured on 100-mm plates for 3 h in a 5% CO2 incubator at 37 C. The nonattached cells were carefully washed away by four times of washing with Hanks’ balanced salt solution and moderately tapping and flicking the plate. The attached cells were resuspended in the same medium as above, counted, and seeded on 60-mm plates with 5 x 106 cells/plate overnight. The second attached cells comprised more than 95% MPM. The MPMs were then depleted in RPMI 1640 medium containing 10% heat-inactive FBS and P/S for 24 h, and cells were then treated with various agonists in RPMI 1640 medium containing 10% heat-inactive FBS and P/S for a certain period of time. In experiments with 12-HETE or 12-HPETE treatment, cells were incubated another 2 h in RPMI 1640 medium containing 0.2% BSA. The animal protocol was approved by the Institutional Animal Safety Committee of the University of Virginia.

Measurement of 12(S)-HETE and 12/15-LO activity
12(S)-HETE was extracted from cells or culture-conditioned medium and measured using a specific RIA (16). Endogenous 12-LO activity was examined as reflected by the capacity of the cells to convert endogenous arachidonic acid (AA) to HETEs, which were measured using our HPLC assay (25) with modified fluorescent labeling (26). The amount of HETEs in the sample was determined by comparing the peak area of HETEs with that of an internal standard, 8-HETE.

RNA extraction and real-time PCR
Total RNA was extracted from cells using the RNeasy kit (QIAGEN), with deoxyribonuclease I treatment. The RNA was processed to cDNA synthesis using the SuperScript II reverse transcriptase and oligo d(T) (Invitrogen) according to the manufacturer’s protocol. The sequences of forward and reverse primers for target gene12-LO and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were selected based on published sequence data from the National Center for Biotechnology Information (Bethesda, MD) database and previous study (27).

Primers for cytokines (see Table 1Go) were synthesized from Integrated DNA technologies Inc. (Coralville, IA). Double-stranded DNA dye, SYBR Green I (Molecular Probes Inc., Eugene, OR) was used along with AmpliTaq Gold and 0.1 µM of each primer. All reactions were performed in triplicate in iCycler iQ real-time PCR detection system (Bio-Rad Laboratory, Hercules, CA). The real-time PCR was carried out in two steps: step 1, 94 C for 10 min, one cycle; step 2, 94 C for 15 sec, followed by 60 C for 60 sec, 45 cycles. GAPDH was used as an endogenous reference to correct for differences in the amount of total RNA added to the reaction and to compensate for different levels of inhibition during reverse transcription of RNA and during PCR. Data are calculated using the 2-{Delta}{Delta}CT method (28, 29) and are presented as fold induction of transcripts for target genes normalized to GAPDH in cells treated with 12(S)-HETE or inhibitors.


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  		TABLE 1. Forward and reverse nucleotide sequences of cytokine/chemokine gene primers

 
In our iCycler iQ real-time PCR detection system, heat dissociation of the PCR consistently produced a single peak for IL-6, TNF-{alpha}, and GAPDH (not shown), demonstrating that only one product was present in the PCR. In addition, the presence of a single product was further verified by gel electrophoresis (not shown).

mRNA expression levels can be measured by relative or absolute quantitative RT-PCR. Absolute quantitation relates the PCR signal to input copy number using a standard curve, whereas relative quantification measures the relative change in mRNA expression. In this study, cytokine mRNA at each point after treatments of 12(S)-HETE, or 12(S)-HPETE was expressed as a fold change in cytokine expression, which was calculated by the formula of 2-({Delta}{Delta}Ct). The results are accorded with that input cDNA copy number and Ct are inversely related, i.e. a sample that contains more copies of template will cross the threshold at an earlier cycle, compared with one containing fewer copies of template. Actually, several reports have shown that relative quantitation of gene expression using the 2-({Delta}{Delta}Ct) method correlates with the absolute gene quantitation obtained with standard curves (28, 29). Therefore, RNA purification method and {Delta}{Delta}Ct--quantitative real-time RT-PCR method used in this study are a valid way of analyzing changes in gene expression.

