Cutting Edge: Novel Role of Lipoxygenases in the Inflammatory Response: Promotion of TNF mRNA Decay by 15-Hydroperoxyeicosatetraenoic Acid i
http://www.100md.com
免疫学杂志 2005年第6期
Abstract
The metabolism of arachidonic acid via the lipoxygenase and cyclooxygenase pathways generates metabolites that regulate the inflammatory response. Although products of lipoxygenase are classically proinflammatory, recently it has been demonstrated that lipoxins, 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 15-hydroxyeicosatetraenoic acid exhibit anti-inflammatory activity. We now demonstrate for the first time that 15-HPETE regulates the production of the proinflammatory cytokine TNF posttranscriptionally by promoting degradation of LPS-induced TNFmRNA in a human monocytic cell line, Mono Mac 6. 15-HPETE causes a significant increase in the rate of TNF but not G3PDHmRNA degradation in the presence of the transcription inhibitor, actinomycin D. The decay of TNFmRNA is accelerated 1.7-fold, and its half-life is decreased by 57%. In view of its chemical and physical properties, we propose that 15-HPETE may function by destabilizing TNFmRNA by interaction with a trans-activating protein bound to the AU-rich element of TNFmRNA.
Introduction
During inflammation, the metabolism of arachidonic acid (AA) 3 generated via the lipoxygenase (LO) and cyclooxygenase (CO) pathways leads to the generation of several metabolites that regulate the inflammatory response. Classically, products of LO are known for their proinflammatory properties; however, we and others (1, 2, 3, 4, 5, 6, 7) have proposed recently that some AA metabolites of the LO pathway exhibit anti-inflammatory activity. The lipoxins (LX) were found to inhibit the neutrophil responses of chemotaxis and respiratory burst (8). We demonstrated that when AA was converted to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), it lost the ability to activate the neutrophil responses of superoxide production, degranulation, and adherence (9) but showed an increased ability to inhibit the production of TNF (6), a cytokine involved in the pathogenesis of a variety of conditions (10, 11). These studies demonstrated that pretreatment of macrophages with 15-HPETE inhibited LPS-induced TNF production at both the protein and mRNA levels (6). Furthermore, 15-HPETE inhibited the ability of LPS to activate protein kinase C (6). 15-HPETE was also highly inhibitory to the TNF-induced up-regulation of endothelial cell adhesion molecule expression compared with AA and omega-3 polyunsaturated fatty acids (7).
These findings reveal that the regulation/control of the inflammatory response needs, first, to consider the down-regulation of the proinflammatory processes by the hydroxyeicosatetraenoic acids (HETEs), LX, and HPETEs and, second, to provide an avenue for developing novel anti-inflammatory agents (3). To enhance our understanding of the mechanisms by which 15-HPETE causes inhibition of inflammatory mediators, we examined whether its effects extend to posttranscriptional events. The data reveal unique effects of this LO product on monocyte TNF production involving promotion of TNFmRNA decay.
Materials and Methods
Reagents
The synthesis of 15-HPETE and its solubilization for micelle presentation to cells were conducted as previously described (6). 5-HPETE and 12-HPETE were obtained from Cayman Chemical. Actinomycin D and LPS were purchased from Sigma-Aldrich.
Mono Mac 6 cells
The Mono Mac 6 cell line was kindly provided by Dr. H. W. L. Ziegler-Heitbrock (Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universitat, Munchen, Germany) and was cultured as described previously (6).
cDNA probes
The human TNF cDNA, an 820-bp EcoRI fragment cloned into a pUC vector was obtained from Genentech. The G3PDH cDNA control probe was a 1.1-kb fragment obtained from Clontech.
RNA isolation and hybridization
Mono Mac 6 cells (106 in total volume of 1 ml) were induced as described in the figures. RNA for slot blots was isolated and prepared by the RNAzol B method for the isolation of total RNA (Cinna/Biotecx) (12). The heat treatment of mRNA samples in formaldehyde solution, application to nylon membrane via a slot blot vacuum manifold system (Hoefer Scientific Instruments), and all subsequent procedures, including the normalization of data to G3PDH in the quantitative analysis of probed slot blots using the Packard instant imager (Canberra-Packard), were conducted as described previously (13).
Results
The results in Fig. 1 show that pretreatment of Mono Mac 6 cells with 10 μM 15-HPETE, a metabolite of AA formed by the LOs, depressed TNFmRNA induced by 1 μg of LPS. 15-HPETE pretreatment times of 60–90 min resulted in complete inhibition of TNFmRNA production. With further decrease in pretreatment time, there was a gradual loss of ability to inhibit TNFmRNA production such that the simultaneous addition of 15-HPETE and LPS resulted in only 48% inhibition (Fig. 1). Unexpectedly, a delay in addition of 15-HPETE following the stimulation of cells by LPS did not lead to a further decrease in inhibition of TNFmRNA but, in fact, resulted in an increased inhibition, reaching 80% inhibition with 15-HPETE addition at 90 min after LPS stimulation. Thus, a depression of TNFmRNA content was observed, irrespective of whether 15-HPETE was added to the cells before LPS induction, simultaneously with LPS induction, or after LPS induction. This result suggested that 15-HPETE might affect TNFmRNA stability.
FIGURE 1. The effect of 15-HPETE on TNFmRNA production when added pre- and poststimulation with LPS. Mono Mac 6 cells (1 x 106 in 1 ml) were incubated at 37°C with 10 μM 15-HPETE for either 90, 60, 30, or 0 min before, or for the same time periods, following their stimulation with 1 μg of LPS and further incubated at 37°C for 90 min at which time RNA was extracted and mRNA quantified as described previously (14 ). The data are presented as mean ± SEM of three experiments. ANOVA, p = 0.0006.
