We studied the effect of tumor necrosis factor (TNF)-α exposure on cysteinyl leukotriene (LT) synthesis by cells of monocyte/macrophage lineage. TNF-α conditioning of monocytic THP-1 cells and primary human monocytes resulted in a decreased capacity for LTC4 release. TNF-α exposure (for 16–24 h) decreased LTC4 synthase mRNA in THP-1 cells, primary mouse bone marrow-derived macrophages, and eosinophilic AML14.3D10 cells. TNF-α downregulated LTC4 synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with downregulation observed as early as 4 h. The effect of TNF-α on LTC4 synthase mRNA expression was mediated via the MEK/ERK pathway, but not via cyclooxygenase or nitric oxide synthase pathways. Conditioning of actinomycin D-treated cells with TNF-α did not accelerate degradation of LTC4 synthase mRNA. TNF-α produced sustained activation of p50 and p65, which were previously reported by our group to decrease LTC4 synthase promoter activity. In transiently transfected THP-1 cells, TNF-α decreased promoter activity via an element located within the first 620 bp of the promoter. We conclude that TNF-α exposure downregulates the synthetic capacity for cysteinyl LT release and LTC4 synthase gene expression in monocytes/macrophages via a transcriptional mechanism.
- Toll-like receptor
the leukotrienes (LTs) are lipid metabolites that are synthesized from arachidonic acid via the initial action of 5-lipoxygenase (5-LO) and its associated 5-lipoxygenase activating protein (FLAP). The subsequent synthesis of LTC4 is mediated by the downstream enzyme LTC4 synthase, which represents the first committed step in the synthesis of the cysteinyl LTs (LTC4, LTD4, and LTE4) by mononuclear phagocytes and other inflammatory cells (9). Extensive data support the role of the cysteinyl LTs, acting via the cysLT1 and cysLT2 receptors, in mediating enhanced vascular permeability, smooth muscle contraction, mucus hypersecretion, bronchial hyperreactivity, and eosinophil chemotaxis (9, 18). The 5-LO pathway-derived LTs, including the cysteinyl LTs and LTB4, have been shown to play prominent roles in host defense by enhancing leukocyte function and microbial killing (1, 3). These mediators have been implicated in the pathogenesis of a number of inflammatory and allergic disorders, including asthma, sepsis, acute lung injury, pulmonary fibrosis, and atherosclerosis (9, 12, 17).
The LTC4 synthase enzyme, a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) enzyme family, catalyzes the conversion of LTA4 to LTC4. The biological distribution of LTC4 synthase activity is limited to eosinophils, basophils, mast cells, platelets, endothelial cells, and cells of monocyte/macrophage lineage (5, 9, 20). The gene for LTC4 synthase has been cloned, sequenced, and mapped to the distal region of chromosome 5 (4, 19). The LTC4 promoter region possesses a functional proximal promoter Sp1 consensus site and an upstream Kruppel-like factor consensus site, which mediate constitutive gene expression (26, 35). Transcriptional induction of the LTC4 synthase gene by TGF-β was previously demonstrated by our group (23).
The role of bacterial antigens, such as lipopolysaccharide (LPS), in modulating the synthesis of 5-LO pathway-derived LTs has been the subject of significant interest. Recent studies indicate that prolonged LPS exposure decreases the synthesis of 5-LO and FLAP by a NO-mediated mechanism (6), possibly representing a mechanism by which bacteria attenuate LT-mediated host defense. While studies indicate that in vivo intraperitoneal LPS exposure increases organ-specific LTC4 synthase gene expression (25), work from our group (28) indicates that prolonged LPS exposure decreases LTC4 synthase activity and gene expression in the monocyte-like cell line THP-1. LPS binds to Toll-like receptor (TLR)-4, the downstream effects of which are mediated by both myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways (31). These pathways culminate in the activation of the transcription factor NF-κB. LPS is also known to induce TNF-α release from mononuclear phagocytes (34). Whereas LPS and TNF-α both activate the downstream transcription factor NF-κB (7, 11), each stimuli appears to operate through distinct mechanisms of action (30). Importantly, our prior work (28) indicates that the downregulatory effect of LPS on the LTC4 synthase gene in mononuclear phagocytes involves a TNF-α-independent mechanism.