Cytokine protein release measurement using ELISA
Cells were seeded on 24-well plates with each well containing 1 x 106 cells of pooled MPM from six to eight C57BL6 mice or 2 x 105 cells of J774A.1 in 1 ml medium. After depletion for 24 h, cells were then treated with 12-HETE, 12-HPETE, or vehicle, DMSO, at 37 C for the indicated time in CO2 incubator. Conditioned medium was collected in a sequential fashion (e.g. 0–16, 16–24, 24–36, and 36–60 h) with change of 1 ml fresh medium containing fresh 12-HETE or 12-HPETE at each time point. The collected culture-conditioned medium was centrifuged to separate floating cells in medium and the supernatant was stored at –80 C. IL-6 and TNF-{alpha} protein levels were measured using specific mouse IL-6 or TNF-{alpha} ELISA kits (R & D Systems) following their instruction.

Kinase activity measurement using Western blot with specific phospho-antibodies
50 µg EtOH- or 12(S)-HETE-treated cellular protein extracts were resolved on 10% SDS-polyacrylamide gel. Electrophoresis was run under constant voltage conditions. The proteins on the gel were transferred onto Immun-Blot polyvinylidene difluoride membrane from Bio-Rad, which was incubated consecutively with specific phospho-antibody and antirabbit or antimouse Ig conjugated with horseradish protein and the protein bands were detected using enhanced chemiluminescence detection reagents from Amersham Biosciences Co. (Piscataway, NJ). The membrane was then stripped using Restore Western blot stripping buffer (Pierce Co., Rockford, IL). The membranes were incubated with ß-actin antibody and detected using enhanced chemiluminescence detection reagents. The result presented in figures was a representative of three experiments using three different batches of primary cultured J774A.1 cells treated with 1 nmol/liter 12(S)-HETE for different times.

Transient transfection in J774A.1 cells
Plasmids, dominant-negative MAPK kinase (dnMKK)-4 was generously provided by Dr. Michael Karin (University of California, San Diego, San Diego, CA) and dnMKK7 was generously provided by Dr. Tse-Hua Tan (Baylor College of Medicine, Houston, TX). The TransFast transfection reagent (Promega, Madison, WI), which is composed of the synthetic cationic lipid, (+)-N,N[bis(2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl]ammonium iodide and the natural lipid, DOPE, was used for DNA transfection followed the manufacturer’s instructions. Briefly, the J774A.1 cells were seeded the day before the transfection experiment at 2.5 x 106 cells per 100-mm dish. The next day, cells were washed with DMEM medium. A 12-µl TransFast transfection reagent per 8 µg plasmid mixture was prepared. After vortexed vigorously and incubated for 15 min at room temperature, the mixture was added to each dish. The cells were incubated for 1 h in 5% CO2 incubator before addition of 10 ml DMEM medium containing 10% FBS. Twenty-four hours later, fresh DMEM medium containing 10% FBS was changed and maintained for 24 h. Cells were then depleted in DMEM medium containing 0.2% BSA for 20–24 h, and the cells were ready to be treated with 12(S)-HETE.

NADPH oxidase activity measurement
NADPH oxidase-dependent superoxide production was measured by superoxide dismutase-inhibitable cytochrome c reduction as described elsewhere (30). J774A.1 cells were treated with vehicle, ethanol, or 12(S)-HETE for different times. After treatment, the J774A.1 cells were lysed in lysis buffer. One hundred micrograms of protein were distributed in 96-well flat-bottom culture plates (final volume 200 µl/well). Cytochrome c (500 µmol/liter) and NADPH (100 µmol/liter) were added in the presence or absence of superoxide dismutase (200 U/ml) and incubated at room temperature for 30 min. Cytochrome c reduction was measured by reading absorbance at 550 nm on a microplate reader.

Data analysis
The results are expressed as mean ± SEM from three batches of cultured MPM with each batch of MPM from six to eight mice as noted in each legend. For J774A.1 cells, results are expressed as mean ± SEM from three batches of cultured cells. For experiments running at one time period, the control and experimental samples are analyzed using the Student’s t test. These comparisons are based on a minimum of three experiments in triplicate per treatment. For multiple time periods of conditions, ANOVA was used with the appropriate correction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
12/15-LO products stimulate mRNA expression of IL-6 and TNF-{alpha} in J774A.1 cells and primary cultured macrophages
We first examined whether direct addition of 12/15-LO products can stimulate cytokine mRNA expression in J774A.1 cells. After cells were serum depleted, they were treated with 12(S)-HETE at 0.1 nmol/liter and 10 nmol/liter or 12(S)-HPETE at 1 pmol/liter and 100 pmol/liter for 2 or 4 h. The results are shown in Fig. 1Go. 12(S)-HETE dose-dependently increased IL-6 mRNA expression at 2 and 4 h, compared with vehicle alone. Interestingly, 12(S)-HPETE, the precursor of 12(S)-HETE, stimulated cytokine gene expression at 100-fold lower concentrations than 12(S)-HETE. 12(S)-HPETE increased IL-6 mRNA expression in a dose-dependent manner. 12(S)-HETE, at a concentration of 10 nmol/liter, significantly increased TNF-{alpha} mRNA expression at 2 and 4 h (Fig. 1AGo). 12(S)-HPETE also dose- and time-dependently activated TNF-{alpha} mRNA expression at concentrations of 1 or 10 pmol/liter (Fig. 1BGo).