The effect of 15-HPETE on the decay of LPS-induced TNFmRNA was therefore examined. Mono Mac 6 cells were stimulated with LPS to a point of maximal TNFmRNA induction, washed, and resuspended in tissue culture medium to remove exogenous LPS to establish steady-state kinetics. The addition of alternative treatments after this time point enabled, in the absence of LPS stimulation, a comparison of the natural rate of decay of TNFmRNA (no treatment) with decay influenced by blocking transcription (+actinomycin D), treatment with 15-HPETE alone, or treatment with actinomycin D and 15-HPETE in combination. There was a decreased level of TNFmRNA in the presence of actinomycin D suggesting that some transcription of TNFmRNA was occurring during this period (Fig. 2). In the absence of actinomycin D, cells treated with 15-HPETE also showed decreased levels of TNFmRNA. In the presence of actinomycin D, 15-HPETE-treated cells showed an accelerated rate of TNFmRNA decay. In fact, the half-life of TNFmRNA measured in cells treated with actinomycin D alone was reduced by half in cells treated with actinomycin D plus 15-HPETE (Fig. 3b). These results show that 15-HPETE caused decreased stability of the TNFmRNA molecule in Mono Mac 6 cells. The inhibition of cytokine message was not related to any toxic effects of 15-HPETE since apart from the lack of inhibition of G3PDH mRNA production, the highest concentration tested, 30 μM, did not cause cell death based on the ability to exclude trypan blue (data not presented). This is consistent with the data in Fig. 1 showing that the effects of the drug varied according to time of LPS stimulation. The effects on TNFmRNA when added post-LPS stimulation were found to occur over a concentration range of 7.5–30.0 μM 15-HPETE (Fig. 3a). In contrast, neither 5-HPETE nor 12-HPETE at 15 μM was able to cause a significant decrease in half-life of TNFmRNA when added to actinomycin D-treated cells. This raises the issue of whether the basis for the 15-HPETE effect is through the formation of downstream products, namely LX (3).
FIGURE 2. a, The effect of 15-HPETE on the decay of TNFmRNA in Mono Mac 6 cells. Mono Mac 6 cells (1 x 106 in 1 ml) were stimulated with 1 μg of LPS for 60 min at 37°C. The cells were pelleted and gently resuspended in 1 ml in the following combinations: RPMI 1640, + actinomycin D (5 μg/ml), +15 μM 15-HPETE, and + actinomycin D (5 μg/ml) + 15 μM 15-HPETE, and incubated at 37°C. Total RNA was extracted at 15, 30, and 45 min and mRNA quantified as described previously (14 ). b, For measurement of half-life, the data are presented as line of best fit of log of mean ± SEM of three experiments. Kruskal-Wallis test, p = 0.0002.
FIGURE 3. a, The effect of varying the concentration of 15-HPETE on TNFmRNA half-life. Mono Mac 6 cells (1 x 106 in 1 ml) were stimulated with 1 μg of LPS for 60 min at 37°C. The cells were pelleted and gently resuspended in 1 ml in the following combinations: RPMI 1640, + actinomycin D (ACT D; 5 μg/ml), + 15-HPETE, and + actinomycin D (5 μg/ml) + 15-HPETE (wherein 15-HPETE final concentration was varied from 0 to 30 μM) and incubated at 37°C. Total RNA was extracted at 45 min, and mRNA quantified as described previously (14 ). The data are presented as the ratio of the mean of half-lives of the + ACT D + 15-HPETE concentration treatments relative to their + ACT D treatment counterparts calculated from three experiments. ANOVA, p = 0.001. b, The effect of 15-HPETE on the half-life of TNFmRNA in Mono Mac 6 cells. Mono Mac 6 cells (1 x 106 in 1 ml) were stimulated with 1 μg of LPS for 60 min at 37°C. The cells were pelleted and gently resuspended in 1 ml in the following combinations: RPMI 1640, + actinomycin D (5 μg/ml), + 15 μM 15-HPETE, and + actinomycin D (5 μg/ml) + 15 μM 15-HPETE, and incubated at 37°C. Total RNA was extracted at 15, 30, and 45 min, and mRNA quantified as described previously (14 ). The data are presented as mean ± SEM of half-lives calculated from three experiments. Student’s t test, p = 0.002.
Discussion
The present findings demonstrate that 15-HPETE mediates two mechanisms of action in effecting the inhibition of TNF production. First, consistent with our previous report, the addition of lipid mediator before the agonist, LPS, resulted in marked inhibition of TNFmRNA production, through the inhibition of protein kinase C activation (6), signals mandatory for the stimulation of TNF gene transcription (6, 7). This effect was specific for this metabolite as neither AA nor 15-hydroxyeicosatetraenoic acid (15-HETE) was active (6, 7). Also as previously reported, when the pre-exposure time was reduced, there was a concomitant reduction in the degree of inhibition caused by this mediator. This was most likely due to the greater opportunity afforded for LPS induction of the relevant intracellular signals necessary for TNF gene transcription. Second, the present data demonstrate that the treatment of cells post-LPS stimulation led to an increased inhibition with increased postaddition time. These kinetics of inhibition are best explained by the degree of transcription occurring and the accelerated increase in TNFmRNA degradation induced by 15-HPETE. When steady-state kinetics prevailed, the effects of 15-HPETE on TNFmRNA stability in the presence of actinomycin D were clearly demonstrated to inhibit any residual transcription. Thus, it is evident that once transcription has been initiated, 15-HPETE is able to inhibit TNF production by promoting the degradation of TNFmRNA.