TNF-α is widely expressed in granulocytes, macrophages, fibroblasts, and epithelial cells. In addition to LPS, induction of TNF-α can occur in response to a variety of stimuli, including cytokines, calcium influx, and oxygen free radicals (7). TNF-α plays a critical role in normal host resistance to infection and tumorigenesis, serving as a key mediator of the inflammatory response (7). We previously reported (22) a pivotal role of TNF-α in upregulating the 5-LO pathway FLAP gene in mononuclear phagocytes. As such, TNF-α may modulate the synthesis of LTs via a distinct, TLR-4-independent mechanism, which may alter the ability of the 5-LO pathway in macrophages to respond to future bacterial (and other) antigenic challenges. The clinical consequences of such altered responsiveness may be relevant in the pathogenesis of asthma (15) and the sepsis syndrome (17).
The purpose of this study was to investigate the independent effect(s) of TNF-α on LTC4 synthase activity and gene expression in primary effector cells known to function in inflammatory diseases, such as asthma and sepsis. We now demonstrate that TNF-α acts to downregulate activity and expression of LTC4 synthase in mononuclear phagocytes, as well as in an eosinophilic cell line. The effect of TNF-α appears to involve transcriptional downregulation, mediated via sustained activation of p50 and p65 NF-κB family members.
MATERIALS AND METHODS
THP-1 cells were obtained from American Type Culture Collection (Manassas, VA). This cell line has been extensively used as a model for the study of 5-LO pathway regulation in previous work from our laboratory (26). THP-1 cells were grown at 37°C with 5% CO2 in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS), 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 250 μg/ml of gentamicin. The eosinophil-like cell line AML14.3D10 (generously provided by Dr. Cassandra Paul, Wright State University, Dayton, OH) has been utilized as an effective model for the study of multiple inflammatory mediator pathways and eosinophil-specific genes (2). AML14.3D10 cells were grown at 37°C with 5% CO2 in RPMI 1640 medium containing 10% FCS, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 1 mM sodium pyruvate. The medium was changed every 2 days for all experiments.
Isolation of primary human monocytes.
Mononuclear cell fractions were isolated from peripheral blood (obtained from normal human subjects) by a previously described technique (24). Mononuclear cells were subjected to positive magnetic selection with CD14 Microbeads, according to the manufacturer's instructions (Miltenyi Biotec; Auburn, CA). The human subject protocol was approved by the IRB at the University of California, San Diego.
Isolation of primary murine bone marrow-derived macrophages.
Wild-type C57BL/6 mice were obtained from Harlan Laboratories (Indianapolis, IN). After euthanasia, bone marrow-derived macrophages (BMDM) were isolated by a previously described technique (14). Briefly, the femurs were dissected and flushed with DMEM containing 10% FCS, 2 mM l-glutamine, 1% penicillin-streptomycin, and 30% L929 cell conditioned medium (consisting of DME high-glucose medium containing 10% FCS, 1% penicillin-streptomycin, 2 mM l-glutamine, and 1 mM sodium pyruvate). The bone marrow cells were washed once with medium, plated, and incubated at 37°C with 5% CO2 for 7 days until confluent. The animal protocol was approved by the Veterans Affairs San Diego Healthcare System.
Cell activity assay for LTC4 release.
THP-1 cells were conditioned for 3 and 7 days. Cell viability was assessed by Trypan blue staining. THP-1 cell viability for control conditions was >90% at both the 3 and 7 day time points, and cell viability for TNF-α conditions was 82% at 3 days and 75% at 7 days. Monocytes were conditioned for 48 h with TNF-α (at 10 ng/ml). Monocyte cell viability for control conditions was 86% at the 48 h time point, and cell viability for TNF-α conditions was 77% at the 48 h time point. The cells were harvested, resuspended in HBSS (at 10 × 106 cells/ml), and stimulated with the calcium ionophore A-23187 (at 1 μM) for 15 min at 37°C. After centrifugation, supernatants were assayed for LTC4 release by ELISA (Oxford Biomed, Oxford, MI and Cayman Chemical, Ann Arbor, MI), per the manufacturers' instructions. LTC4 concentrations are expressed as picograms per million cells.