Figure 1
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  		FIG. 1. The stimulatory effects of 12(S)-HETE and 12(S)-HPETE on IL-6 and TNF-{alpha} mRNA levels in J774A.1 cells and MPMs. Depleted cells were treated with 0.1 or 10 nmol/liter 12(S)-HETE or 1 or 100 pmol/liter 12(S)-HPETE for 2 or 4 h with the control treated with the same volume of vehicle, ethanol. Total cellular RNA was extracted, cDNA was synthesized, and aliquots of cDNA were used as template reactions containing either primers for IL-6, TNF-{alpha}, or GAPDH. cDNA derived from 25 ng of RNA was used for real-time RT-PCR. Data were presented as fold induction of transcripts for cytokine genes normalized to GAPDH in cells treated with 12(S)-HETE or 12(S)HPETE over that in untreated cells. Mean ± SE was calculated based on three individual experiments with samples from three batches of J774A.1 cells or primary cultured MPMs. In each experiment, each sample was run in three wells in PCR. IL-6 mRNA expression changes in J774A.1 cells are shown (A) and TNF-{alpha} mRNA expression changes in J774A.1 cells are shown (B). IL-6 mRNA expression changes in MPMs are shown (C), and TNF-{alpha} mRNA expression changes in MPM are shown (D). *, P < 0.05 vs. control; **, P < 0.01 vs. control; ***, P < 0.001 vs. control

 
We next conducted experiments examining the effects of 12/15-LO products on cytokine expression in MPM from C57BL6 mice. The data are shown in Fig. 1CGo. IL-6 mRNA expression was stimulated when cells were treated with 0.1 or 10 nmol/liter 12(S)-HETE for 2 h, compared with cells treated with vehicle alone. IL-6 expression reminded significantly increased for 4 h. IL-6 mRNA expression was also increased with 12(S)-HPETE treatment and levels remained significantly increased in cells treated with 100 pmol/liter 12(S)-HPETE for 4 h. mRNA expression of TNF-{alpha} was significantly increased when MPMs were treated with 12(S)-HETE or 12(S)-HPETE in a dose-dependent manner (Fig. 1DGo). In contrast, 12(R)-HETE, an inactive analog of 12(S)-HETE, did not stimulate IL-6 or TNF-{alpha} expression (data not shown). 12(S)-HETE and 12(S)-HPETE also increased the protein expression of proinflammatory cytokines of IL-6 and TNF-{alpha} in J774A.1 cells and MPMs. 12(S)-HETE (0.1 nmol/liter) increased IL-6 and TNF-{alpha} protein secretion from J774A.1 cells 2.0- (P < 0.01, n = 3) and 1.4-fold (P < 0.05, n = 3), whereas 1 pmol/liter 12(S)-HPETE increased them 1.7-fold (P < 0.01, n = 3) and 1.3-fold (P < 0.05, n = 3), respectively, over basal. Increases of cytokine protein secretion were also greater in MPMs. 12(S)-HETE (0.1 nmol/liter) increased IL-6 and TNF-{alpha} protein secretion 2.3- (P < 0.01, n = 3) and 1.4-fold (P < 0.05, n = 3) over basal, respectively. Whereas 1 pmol/liter 12(S)-HPETE increase IL-6 and TNF-{alpha} protein secretion from MPM 3.6- (P < 0.01, n = 3) and 1.9-fold (P < 0.01, n = 3) over basal.