Posttranscriptional control has been shown to play a vital role in the rapid turnover of TNF in monocytes and its regulation by known trans-acting proteins that bind the AU-rich element and stabilize or destabilize the transcript has been established (14, 15). In particular, of these proteins, one (AUH) (an AU-specific RNA binding protein with intrinsic enoyl-CoA hydratase activity; EC 4.2.1.17) (14), has been shown to possess simultaneous enoyl-CoA hydratase and TNFmRNA binding activities. The mechanism of how 15-HPETE exercises control at the mRNA level remains to be established, but it is of interest in this regard that this fatty acid is a substrate for and up-regulates the second enzyme in the fatty acid -oxidation pathway, viz enoyl-CoA hydratase. Although AUH possesses simultaneous mRNA binding and hydratase activities with comparatively short chain fatty acids, these activities are mutually exclusive with molecules of the length of 15-HPETE (16, 17). In this instance, it may be proposed that 15-HPETE may adopt a role as a substrate analog to force a competition with TNFmRNA for binding to the AUH protein. Thus, because of its length, 15-HPETE would be able to act effectively as a noncompetitive inhibitor and selectively deplete bound AUH from the mRNA, rendering the unprotected nucleotide sequence available to the normal degradative enzyme activities and promoting accelerated TNFmRNA decay. We observe that 15-HPETE alone produces an effect similar to actinomycin D and suggest that the latter may be preventing the appearance of the same protective protein as we believe the 15-HPETE to be removing from the TNFmRNA.
Products of AA metabolism via the LO pathway, in contrast to those of the CO pathway, have been well established as promoters of inflammation. Our finding (6) that 15-HPETE inhibits TNF production by macrophages as well as reports that 15-HETE (5) and LX (1) have anti-inflammatory activity have modernized our concepts about the role of LO enzymes in the regulation of the inflammatory response. Interestingly, we now demonstrate that a product of the LO enzymes, 15-HPETE, inhibits production of the proinflammatory cytokine, TNF, by promoting degradation of its mRNA. It is of interest that the concentration of 15-HPETE that was found inhibitory to TNF production is found in pathophysiologic fluids (18, 19). Thus, the lipid mediators generated during the metabolism of AA by 15-LO represent a unique anti-inflammatory response. In this concept, 15-HETE inhibits 5-LO and leukotriene B4 production (20) and the neutrophil respiratory burst (5). Lipoxin A4 (LXA4) and LXB4 inhibit neutrophil emigration in the microcirculation and chemotaxis and reduce neutrophil endothelium interactions mediated by 2 integrins (21). A stable analog of LXA4 was shown to be a potent inhibitor of neutrophil transmigration and adhesion (22). Furthermore, aspirin enhances LX production and triggers the generation of 15-epi-LXs such as 15-epi-LXA4, which blocks neutrophil adherence to endothelial cells (23) and neutrophil degranulation (2). In addition, 15-HPETE has been shown to inhibit the expression of surface adhesion molecules on endothelial cells and the binding of leukocytes to these cells (7) as well as the inhibition of TNF production by monocytes and monocytic cell lines (6). These effects are unique to this metabolite and are neither a function of AA itself nor of other metabolites such as 15-HETE (6, 7).
TNF is a pleiotropic cytokine playing key roles in inflammation, resistance to infection, and cancer. Besides its beneficial actions, the pathogenetic effects of TNF are evident in a wide variety of conditions such as septic shock, meningitis, cerebral malaria, cerebral ischemia, cancer, AIDS, obesity, and autoimmune diseases (multiple sclerosis, rheumatoid arthritis, and diabetes). While anti-cytokine therapy remains in its infancy, anti-TNF Ab medication is currently used in the treatment of rheumatoid arthritis (24). However, it is evident that efforts are being made to identify mechanisms regulating cytokine production, especially at the mRNA stability level (25, 26). In the current context, in consideration of its use for therapeutic purposes, 15-HPETE can be delivered as a pro-drug in which the reactive hydroperoxy group is protected by methyl groups (27).
The concentrations of AA and its metabolites under current consideration are found in pathophysiological fluids. In human malaria patients, the free plasma AA levels have been shown to be >100 μM (28). The brain free AA levels rise from 50 to 500 μM under ischemic conditions (29). The HETE levels increase to 40 μM in blood stimulated with various agents (30). HPETE has also been reported to reach levels that we found to be active in inhibiting TNF production. Under certain conditions or diseases where a reduced or compromised concentration of reductases occurs, 15-HPETE accumulates, as seen in HIV infections (18). Indeed, there are pathological conditions where HPETE levels of 20 μM have been measured (19).
The ability to target TNF production at the mRNA level is of primary importance in intervention against TNF-induced disease since it can confer significant advantage over therapeutics that target TNF at either the protein or transcriptional levels. Thus, we have identified a novel pathway that generates a lipid mediator promoting this degradation. Not only is this finding consistent with our previous views that these products possess therapeutic potential as anti-inflammatory agents (1, 6), but it also constitutes the first report of a LO metabolite of AA promoting the decay of TNFmRNA. Most interesting will be the characterization of the mechanisms by which 15-HPETE effects accelerated TNFmRNA decay.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia.
2 Address correspondence and reprint requests to Dr. Antonio Ferrante, Department of Immunopathology, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia. E-mail address: antonio.ferrante{at}adelaide.edu.au
3 Abbreviations used in this paper: AA, arachidonic acid; LO, lipoxygenase; CO, cyclooxygenase; 15-HPETE, 15-hydroperoxyeicosatetraenoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; AUH, enoyl-CoA hydratase (EC No. 4.2.1.17); LX, lipoxin.
Received for publication July 28, 2004. Accepted for publication January 18, 2005.
References
Serhan, C. N., J. M. Drazen. 1997. Anti-inflammatory potential of lipoxygenase-derived eicosanoids: a molecular switch at 5 and 15 positions. J. Clin. Invest. 99:1147.
Gewirtz, A. T., V. V. Fokin, N. A. Petasis, C. N. Serhan, J. L. Madara. 1999. LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively down-regulate PMN azurophilic degranulation. Am. J. Physiol. 276:C988.