Northern blot analysis for LTC4 synthase mRNA.
THP-1 cells, AML14.3D10 cells, and primary murine BMDM were conditioned with TNF-α (at 10 ng/ml), the MEK/ERK inhibitor PD-98059 (at 30 μM), JNK inhibitor II (at 15 μM), the p38 MAP kinase inhibitor SB-202190 (at 10 μM), the cyclooxygenase (COX)-2 inhibitor SC-236 (at 1.5 μg/ml), the inducible nitric oxide synthase (iNOS) inhibitor l-N6-(1-iminoethyl)lysine (l-NIL; at 25 μM), and/or the transcription inhibitor actinomycin D (at 2 μg/ml). SC-236 has been demonstrated to inhibit COX-2 activity at a concentration of 0.8 μg/ml in previous studies from our group (13). At 25 μM, l-NIL has been shown to inhibit >90% of iNOS activity (16). After conditioning, total cellular RNA was isolated and subjected to electrophoresis on a 1% agarose-2.2 M formaldehyde gel. RNA was then blotted overnight onto nylon membranes (Zeta-Probe; Bio-Rad; Hercules, CA). The blots were probed with a 32P-labeled full-length cDNA probe for human LTC4 synthase (4) or murine LTC4 synthase, washed under high-stringency conditions, and exposed to autoradiographic film. Loading equivalence and transfer efficiency were assessed by probing with 32P-labeled full-length cDNA probes for human or murine β-actin.
Nuclear extracts were harvested from control, LPS, and TNF-α-conditioned THP-1 cells and BMDM after various periods of incubation with a Nuclear Extract Kit, according to the manufacturer's instructions (Active Motif; Carlsbad, CA). The activation of NF-κB family members p50, p65, p52, c-Rel, and RelB were quantified in the extracts by ELISA (Active Motif).
LTC4 synthase promoter segments were ligated into a pGL3 Basic firefly luciferase reporter vector as previously described to create the p1.35LTC4S and p0.62LTC4S constructs (26). The constructs were purified with an endotoxin-free Qiagen-tip 500 column (Qiagen; Chatsworth, CA) according to the manufacturer's instructions. THP-1 cells (2 × 106 cells per condition) were transiently transfected (at a DNA-to-Effectene ratio of 1:10) with 900 ng of a pGL3 LTC4 synthase promoter-reporter construct utilizing Effectene reagent (Qiagen) by a previously described technique (26, 28). After transfection for 4 h, the cells were resuspended in RPMI 1640 medium containing 10% FCS and incubated in the presence of TNF-α (at 10 ng/ml) for 12 h at 37°C with 5% CO2.
Reporter gene assays.
Transfected cells were harvested and lysed with 100 μl of Promega passive lysis buffer (Promega; Madison, WI). The extracts were assayed for firefly luciferase activity with the Promega Dual Luciferase Assay System (Promega) according to the manufacturer's instructions. Measurements were made with an Optocomp I luminometer (MGM Instruments; Hamden, CT). Firefly luciferase activity was normalized to the luciferase activity of pGL3 Basic (empty vector) and expressed as a percentage of maximal activity of the untreated cells.
FCS, penicillin, streptomycin, sodium pyruvate, and gentamicin were obtained from the Cell Culture Facility, University of California (San Diego, CA). RPMI 1640 medium was obtained from BioWhittaker (Walkersville, MD). Salmonella minnesota Re595 LPS was generously provided by Dr. Theo Kirkland, Department of Veterans Affairs (VA) San Diego Healthcare System (San Diego, CA). PD-98059, SB-202190, JNK inhibitor II, SC-236, l-NIL, and A-23187 were obtained from Calbiochem (La Jolla, CA). TNF-α was obtained from R&D Systems (Minneapolis, MN). All restriction enzymes were obtained from GIBCO (Gaithersburg, MD). All synthesized oligonucleotides were obtained from Cruachem (Dulles, VA). Autoradiographic film was purchased from Eastman Kodak (Rochester, NY). The pGEM-T, pGL3 Basic, and pGL3 Control vectors were obtained from Promega. All other reagents were from Sigma (St. Louis, MO) and were of the finest grade available.