Overexpression of 12/15-LO induces an increase in mRNA expression of IL-6 and TNF-{alpha} in J774A.1 cells
We next used Plox-86 cells, a cell line stably overexpressing porcine leukocyte-type 12/15-LO, to evaluate whether overexpression of 12/15-LO itself could directly activate cytokine gene expression. We first evaluated 12/15-LO mRNA expression and 12-LO activity in these cells. We designed primers that specifically target the porcine leukocyte-type 12/15-LO and not the murine leukocyte-type 12/15-LO. Figure 2AGo shows a clear increase of porcine leukocyte-type of 12-LO mRNA expression in Plox-86 cells, whereas no porcine type of 12-LO expression was seen in mock-transfected cells using RT-PCR assay. 12/15-LO activity was also examined as reflected by the capacity of cells to convert exogenous AA to12-HETE and 15-HETE using fluorescent HPLC. The maximal amount of 12-HETE produced by Plox-86 cells in the presence of exogenous AA substrate was significantly higher than that in mock-transfected cells (2.57 ± 0.33 ng/mg protein in mock cells vs. 554.8 ± 15 ng/mg protein in Plox-86 cells). Plox-86 cells in the presence of exogenous substrate also produced higher 15-HETE levels than that in mock-transfected cells (data not shown). The absolute values of the15-HETE increases were less than that for 12-HETE. In contrast, no 5-HETE could be detected in the Plox-86 cells. These data indicate that pig leukocyte-type 12-LO is active in the Plox-86 cells. The 12/15-LO enzymatic activity of Plox-86 with endogenous substrates, AA, was also examined using RIA. The results show that 12(S)-HETE release into the medium from Plox-86 cells was 3.6-fold (n = 3) greater than that from the mock-transfected cells (0.385 ± 0.015 ng/ml in mock cells vs. 1.37 ± 0.1 ng/ml in Plox-86 cells, P < 0.01, n = 3).


Figure 2
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  		FIG. 2. Stable overexpression of 12/15-LO increases IL-6 and TNF-{alpha} mRNA levels in Plox-86 macrophages. After depletion, total cellular RNA was extracted for real-time RT-PCR analysis. Data were calculated as described in Fig. 1Go with three experiments with three batches of Plox-86 cells paired with three batches of mock-transfected cells. PCR products were analyzed on 2% agarose gel. A representative agarose gel of 12/15-LO mRNA levels was shown (A). IL-6 mRNA expression changes are shown (B), and TNF-{alpha} mRNA expression changes are shown (C). *, P < 0.05 vs. in mock cells; **, P < 0.01 vs. in mock cells.

 
To explore whether overexpression of 12/15-LO can stimulate mRNA expression of IL-6 and TNF-{alpha}, we compared cytokine expression in Plox-86 cells with that in mock transfected cells. As shown in Fig. 2BGo, IL-6 expression was 7-fold (n = 3) higher in Plox-86 cells compared with that in mock-transfected cells. TNF-{alpha} mRNA expression was increased about 1.8-fold (n = 3) in Plox-86 cells compared with that in mock cells (Fig. 2CGo). Interestingly, overexpression of 12/15-LO also increased IL-1ß (2.3 ± 0.4-fold, P < 0.02, n = 3), IL-12 (2.0 ± 0.03-fold, P < 0.01, n = 3), and monocyte chemoattractant protein (MCP-1) (3.8 ± 0.3-fold, P < 0.01, n = 5) mRNA expression compared with that in mock cells. However, overexpression of 12/15-LO did not increase IL-18 mRNA expression. These results suggest a clear link between 12/15-LO overexpression and the expression of several proinflammatory cytokines.

To further explore the role of endogenous 12/15-LO in cytokine expression, MPM were isolated from 12/15-LO Tg mice. Consistent with the data in Plox-86 cells, the expression of IL-6 was 5.5-fold increased (5.50 ± 0.45-fold, P = 0.023, n = 3), and TNF-{alpha} mRNA expression was 3-fold increased (2.94 ± 0.50, P = 0.03, n = 3) in MPMs from 12/15-LO Tg mice compared with that in MPMs from wild-type C57BL6 mice.

These consistent results from cultured macrophage-like cells as well as from primary cultured MPMs isolated from mice clearly suggest that 12/15-LO products can directly stimulate cytokine gene and protein expression in macrophage cells. The next set of studies was designed to identify the major signaling mechanisms of 12-LO product action on cytokine expression.