Serhan, C. N.. 2002. Lipoxins and aspirin-triggered 15-epi-lipoxin biosynthesis: an update and role in anti-inflammation and pro-resolution. Prostaglandins Other Lipid Mediat. 68–69:433.
Serhan, C. N., A. Jain, S. Malean, C. Clish, A. Kantarci, B. Behbehani, S.P. Colgan, G.L. Stahl, A. Merched, N.A. Petasis, et al 2003. Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators. J. Immunol. 171:6856.
Smith, R. J., J. M. Justen, E. G. Nidy, L. M. Sam, J. E. Bleasdale. 1993. Transmembrane signalling in human polymorphonuclear neutrophils: 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid modulates receptor agonist-triggered cell activation. Proc. Natl. Acad. Sci. USA 90:7270.
Ferrante, J. V., Z. H. Huang, M. Nandoskar, C. S. T. Hii, B. S. Robinson, D. A. Rathjen, A. Poulos, C. P. Morris, A. Ferrante. 1997. Altered responses of human macrophages to lipopolysaccharide by hydroperoxyeicosatetraenoic acid and arachidonic acid. J. Clin. Invest. 99:1445.
Huang, Z. H., E. J. Bates, J. V. Ferrante, C. S. T. Hii, A. Poulos, B. S. Robinson, A. Ferrante. 1997. Inhibition of stimulus-induced endothelial cell intercellular adhesion molecule-1, E-selectin and vascular cellular adhesion molecule-1 expression by arachidonic acid and its hydroxy and hydroperoxy derivatives. Circ. Res. 80:149.
Hachicha, M., M. Pouliot, N. A. Petasis, C. N. Serhan. 1999. Lipoxin (LX)A4 and aspirin-triggered 15-epi-LXA4 inhibit tumor necrosis factor 1-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis. J. Exp. Med. 189:1923.
Ferrante, A., C. S. T. Hii, Z. H. Huang, D. A. Rathjen. 1999. Regulation of neutrophil functions by fatty acids. D. I. Gabrilovich, ed. The Neutrophils: New Outlook For Old Cells 79. Imperial College Press, London, U.K.
Hunt, N. H., G. E. Grau. 2003. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol. 24:491.
Aggarwal, B. B., S. Shishodia, K. Ashikawa, A.C. Bharti. 2002. The role of TNF and its family members in inflammation and cancer: lessons from gene deletion. Curr. Drug Targets Inflamm. Allergy 1:327.
Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid-guanidinium thiocyanate-phenol-chloroform. Anal. Biochem. 162:156.
Jersmann, H. P., C. S. T. Hii, J. V. Ferrante, A. Ferrante. 2001. Bacterial lipopolysaccharide and tumor necrosis factor synergistically increase expression of human endothelial adhesion molecules through activation of NF-B and p38 mitogen-activated protein kinase signalling pathways. Infect. Immun. 69:1273.
Nakagawa, J., H. Waldner, S. Meyer-Monard, J. Hofsteenge, P. Jeno, C. Moroni. 1995. AUH, a gene encoding an AU-specific RNA binding protein with intrinsic enoyl-CoA hydratase activity. Proc. Natl. Acad. Sci. USA 92:2051.[Abstract/Free Full Text]
Brennan, L. E., J. Nakagawa, D. Egger, K. Bienz, C. Moroni. 1999. Characterisation and mitochondrial localisation of AUH, an AU-specific RNA-binding enoyl-CoA hydratase. Gene 228:85.
Engel, C. K., T. R. Kiema, J. K. Hiltunen, R. K. Wierenga. 1998. The crystal structure of enoyl-CoA hydratase complexed with octanoyl-CoA reveals the structural adaptations required for binding of a long chain fatty acid-CoA molecule. J. Mol. Biol. 275:847.
Kurimoto, K., S. Fukai, O. Nureki, Y. Muto, S. Yokoyama. 2001. Crystal structure of human AUH protein, a single-stranded RNA binding homolog of enoyl-CoA hydratase. Structure 9:1253.
Filep, J. G., C. Zouki, N. A. Petasis, M. Hachicha, C. N. Serhan. 1999. Anti-inflammatory actions of lipoxin A(4) stable analogs are demonstrable in human whole blood: modulation of leukocyte adhesion molecules and inhibition of neutrophil-endothelial interactions. Blood 94:4132.
Claria, J., C. N. Serhan. 1995. Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc. Natl. Acad. Sci. USA 92:9475
Vilcek, J., M. Feldman. 2004. Historical review: cytokines as therapeutics and targets of therapeutics. Trends Pharmacol. Sci. 25:201.
Lee, S. W., A. P. Tsou, H. Chan, J. Thomas, K. Petrie, E. M. Eugui, A. C. Allison. 1988. Glucocorticoids selectively inhibit the transcription of the interleukin 1 gene and decrease the stability of interleukin 1 mRNA. Proc. Natl. Acad. Sci. USA 85:1204
Moreira, A. L., E. P. Sampaio, A. Zmuidzinas, P. Frindt, K. A. Smith, G. Kaplan. 1993. Thalidomide exerts its inhibitory action on tumor necrosis factor by enhancing mRNA degradation. J. Exp. Med. 177:1675.
Easton, C. J., T. A. Robertson, M. J. Pitt, D. A. Rathjen, A. Ferrante, A. Poulos. 2001. Oxidation of oxa and thia fatty acids and related compounds catalysed by 5- and 15-lipoxygenase. Bioorg. Med. Chem. 9:317.
Eissen, E. U.. 1993. Significance of plasma free fatty acid levels in human malaria with parasitaemia. Med. Sci. Res. 21:405.
Yasuda, H., K. Kishiro, N. Izumi, M. Nakanishi. 1985. Biphasic liberation of arachidonic and stearic acids during cerebral ischemia. J. Neurochem. 45:168.