Data are expressed as means ± SE in all circumstances where mean values are compared. Data were analyzed by unpaired Student's t-test (InStat, version 2.03, GraphPad Software, San Diego, CA). Differences were considered significant when P < 0.05.
TNF-α decreases calcium ionophore-stimulated LTC4 release from THP-1 cells and primary human monocytes.
To determine the effect of TNF-α on the capacity for cysteinyl LT synthesis and release, THP-1 cells were conditioned for 3 and 7 days and human monocytes were conditioned for 48 h with TNF-α (at 10 ng/ml). The cells were harvested and stimulated with the calcium ionophore A-23187 (at 1 μM), with LTC4 release quantitated by ELISA. At the time point of 3 days, TNF-α conditioning resulted in a decrease in ionophore-stimulated LTC4 release from THP-1 cells to 45% of control [n = 3, P = not significant (NS)]. At the time point of 7 days, TNF-α conditioning resulted in a decrease in ionophore-stimulated LTC4 release from THP-1 cells to 42% of control (n = 4, P < 0.0001) (Fig. 1A). Similarly, at the time point of 48 h, TNF-α conditioning resulted in a significant decrease in ionophore-stimulated LTC4 release from monocytes to 58% of control (n = 3, P < 0.01) (Fig. 1B).
TNF-α downregulates LTC4 synthase mRNA accumulation in THP-1 cells, AML14.3D10 cells, and primary murine BMDM.
To determine the effect of TNF-α on LTC4 synthase mRNA accumulation, THP-1 cells, AML14.3D10 cells, and primary murine BMDM were conditioned for 24 h (THP-1 and AML14.3D10 cells) and 16 h (BMDM) with TNF-α (at 10 ng/ml). Total RNA was extracted, and LTC4 synthase mRNA was analyzed by Northern blotting. Treatment with TNF-α resulted in a significant suppression of LTC4 synthase mRNA accumulation in THP-1 cells (0.34 ± 0.02 densitometric units normalized to control; mean ± SE; n = 3, P < 0.0001) (Fig. 2, A and B), AML14.3D10 cells (0.36 ± 0.03 densitometric units normalized to control; mean ± SE; n = 3, P < 0.0001) (Fig. 2, C and D), and BMDM (0.15 ± 0.04 densitometric units normalized to control; mean ± SE; n = 3, P < 0.0001) (Fig. 2, E and F).
TNF-α downregulates LTC4 synthase mRNA accumulation in a dose-dependent manner in THP-1 cells.
To determine whether the observed downregulatory effect of TNF-α on LTC4 synthase mRNA accumulation is dose dependent, THP-1 cells were conditioned for 16 h with TNF-α (at doses ranging from 0.1 to 25 ng/ml). The addition of TNF-α resulted in a dose-dependent decrease in LTC4 synthase mRNA accumulation with an IC50 of ∼1 ng/ml (Fig. 3, A and B).
TNF-α induces a time-dependent, biphasic pattern of LTC4 synthase mRNA accumulation in THP-1 cells.
To determine the time course of the downregulatory effect of TNF-α on LTC4 synthase mRNA accumulation, THP-1 cells were conditioned for up to 24 h with TNF-α (at 10 ng/ml). Treatment with TNF-α resulted in a transient 17% increase in LTC4 synthase mRNA accumulation within 1 h, followed by a progressive decrement in mRNA to 31% of control by 24 h (Fig. 3, C and D).
COX-2 and iNOS do not mediate the effect of TNF-α on LTC4 synthase mRNA accumulation in THP-1 cells.
To determine whether COX-2 and iNOS play a role in mediating the downregulatory effect of TNF-α on LTC4 synthase mRNA accumulation, THP-1 cells were preconditioned for 2 h with the COX-2 inhibitor SC-236 (at 1.5 μg/ml) or the iNOS inhibitor l-NIL (at 25 μM), followed by conditioning with TNF-α (at 10 ng/ml) for an additional 16 h. Compared with control, inhibition of neither COX-2 (0.26 ± 0.09 densitometric units normalized to control; mean ± SE; n = 3, P < 0.05) nor iNOS (0.25 ± 0.04 densitometric units normalized to control; mean ± SE; n = 3, P < 0.01) blocked the effect of TNF-α (0.3 ± 0.05 densitometric units normalized to control; mean ± SE; n = 3, P < 0.001) (Fig. 4, A and B).