Role of protein kinase C (PKC) in 12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression
We first examined whether 12-HETE increased PKC activity in J774A.1 cells. PKC activation was examined using a specific antiphosphorylated myristoylated alanine-rich C-kinase substrate (MARCKS) antibody. Figure 3AGo shows a time-dependent increase in PKC activity, illustrating that the phosphorylated MARCKS was increased at 5–10 min and reached a peak at 2 h with 1 nmol/liter of 12(S)-HETE treatment. PKC activity remained activated until 4 h. We then tested whether two structurally distinct PKC inhibitors, GF-109203X or calphostin C, attenuated 12-HETE effects on IL-6 and TNF-{alpha} mRNA expression. J774A.1 cells were pretreated with GF-109203X, calphostin C, or vehicle control DMSO for 30 min. J774A.1 cells were then treated with 1 nmol/liter 12(S)-HETE or the same volume of vehicle, EtOH, for 4 h in the presence of the inhibitor. Twenty-five nanomoles per liter GF-109203X or 25 nmol/liter calphostin C completely inhibited 12(S)-HETE-induced IL-6 (Fig. 3CGo) with no effect on basal IL-6 mRNA expression. In contrast, 100 nmol/liter GF-109203X or 100 nmol/liter calphostin C was needed to completely inhibit 12(S)-HETE-induced TNF-{alpha} (Fig. 3DGo) mRNA expression, 100 nmol/liter GF-109203X, or 100 nmol/liter calphostin C alone had no effect on TNF-{alpha} mRNA expression under EtOH conditions. These results suggest that PKC activation is important in 12(S)-HETE effects on IL-6 and TNF-{alpha} mRNA expression.


Figure 3
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  		FIG. 3. Effect of 12-LO products on PKC and p38 activity and effect of PKC and p38 inhibitors on 12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression. A, PKC and p38 activity was measured by detecting the density changes of phospho-proteins using a Western blot method. Fifty micrograms of J774A.1 cell protein were resolved on a 10% SDS-polyacrylamide gel and the proteins were detected by a specific phospho antibody. A, Time course of 12(S)-HETE stimulatory effect on the amount of phospho-MARCKS. B, Time course of 12(S)-HETE stimulatory effect on the amount of phospho-p38. C, The inhibitory effects of PKC and p38 inhibitors on 12(S)-HETE-induced IL-6 mRNA expression. J774A.1 cells were pretreated with calphostin C (25 nmol/liter) or GF-109203X (25 nmol/liter) for 30 min. The cells were then incubated in EtOH or 12(S)-HETE in the presence of inhibitors for an additional 4 h. D, The inhibitory effects of PKC and p38 inhibitors on 12(S)-HETE-induced TNF-{alpha} mRNA expression. J774A.1 cells were pretreated with calphostin C (100 nmol/liter) or GF-109203X (100 nmol/liter) for 30 min. The cells were then incubated in EtOH or 12(S)-HETE for an additional 4 h in the presence of inhibitors. Total cellular RNA was extracted for real-time RT-PCR analysis as in Fig. 1Go. Results are mean ± SE of three independent experiments run in triplicate. **, P < 0.01 vs. EtOH; #, P < 0.03 vs.12(S)-HETE; ##, P < 0.01 vs. 12(S)-HETE.

 
Role of P38 MAPK in 12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression
12(S)-HETE activates p38 MAPK activity as detected by Western blot using a specific antiphospho-p38 antibody (Fig. 3BGo). 12(S)-HETE-induced p38 activity occurred as early as at 5 min, declined at 10 and 30 min, and then increased again at 1 h. We also tested the effect of the relatively specific p38 MAPK inhibitor, SB 202190. 0.5 µmol/liter. SB 202190 almost completely inhibited 12(S)-HETE-induced IL-6 expression; 0.5 µmol/liter SB 202190 alone had no effect on basal IL-6 mRNA expression (Fig. 3CGo). However, we needed 2 µmol/liter SB 202190 to completely inhibit 12(S)-HETE-induced TNF-{alpha} mRNA expression (Fig. 3DGo). These results suggest that p38 MAPK is also an important signal for 12(S)-HETE action.