Walenga, R. W., S. Boone, M. J. Stuart. 1987. Analysis of blood HETE levels by selected ion monitoring with ricinoleic acid as the internal standard. Prostaglandins 34:733.(Judith V. Ferrante and An)
The metabolism of arachidonic acid via the lipoxygenase and cyclooxygenase pathways generates metabolites that regulate the inflammatory response. Although products of lipoxygenase are classically proinflammatory, recently it has been demonstrated that lipoxins, 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 15-hydroxyeicosatetraenoic acid exhibit anti-inflammatory activity. We now demonstrate for the first time that 15-HPETE regulates the production of the proinflammatory cytokine TNF posttranscriptionally by promoting degradation of LPS-induced TNFmRNA in a human monocytic cell line, Mono Mac 6. 15-HPETE causes a significant increase in the rate of TNF but not G3PDHmRNA degradation in the presence of the transcription inhibitor, actinomycin D. The decay of TNFmRNA is accelerated 1.7-fold, and its half-life is decreased by 57%. In view of its chemical and physical properties, we propose that 15-HPETE may function by destabilizing TNFmRNA by interaction with a trans-activating protein bound to the AU-rich element of TNFmRNA.
Introduction
During inflammation, the metabolism of arachidonic acid (AA) 3 generated via the lipoxygenase (LO) and cyclooxygenase (CO) pathways leads to the generation of several metabolites that regulate the inflammatory response. Classically, products of LO are known for their proinflammatory properties; however, we and others (1, 2, 3, 4, 5, 6, 7) have proposed recently that some AA metabolites of the LO pathway exhibit anti-inflammatory activity. The lipoxins (LX) were found to inhibit the neutrophil responses of chemotaxis and respiratory burst (8). We demonstrated that when AA was converted to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), it lost the ability to activate the neutrophil responses of superoxide production, degranulation, and adherence (9) but showed an increased ability to inhibit the production of TNF (6), a cytokine involved in the pathogenesis of a variety of conditions (10, 11). These studies demonstrated that pretreatment of macrophages with 15-HPETE inhibited LPS-induced TNF production at both the protein and mRNA levels (6). Furthermore, 15-HPETE inhibited the ability of LPS to activate protein kinase C (6). 15-HPETE was also highly inhibitory to the TNF-induced up-regulation of endothelial cell adhesion molecule expression compared with AA and omega-3 polyunsaturated fatty acids (7).
These findings reveal that the regulation/control of the inflammatory response needs, first, to consider the down-regulation of the proinflammatory processes by the hydroxyeicosatetraenoic acids (HETEs), LX, and HPETEs and, second, to provide an avenue for developing novel anti-inflammatory agents (3). To enhance our understanding of the mechanisms by which 15-HPETE causes inhibition of inflammatory mediators, we examined whether its effects extend to posttranscriptional events. The data reveal unique effects of this LO product on monocyte TNF production involving promotion of TNFmRNA decay.
Materials and Methods
Reagents
The synthesis of 15-HPETE and its solubilization for micelle presentation to cells were conducted as previously described (6). 5-HPETE and 12-HPETE were obtained from Cayman Chemical. Actinomycin D and LPS were purchased from Sigma-Aldrich.
Mono Mac 6 cells
The Mono Mac 6 cell line was kindly provided by Dr. H. W. L. Ziegler-Heitbrock (Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universitat, Munchen, Germany) and was cultured as described previously (6).
cDNA probes
The human TNF cDNA, an 820-bp EcoRI fragment cloned into a pUC vector was obtained from Genentech. The G3PDH cDNA control probe was a 1.1-kb fragment obtained from Clontech.
RNA isolation and hybridization
Mono Mac 6 cells (106 in total volume of 1 ml) were induced as described in the figures. RNA for slot blots was isolated and prepared by the RNAzol B method for the isolation of total RNA (Cinna/Biotecx) (12). The heat treatment of mRNA samples in formaldehyde solution, application to nylon membrane via a slot blot vacuum manifold system (Hoefer Scientific Instruments), and all subsequent procedures, including the normalization of data to G3PDH in the quantitative analysis of probed slot blots using the Packard instant imager (Canberra-Packard), were conducted as described previously (13).
Results
The results in Fig. 1 show that pretreatment of Mono Mac 6 cells with 10 μM 15-HPETE, a metabolite of AA formed by the LOs, depressed TNFmRNA induced by 1 μg of LPS. 15-HPETE pretreatment times of 60–90 min resulted in complete inhibition of TNFmRNA production. With further decrease in pretreatment time, there was a gradual loss of ability to inhibit TNFmRNA production such that the simultaneous addition of 15-HPETE and LPS resulted in only 48% inhibition (Fig. 1). Unexpectedly, a delay in addition of 15-HPETE following the stimulation of cells by LPS did not lead to a further decrease in inhibition of TNFmRNA but, in fact, resulted in an increased inhibition, reaching 80% inhibition with 15-HPETE addition at 90 min after LPS stimulation. Thus, a depression of TNFmRNA content was observed, irrespective of whether 15-HPETE was added to the cells before LPS induction, simultaneously with LPS induction, or after LPS induction. This result suggested that 15-HPETE might affect TNFmRNA stability.
FIGURE 1. The effect of 15-HPETE on TNFmRNA production when added pre- and poststimulation with LPS. Mono Mac 6 cells (1 x 106 in 1 ml) were incubated at 37°C with 10 μM 15-HPETE for either 90, 60, 30, or 0 min before, or for the same time periods, following their stimulation with 1 μg of LPS and further incubated at 37°C for 90 min at which time RNA was extracted and mRNA quantified as described previously (14 ). The data are presented as mean ± SEM of three experiments. ANOVA, p = 0.0006.