MEK/ERK pathway activity mediates downregulatory effect of TNF-α on LTC4 synthase mRNA accumulation and enzymatic activity in THP-1 cells.
To determine the role of MAP kinase signaling pathways in mediating the downregulatory effect of TNF-α on LTC4 synthase mRNA expression, THP-1 cells were preconditioned for 2 h with the MEK/ERK inhibitor PD-98059 (at 30 μM), JNK inhibitor II (at 15 μM), or the p38 inhibitor SB-202190 (at 10 μM), followed by conditioning with TNF-α (at 10 ng/ml) for an additional 16 h. Inhibition of the MEK/ERK pathway abrogated the downregulatory effect of TNF-α on LTC4 synthase mRNA (Fig. 4, C and D). Consistent with previous studies from our group (28), inhibition of the MEK/ERK pathway resulted in an induction of LTC4 synthase mRNA expression by an undefined mechanism.
To determine whether MEK/ERK inhibition blocks the TNF-α-associated downregulation of LTC4 synthase enzymatic activity as well, THP-1 cells were conditioned for 7 days with PD-98059 (at 30 μM) and TNF-α (at 10 ng/ml). Conditioning with TNF-α resulted in a decrease in stimulated LTC4 release to 54% of control (n = 3, P < 0.05), similar to the findings in Fig. 1. In the presence of both TNF-α and PD-98059, stimulated LTC4 release was 111% of control (n = 3, P = NS), indicating that MEK/ERK inhibition abrogates the downregulatory effect of TNF-α on LTC4 synthase enzymatic activity.
TNF-α does not accelerate LTC4 synthase mRNA degradation in THP-1 cells.
To determine whether the downregulatory effect of TNF-α on LTC4 synthase mRNA was associated with enhanced RNA degradation, THP-1 cells were conditioned with actinomycin D (at 2 ng/μl) in the presence or absence of TNF-α (at 10 ng/ml) for up to 10 h. Conditioning with TNF-α does not shorten LTC4 synthase mRNA half-life, as determined by the slope of mRNA decay (from the peak expression level) of the TNF-α-treated cells (−1,421) compared with the slope for control cells (−1,371) (Fig. 5). These data suggest that TNF-α does not accelerate the rate of LTC4 synthase mRNA decay. Interestingly, the decrease in LTC4 synthase mRNA observed at 4 h in the dose dependence experiment (Fig. 3, C and D) was inhibited by actinomycin D at the 4 h time point (Fig. 5). These findings suggest that the early LTC4 synthase mRNA induction event (at 0.25 h) and the subsequent progressive inhibition of LTC4 synthase mRNA (at least at 4 h) require new gene transcription.
TNF-α induces sustained activation of NF-κB component proteins p50 and p65 in THP-1 cells.
To determine the effect of TNF-α on the activation of the NF-κB family members, these proteins were quantified in nuclear extracts obtained from TNF-α- or LPS-treated THP-1 cells. TNF-α resulted in sustained activation of p50 and p65 (as well as p52 and RelB) in THP-1 cells as early as 4 h, persisting through 48 h (Fig. 6, A and E). Similarly, sustained activation of p65 was observed in BMDM (Fig. 6B). In contrast, LPS treatment resulted in only transient induction of p50 and p65 activation in THP-1 cells (Fig. 6C) and transient induction of p65 activation in BMDM (Fig. 6D), with normalization by 48 h. TNF-α treatment resulted in no induction of c-Rel (Fig. 6E).
TNF-α downregulates LTC4 synthase promoter activity in THP-1 cells.