Role of JNK in12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression
12(S)-HETE addition as early as 5 min activates c-Jun amino terminal kinase (JNK) (Fig. 4AGo) To explore whether the JNK pathway is involved in 12(S)-HETE action on IL-6 and TNF-{alpha} gene expression, we tested the effect of dnMKK-4 or dnMKK-7, which specifically inhibits JNK activation. The control was cells transfected with the backbone of dnMKK plasmids. 12(S)-HETE activated IL-6 (Fig. 4BGo) and TNF-{alpha} (Fig. 4CGo) mRNA expression in the J774A.1 cells expressing the plasmid backbone. The transfection of dnMKK4 partially inhibited 12(S)-HETE-induced IL-6 (Fig. 4BGo) but no effect on TNF-{alpha} mRNA expression (Fig. 4CGo). However, the transfection of dnMKK7 completely suppressed 12(S)-induced IL-6 (Fig. 4BGo) and TNF-{alpha} (Fig. 4CGo) mRNA expression. These data suggest that the JNK pathway is involved in 12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression.


Figure 4
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  		FIG. 4. 12(S)-HETE activates JNK activity and effect of transfection of dnMKK4 or dnMKK7 on 12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression. A, Time course of 12(S)-HETE stimulatory effects on phospho-JNK protein detected by Western blot. The Western blot conditions were the same as in Fig. 3AGo. B, Inhibitory effects of transfection of dnMKK4 and dnMKK7 on 12(S)-HETE-induced IL-6 mRNA expression. C, Inhibitory effects of transfection of dnMKK4 and dnMKK7 on 12(S)-HETE-induced TNF-{alpha} mRNA expression. J774A.1 cells were transfected with dnMKK4 or dnMKK7 or the backbone of plasmids as described in Materials and Methods. The cells were then incubated in EtOH or 12(S)-HETE 1 nmol/liter for 4 h. Total cellular RNA was extracted for real-time RT-PCR analysis as in Fig. 1Go. Results are shown as mean ± SE of three experiments running in triplicate. *, P < 0.05 vs. EtOH; **, P < 0.01 vs. EtOH; #, P < 0.05 vs. 12(S)-HETE; ##, P < 0.01 vs. 12(S)-HETE.

 
NADPH oxidase activity implicates in 12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression
Four hours of treatment of 12(S)-HETE (1 nM) significantly increased NADPH oxidase activity 3.0-fold (2.93 ± 0.32-fold vs. EtOH, P < 0.01, n = 3) in J774A.1 cells. Pretreatment with 0.1 mM apocynin significantly inhibited 12-HETE-induced NADPH oxidase activity (P < 0.05 vs. 12-HETE). To determine whether oxidative stress plays a role in 12(S)-HETE-induced IL-6 and TNF-{alpha} mRNA expression, two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium chloride (DPI), were used. Figure 5AGo shows that 0.1 mM apocynin did not significantly alter 12(S)-HETE-induced IL-6 mRNA expression. However, when the concentration was increased to 1 mM, apocynin completely suppressed 12(S)-HETE-induced IL-6 mRNA expression but also suppressed the basal IL-6 expression. Therefore, we also tested another NADPH oxidase inhibitor, DPI. Figure 5BGo indicates that 0.1 µM significantly inhibited 12(S)-HETE-induced IL-6 mRNA expression (P = 0.014) without affecting basal IL-6 mRNA expression. Increasing DPI to 1 µM tended to increase 12(S)-HETE-induced as well as basal IL-6 mRNA. These two inhibitors also suppressed 12(S)-HETE-induced TNF-{alpha} mRNA expression. Figure 5CGo shows that 1 mM apocynin completely inhibited 12(S)-HETE-induced TNF-{alpha} mRNA expression. In addition, 0.1 µM DPI treatment completely inhibited 12(S)-HETE-induced TNF-{alpha} expression with no effect on basal TNF-{alpha} mRNA expression (Fig. 5DGo). When DPI concentration was increased to 1 µM, both 12(S)-HETE and basal TNF-{alpha} mRNA expression were suppressed (Fig. 5DGo).