The effect of 15-HPETE on the decay of LPS-induced TNFmRNA was therefore examined. Mono Mac 6 cells were stimulated with LPS to a point of maximal TNFmRNA induction, washed, and resuspended in tissue culture medium to remove exogenous LPS to establish steady-state kinetics. The addition of alternative treatments after this time point enabled, in the absence of LPS stimulation, a comparison of the natural rate of decay of TNFmRNA (no treatment) with decay influenced by blocking transcription (+actinomycin D), treatment with 15-HPETE alone, or treatment with actinomycin D and 15-HPETE in combination. There was a decreased level of TNFmRNA in the presence of actinomycin D suggesting that some transcription of TNFmRNA was occurring during this period (Fig. 2). In the absence of actinomycin D, cells treated with 15-HPETE also showed decreased levels of TNFmRNA. In the presence of actinomycin D, 15-HPETE-treated cells showed an accelerated rate of TNFmRNA decay. In fact, the half-life of TNFmRNA measured in cells treated with actinomycin D alone was reduced by half in cells treated with actinomycin D plus 15-HPETE (Fig. 3b). These results show that 15-HPETE caused decreased stability of the TNFmRNA molecule in Mono Mac 6 cells. The inhibition of cytokine message was not related to any toxic effects of 15-HPETE since apart from the lack of inhibition of G3PDH mRNA production, the highest concentration tested, 30 μM, did not cause cell death based on the ability to exclude trypan blue (data not presented). This is consistent with the data in Fig. 1 showing that the effects of the drug varied according to time of LPS stimulation. The effects on TNFmRNA when added post-LPS stimulation were found to occur over a concentration range of 7.5–30.0 μM 15-HPETE (Fig. 3a). In contrast, neither 5-HPETE nor 12-HPETE at 15 μM was able to cause a significant decrease in half-life of TNFmRNA when added to actinomycin D-treated cells. This raises the issue of whether the basis for the 15-HPETE effect is through the formation of downstream products, namely LX (3).
FIGURE 2. a, The effect of 15-HPETE on the decay of TNFmRNA in Mono Mac 6 cells. Mono Mac 6 cells (1 x 106 in 1 ml) were stimulated with 1 μg of LPS for 60 min at 37°C. The cells were pelleted and gently resuspended in 1 ml in the following combinations: RPMI 1640, + actinomycin D (5 μg/ml), +15 μM 15-HPETE, and + actinomycin D (5 μg/ml) + 15 μM 15-HPETE, and incubated at 37°C. Total RNA was extracted at 15, 30, and 45 min and mRNA quantified as described previously (14 ). b, For measurement of half-life, the data are presented as line of best fit of log of mean ± SEM of three experiments. Kruskal-Wallis test, p = 0.0002.
FIGURE 3. a, The effect of varying the concentration of 15-HPETE on TNFmRNA half-life. Mono Mac 6 cells (1 x 106 in 1 ml) were stimulated with 1 μg of LPS for 60 min at 37°C. The cells were pelleted and gently resuspended in 1 ml in the following combinations: RPMI 1640, + actinomycin D (ACT D; 5 μg/ml), + 15-HPETE, and + actinomycin D (5 μg/ml) + 15-HPETE (wherein 15-HPETE final concentration was varied from 0 to 30 μM) and incubated at 37°C. Total RNA was extracted at 45 min, and mRNA quantified as described previously (14 ). The data are presented as the ratio of the mean of half-lives of the + ACT D + 15-HPETE concentration treatments relative to their + ACT D treatment counterparts calculated from three experiments. ANOVA, p = 0.001. b, The effect of 15-HPETE on the half-life of TNFmRNA in Mono Mac 6 cells. Mono Mac 6 cells (1 x 106 in 1 ml) were stimulated with 1 μg of LPS for 60 min at 37°C. The cells were pelleted and gently resuspended in 1 ml in the following combinations: RPMI 1640, + actinomycin D (5 μg/ml), + 15 μM 15-HPETE, and + actinomycin D (5 μg/ml) + 15 μM 15-HPETE, and incubated at 37°C. Total RNA was extracted at 15, 30, and 45 min, and mRNA quantified as described previously (14 ). The data are presented as mean ± SEM of half-lives calculated from three experiments. Student’s t test, p = 0.002.
Discussion
The present findings demonstrate that 15-HPETE mediates two mechanisms of action in effecting the inhibition of TNF production. First, consistent with our previous report, the addition of lipid mediator before the agonist, LPS, resulted in marked inhibition of TNFmRNA production, through the inhibition of protein kinase C activation (6), signals mandatory for the stimulation of TNF gene transcription (6, 7). This effect was specific for this metabolite as neither AA nor 15-hydroxyeicosatetraenoic acid (15-HETE) was active (6, 7). Also as previously reported, when the pre-exposure time was reduced, there was a concomitant reduction in the degree of inhibition caused by this mediator. This was most likely due to the greater opportunity afforded for LPS induction of the relevant intracellular signals necessary for TNF gene transcription. Second, the present data demonstrate that the treatment of cells post-LPS stimulation led to an increased inhibition with increased postaddition time. These kinetics of inhibition are best explained by the degree of transcription occurring and the accelerated increase in TNFmRNA degradation induced by 15-HPETE. When steady-state kinetics prevailed, the effects of 15-HPETE on TNFmRNA stability in the presence of actinomycin D were clearly demonstrated to inhibit any residual transcription. Thus, it is evident that once transcription has been initiated, 15-HPETE is able to inhibit TNF production by promoting the degradation of TNFmRNA.
Posttranscriptional control has been shown to play a vital role in the rapid turnover of TNF in monocytes and its regulation by known trans-acting proteins that bind the AU-rich element and stabilize or destabilize the transcript has been established (14, 15). In particular, of these proteins, one (AUH) (an AU-specific RNA binding protein with intrinsic enoyl-CoA hydratase activity; EC 4.2.1.17) (14), has been shown to possess simultaneous enoyl-CoA hydratase and TNFmRNA binding activities. The mechanism of how 15-HPETE exercises control at the mRNA level remains to be established, but it is of interest in this regard that this fatty acid is a substrate for and up-regulates the second enzyme in the fatty acid -oxidation pathway, viz enoyl-CoA hydratase. Although AUH possesses simultaneous mRNA binding and hydratase activities with comparatively short chain fatty acids, these activities are mutually exclusive with molecules of the length of 15-HPETE (16, 17). In this instance, it may be proposed that 15-HPETE may adopt a role as a substrate analog to force a competition with TNFmRNA for binding to the AUH protein. Thus, because of its length, 15-HPETE would be able to act effectively as a noncompetitive inhibitor and selectively deplete bound AUH from the mRNA, rendering the unprotected nucleotide sequence available to the normal degradative enzyme activities and promoting accelerated TNFmRNA decay. We observe that 15-HPETE alone produces an effect similar to actinomycin D and suggest that the latter may be preventing the appearance of the same protective protein as we believe the 15-HPETE to be removing from the TNFmRNA.
Products of AA metabolism via the LO pathway, in contrast to those of the CO pathway, have been well established as promoters of inflammation. Our finding (6) that 15-HPETE inhibits TNF production by macrophages as well as reports that 15-HETE (5) and LX (1) have anti-inflammatory activity have modernized our concepts about the role of LO enzymes in the regulation of the inflammatory response. Interestingly, we now demonstrate that a product of the LO enzymes, 15-HPETE, inhibits production of the proinflammatory cytokine, TNF, by promoting degradation of its mRNA. It is of interest that the concentration of 15-HPETE that was found inhibitory to TNF production is found in pathophysiologic fluids (18, 19). Thus, the lipid mediators generated during the metabolism of AA by 15-LO represent a unique anti-inflammatory response. In this concept, 15-HETE inhibits 5-LO and leukotriene B4 production (20) and the neutrophil respiratory burst (5). Lipoxin A4 (LXA4) and LXB4 inhibit neutrophil emigration in the microcirculation and chemotaxis and reduce neutrophil endothelium interactions mediated by 2 integrins (21). A stable analog of LXA4 was shown to be a potent inhibitor of neutrophil transmigration and adhesion (22). Furthermore, aspirin enhances LX production and triggers the generation of 15-epi-LXs such as 15-epi-LXA4, which blocks neutrophil adherence to endothelial cells (23) and neutrophil degranulation (2). In addition, 15-HPETE has been shown to inhibit the expression of surface adhesion molecules on endothelial cells and the binding of leukocytes to these cells (7) as well as the inhibition of TNF production by monocytes and monocytic cell lines (6). These effects are unique to this metabolite and are neither a function of AA itself nor of other metabolites such as 15-HETE (6, 7).
TNF is a pleiotropic cytokine playing key roles in inflammation, resistance to infection, and cancer. Besides its beneficial actions, the pathogenetic effects of TNF are evident in a wide variety of conditions such as septic shock, meningitis, cerebral malaria, cerebral ischemia, cancer, AIDS, obesity, and autoimmune diseases (multiple sclerosis, rheumatoid arthritis, and diabetes). While anti-cytokine therapy remains in its infancy, anti-TNF Ab medication is currently used in the treatment of rheumatoid arthritis (24). However, it is evident that efforts are being made to identify mechanisms regulating cytokine production, especially at the mRNA stability level (25, 26). In the current context, in consideration of its use for therapeutic purposes, 15-HPETE can be delivered as a pro-drug in which the reactive hydroperoxy group is protected by methyl groups (27).
The concentrations of AA and its metabolites under current consideration are found in pathophysiological fluids. In human malaria patients, the free plasma AA levels have been shown to be >100 μM (28). The brain free AA levels rise from 50 to 500 μM under ischemic conditions (29). The HETE levels increase to 40 μM in blood stimulated with various agents (30). HPETE has also been reported to reach levels that we found to be active in inhibiting TNF production. Under certain conditions or diseases where a reduced or compromised concentration of reductases occurs, 15-HPETE accumulates, as seen in HIV infections (18). Indeed, there are pathological conditions where HPETE levels of 20 μM have been measured (19).
The ability to target TNF production at the mRNA level is of primary importance in intervention against TNF-induced disease since it can confer significant advantage over therapeutics that target TNF at either the protein or transcriptional levels. Thus, we have identified a novel pathway that generates a lipid mediator promoting this degradation. Not only is this finding consistent with our previous views that these products possess therapeutic potential as anti-inflammatory agents (1, 6), but it also constitutes the first report of a LO metabolite of AA promoting the decay of TNFmRNA. Most interesting will be the characterization of the mechanisms by which 15-HPETE effects accelerated TNFmRNA decay.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia.
2 Address correspondence and reprint requests to Dr. Antonio Ferrante, Department of Immunopathology, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia. E-mail address: antonio.ferrante{at}adelaide.edu.au
3 Abbreviations used in this paper: AA, arachidonic acid; LO, lipoxygenase; CO, cyclooxygenase; 15-HPETE, 15-hydroperoxyeicosatetraenoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; AUH, enoyl-CoA hydratase (EC No. 4.2.1.17); LX, lipoxin.
Received for publication July 28, 2004. Accepted for publication January 18, 2005.
References
Serhan, C. N., J. M. Drazen. 1997. Anti-inflammatory potential of lipoxygenase-derived eicosanoids: a molecular switch at 5 and 15 positions. J. Clin. Invest. 99:1147.
Gewirtz, A. T., V. V. Fokin, N. A. Petasis, C. N. Serhan, J. L. Madara. 1999. LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively down-regulate PMN azurophilic degranulation. Am. J. Physiol. 276:C988.
Serhan, C. N.. 2002. Lipoxins and aspirin-triggered 15-epi-lipoxin biosynthesis: an update and role in anti-inflammation and pro-resolution. Prostaglandins Other Lipid Mediat. 68–69:433.