Figure 5
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  		FIG. 5. An involvement of oxidative stress in 12(S)-HETE-activated IL-6 or TNF-{alpha} mRNA expression in J774A.1 cells. J774A.1 cells were pretreated with 0.1 or 1 mM apocynin or 0.1 or 1 µM DPI for 30 min, whereas control was treated with dimethylsulfoxide. Cells were then treated with EtOH or 12(S)-HETE in the absence or presence of 12(S)-HETE for 4 h. RNA extraction, cDNA synthesis, and RT-PCR were performed as described in Fig. 1Go. Effects of apocynin on 12(S)-HETE-activated IL-6 mRNA expression are shown (A). Effects of DPI on 12(S)-HETE-activated IL-6 mRNA expression are shown (B). Effects of apocynin on 12(S)-HETE-activated TNF-{alpha} mRNA expression are shown (C). Effects of DPI on 12(S)-HETE-activated TNF-{alpha} mRNA expression are shown (D). **, P < 0.01 vs. EtOH; #, P < 0.05 vs. 12(S)-HETE; ##, P < 0.01 vs. 12(S)-HETE.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The 12/15-LO products, 12(S)-HPETE and 12(S)-HETE, have been recognized as potent inflammatory compounds (31, 32). To examine the role of 12/15-LO pathway in the mRNA expression of IL-6 and TNF-{alpha} in macrophages, experiments were performed with a direct addition of 12(S)-HPETE or 12(S)-HETE to either J774A.1 cells or primary cultured MPMs. Results showed that 12(S)-HPETE or 12(S)-HETE induced a significant increase in IL-6 and TNF-{alpha} gene and protein expression, whereas the inactive 12(R)-HETE had no effect on IL-6 and TNF-{alpha} gene, clearly suggesting a specific effect of 12(S)-HPETE or 12(S)-HETE on IL-6 and TNF-{alpha} mRNA expression in macrophages.

We next studied whether overexpression of 12-LO activates cytokine gene expression. The experiments were conducted in Plox-86 cells, which stably overexpress active porcine leukocyte-type 12-LO. The Plox-86 cells contain no 5-LO activity, which allows specific evaluation of the contribution of 12/15-LO. The products of 12/15-LO in the presence of endogenous substrate in Plox-86 cells were 3.6-fold increased, compared with the in mock-transfected cells. The 3.6-fold increase in endogenous 12/15-LO enzymatic activity closely matched the 2- to 7-fold increase in the expression of cytokine genes in Plox-86 cells.

Consistent with the data in Plox-86 cells, we have provided data showing that the gene expression of IL-6 and TNF-{alpha} was markedly increased in MPMs from 12/15-LO transgenic (12/15-LO-Tg) mice, compared with that in MPMs from C57BL6 mice. In preliminary results, the expression of cytokine genes in heart tissues from 12/15-LO-Tg was also markedly increased, compared with that in heart tissues from normal mice (33). These results in 12/15-LO-Tg mice suggest the increase in the expression of cytokine genes in Plox-86 cells was not induced by nonspecific genome integration.

These data clearly demonstrate that products of the12/15-LO pathway have direct regulatory effects on cytokine gene expression. To our knowledge, this is the first study demonstrating that 12/15-LO products possess a novel mechanism triggering a cascade of inflammatory reactions. As discussed earlier, 12/15-LO can also convert linoleic acid to 9-, and 13-HPODEs and lead to increased oxidation of LDL. Further studies will be needed to evaluate whether the 9- or 13-HPODE can directly stimulate cytokine gene expression in macrophages. 9-, and 13-HPODE have already been reported to be potent inflammatory agents (34, 35).

We also carried out studies on possible mechanisms of 12(S)-HETE-induced cytokine gene expression. The previous data from our laboratory have already shown that 12(S)-HETE activated PKC (36), p38 MAPK (37), and JNK (38) in adrenal cells and cardiac fibroblasts as well as CHOAT1 cells (CHO cells overexpressing angiotensin II AT1 receptors). 12(S)-HETE at 1 nM activated all these three kinases in J744A.1 cells. To study the role of these kinases in regulating 12(S)-HETE activation of IL-6 and TNF {alpha} gene expression, we used accepted pharmacological inhibitors and dominant-negative mutants. The data show that the blockade of these kinases suppressed 12(S)-HETE-induced IL-6 and TNF-{alpha} gene expression. IL-6 expression was particularly sensitive to blockade of PKC and p38. Lower concentration of PKC or p38 inhibitors were needed to block 12(S)-HETE-induced IL-6 expression than needed for inhibition of TNF-{alpha} mRNA expression in J774A.1 cells. Our data also show that JNK activation plays a role in 12(S)-HETE-induced IL-6 and TNF-{alpha} gene expression. The blockade of MKK4 partially inhibited 12(S)-HETE-induced IL-6 gene expression but only a minor effect on inhibition of TNF-{alpha} gene expression. In contrast, blockade of MKK7 completely suppressed both 12(S)-HETE-induced IL-6 and TNF-{alpha} gene expression.

Lipoxygenase has also been shown to increase reactive oxygen species in the presence of nicotinamide adenine dinucleotide reduced and NADPH (39), suggesting an involvement of NADPH oxidase in 12/15-LO action. We have data showing a 3-fold increase of NADPH oxidase activity measured with cytochrome C reduction in response to stimulation of 1 nM 12(S)-HETE for 4 h in J774A.1 cells. Therefore, we examined the effects of apocynin, which is a well-characterized inhibitor of NADPH oxidase. Our result showed that apocynin inhibited 12(S)-HETE-induced activation of NADPH oxidase in J774A.1 cells. We also showed that 12(S)-HETE effect on expression of IL-6 and TNF-{alpha} mRNA was blocked in the presence of apocynin. DPI, another NADPH oxidase inhibitor, was also used and the results showed that DPI could inhibit 12(S)-HETE-induced expression of IL-6 as well as TNF-{alpha} mRNA at lower concentrations. It is possible that these inhibitors could have other effects. We therefore used two inhibitors with different properties to enhance the ability to interpret the finding. These data clearly suggest a role of NADPH oxidase (oxidative stress) in 12(S)-HETE-induced cytokine expression. There are data showing either a linear signaling cascade or a cross talk among some of these signaling cascades; however, we have not pursued the relationship among these signaling cascades in this study.

Recent elegant studies have shown that lipoxins and related lipids are potent endogenous antiinflammatory compounds that can be derived by cross-cellular metabolism. Lipoxins can in part be formed via the 15-LO enzyme. Therefore, under certain conditions associated with acute inflammation and neutrophil infiltration, the LO enzyme can participate in forming lipids that can resolve the acute inflammatory response (40). Additional studies would therefore be needed to evaluate the interaction of 12/15-LO and lipoxins in cytokine generation.

Summary
Our data indicate that 12/15-LO can directly activate proinflammatory cytokine expression in macrophages. This is the first report demonstrating that 12/15-LO products of AA, 12(S)-HPETE and 12(S)-HETE, can activate inflammatory cytokine expression. Recent studies have shown a critical role of macrophage 12/15-LO for atherosclerosis in the apolipoprotein-deficient mouse model (32). In addition to the well-known effect of 12/15-LO to oxidize LDL through the conversion of linoleic acid to 13-HPODE in macrophages, 12/15-LO products of AA induce inflammatory cytokine expression. This current study provides a new mechanism whereby 12/15-LO participates in the acceleration of atherogenesis (Fig. 6Go).


Figure 6
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  		FIG. 6. Proposed mechanism of 12/15-LO actions in macrophages to induce chronic inflammation and atherosclerosis. Under certain acute inflammatory conditions, particularly associated with neutrophil infiltration, 12/15-LO may participate in forming lipoxin and related antiinflammatory lipids to help resolve the acute inflammatory response.

 


   Acknowledgments
 
The authors express their deep thanks to Mr. George E. Vandenhoff for HETE assay and Micah Nadler for preparing the figures and table. The authors also deeply thank Drs. Michael Karin and Tse-Hua Tan for the generous gift of dnMKK4 and MKK7 plasmids.


   Footnotes
 
This work was supported by National Institutes of Health g P01HL 55798 and DK 55240.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 14, 2006

Abbreviations: AA, Arachidonic acid; dnMKK, dominant-negative MAPK kinase; DPI, diphenyleneiodonium chloride; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 13-HPODE, 13-(S)-hydroperoxyoctadecadienoic acid; JNK, c-jun NH2-terminal kinase; LDL, low-density lipoprotein; LO, lipoxygenase; 12/15-LO–/– or 12/15-LO KO, 12/15-LO knockout; 12/15-LO Tg, 12/15-LO transgenic; MARCKS, myristoylated alanine-rich C kinase substrate; MCP, monocyte chemoattractant protein; MPM, mouse peritoneal macrophage; NADPH, nicotinamide adenine dinucleotide phosphate; p38, p38 MAPK; PKC, protein kinase C; Plox-86, a macrophage-like cell line, J774A.1 stably overexpressing porcine leukocyte type of 12-LO; P/S, penicillin/streptomycin; 12(S)-HETE, 12-(S)-hydroxyeicosatetraenoic acid; 12(S)-HPETE, 12-(S)-hydroperoxyeicosa-5Z, 8Z, 10E, 14Z-tetraenoic acid; Tg, transgenic.

Received May 18, 2006.

Accepted for publication December 5, 2006.


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