Serhan, C. N., A. Jain, S. Malean, C. Clish, A. Kantarci, B. Behbehani, S.P. Colgan, G.L. Stahl, A. Merched, N.A. Petasis, et al 2003. Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators. J. Immunol. 171:6856.
Smith, R. J., J. M. Justen, E. G. Nidy, L. M. Sam, J. E. Bleasdale. 1993. Transmembrane signalling in human polymorphonuclear neutrophils: 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid modulates receptor agonist-triggered cell activation. Proc. Natl. Acad. Sci. USA 90:7270.
Ferrante, J. V., Z. H. Huang, M. Nandoskar, C. S. T. Hii, B. S. Robinson, D. A. Rathjen, A. Poulos, C. P. Morris, A. Ferrante. 1997. Altered responses of human macrophages to lipopolysaccharide by hydroperoxyeicosatetraenoic acid and arachidonic acid. J. Clin. Invest. 99:1445.
Huang, Z. H., E. J. Bates, J. V. Ferrante, C. S. T. Hii, A. Poulos, B. S. Robinson, A. Ferrante. 1997. Inhibition of stimulus-induced endothelial cell intercellular adhesion molecule-1, E-selectin and vascular cellular adhesion molecule-1 expression by arachidonic acid and its hydroxy and hydroperoxy derivatives. Circ. Res. 80:149.
Hachicha, M., M. Pouliot, N. A. Petasis, C. N. Serhan. 1999. Lipoxin (LX)A4 and aspirin-triggered 15-epi-LXA4 inhibit tumor necrosis factor 1-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis. J. Exp. Med. 189:1923.
Ferrante, A., C. S. T. Hii, Z. H. Huang, D. A. Rathjen. 1999. Regulation of neutrophil functions by fatty acids. D. I. Gabrilovich, ed. The Neutrophils: New Outlook For Old Cells 79. Imperial College Press, London, U.K.
Hunt, N. H., G. E. Grau. 2003. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol. 24:491.
Aggarwal, B. B., S. Shishodia, K. Ashikawa, A.C. Bharti. 2002. The role of TNF and its family members in inflammation and cancer: lessons from gene deletion. Curr. Drug Targets Inflamm. Allergy 1:327.
Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid-guanidinium thiocyanate-phenol-chloroform. Anal. Biochem. 162:156.
Jersmann, H. P., C. S. T. Hii, J. V. Ferrante, A. Ferrante. 2001. Bacterial lipopolysaccharide and tumor necrosis factor synergistically increase expression of human endothelial adhesion molecules through activation of NF-B and p38 mitogen-activated protein kinase signalling pathways. Infect. Immun. 69:1273.
Nakagawa, J., H. Waldner, S. Meyer-Monard, J. Hofsteenge, P. Jeno, C. Moroni. 1995. AUH, a gene encoding an AU-specific RNA binding protein with intrinsic enoyl-CoA hydratase activity. Proc. Natl. Acad. Sci. USA 92:2051.[Abstract/Free Full Text]
Brennan, L. E., J. Nakagawa, D. Egger, K. Bienz, C. Moroni. 1999. Characterisation and mitochondrial localisation of AUH, an AU-specific RNA-binding enoyl-CoA hydratase. Gene 228:85.
Engel, C. K., T. R. Kiema, J. K. Hiltunen, R. K. Wierenga. 1998. The crystal structure of enoyl-CoA hydratase complexed with octanoyl-CoA reveals the structural adaptations required for binding of a long chain fatty acid-CoA molecule. J. Mol. Biol. 275:847.
Kurimoto, K., S. Fukai, O. Nureki, Y. Muto, S. Yokoyama. 2001. Crystal structure of human AUH protein, a single-stranded RNA binding homolog of enoyl-CoA hydratase. Structure 9:1253.
Filep, J. G., C. Zouki, N. A. Petasis, M. Hachicha, C. N. Serhan. 1999. Anti-inflammatory actions of lipoxin A(4) stable analogs are demonstrable in human whole blood: modulation of leukocyte adhesion molecules and inhibition of neutrophil-endothelial interactions. Blood 94:4132.
Claria, J., C. N. Serhan. 1995. Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc. Natl. Acad. Sci. USA 92:9475
Vilcek, J., M. Feldman. 2004. Historical review: cytokines as therapeutics and targets of therapeutics. Trends Pharmacol. Sci. 25:201.
Lee, S. W., A. P. Tsou, H. Chan, J. Thomas, K. Petrie, E. M. Eugui, A. C. Allison. 1988. Glucocorticoids selectively inhibit the transcription of the interleukin 1 gene and decrease the stability of interleukin 1 mRNA. Proc. Natl. Acad. Sci. USA 85:1204
Moreira, A. L., E. P. Sampaio, A. Zmuidzinas, P. Frindt, K. A. Smith, G. Kaplan. 1993. Thalidomide exerts its inhibitory action on tumor necrosis factor by enhancing mRNA degradation. J. Exp. Med. 177:1675.
Easton, C. J., T. A. Robertson, M. J. Pitt, D. A. Rathjen, A. Ferrante, A. Poulos. 2001. Oxidation of oxa and thia fatty acids and related compounds catalysed by 5- and 15-lipoxygenase. Bioorg. Med. Chem. 9:317.
Eissen, E. U.. 1993. Significance of plasma free fatty acid levels in human malaria with parasitaemia. Med. Sci. Res. 21:405.
Yasuda, H., K. Kishiro, N. Izumi, M. Nakanishi. 1985. Biphasic liberation of arachidonic and stearic acids during cerebral ischemia. J. Neurochem. 45:168.
Walenga, R. W., S. Boone, M. J. Stuart. 1987. Analysis of blood HETE levels by selected ion monitoring with ricinoleic acid as the internal standard. Prostaglandins 34:733.(Judith V. Ferrante and An)