To determine whether TNF-α downregulates LTC4 synthase gene transcription, THP-1 cells were transiently transfected with a luciferase reporter construct containing the first 1.35 kb of the LTC4 synthase promoter (p1.35LTC4S) or the first 620 bp of the promoter (p0.62LTC4S) and subsequently conditioned with TNF-α (at 10 ng/ml). TNF-α significantly decreased reporter activity, indicating that the downregulatory effect of TNF-α is mediated by an inhibition of LTC4 synthase gene transcription (Fig. 7).
In this study, we demonstrate that prolonged TNF-α exposure decreases the capacity for cysteinyl LT release from a monocytic cell line and primary human peripheral blood monocytes. Our findings elucidate a distinct, TNF-α-specific pathway by which inflammatory stimuli, such as bacterial products, may modulate the activity of LTC4 synthase and synthesis of cysteinyl LTs in inflammatory cells. The effect of TNF-α on LTC4 synthase is not mediated via COX or iNOS pathways but is mediated via the MEK/ERK signaling pathway. TNF-α dose-dependently downregulates LTC4 synthase mRNA expression, exhibiting an IC50 of ∼1 ng/ml. Our studies further demonstrate a biphasic effect of TNF-α, with a transient induction in LTC4 synthase mRNA (occurring within 15 min to 1 h), followed by a progressive decrement in mRNA. TNF-α suppresses LTC4 synthase mRNA expression via decreased promoter activity, but not via enhanced RNA degradation. In addition, TNF-α induces a pattern of sustained p50 and p65 activation, which is distinct from the transient activation induced by LPS. These findings are consistent with our previous work (28) indicating that expression of these NF-κB component proteins is an important downstream event involved in the inhibition of LTC4 synthase gene transcription in THP-1 cells.
Our present findings and those from prior studies support the independent role of TNF-α in coordinating and directing the synthesis of select 5-LO pathway-derived LTs, each of which is known to possess unique biological properties. We previously demonstrated (22) that TNF-α functions to upregulate FLAP gene expression in THP-1 cells. The observed effect(s) of TNF-α in the present study suggests a crucial parallel regulatory pathway, independent of the LPS/TLR-4-mediated mechanism, by which inflammatory stimuli modulate LTC4 synthesis in both mononuclear phagocytes and eosinophilic cells. Such a pathway may represent an alternate mode of amplification of the acute inflammatory response to the presence of bacteria. This is suggested by the acute transient induction of LTC4 synthase mRNA (within 15 min to 1 h) that we observe in this study. Such an early event appears to involve a preformed transcription factor (as it is not inhibited by actinomycin D) and may allow an acute increase in LT synthetic capacity in response to invading bacteria. However, accumulating data suggest that prolonged exposure to bacterial components and the cytokines/factors that they induce, such as TNF-α, may attenuate the capacity of inflammatory cells to generate cysteinyl LTs in response to future stimuli. Whether this phenomenon serves as a means by which persistent bacterial exposure acts to subvert the host defense or an intrinsic regulatory mechanism to moderate an ongoing inflammatory response is not clear. Such theories seem plausible, as 5-LO pathway metabolites have been increasingly implicated in host defense (17, 21). Such a mechanism may also account for the suppression of cysteinyl LT generation in allergic disease by persistent exposure to environmental antigens such as bacterial components. Such an assertion would be consistent with the “hygiene hypothesis,” as has been proposed to describe the role of bacterial antigen exposure in the pathogenesis of asthma (15).
Our present findings expand a body of work indicating that prolonged exposure to LPS modulates 5-LO pathway activity (6). Previous work from our group indicates that LPS induces FLAP expression (29) and suppresses cysteinyl LT synthesis and LTC4 synthase expression in THP-1 cells (28). In our prior studies using TNF-α neutralization experiments (28), we demonstrated that LPS suppresses cysteinyl LT synthesis via a TNF-α-independent mechanism. In contrast to our present data, the effect of LPS on LTC4 synthase gene expression in the prior study occurred in a cell-specific manner, being observed in monocytic cells but not in eosinophilic cells (27). The exact significance of the confinement of the effect of LPS to cells of monocyte/macrophage lineage, with TNF-α acting in a less cell-specific manner, is unclear. Although the overlapping biological functions of LPS and TNF-α have not yet been fully defined, it is evident that significant cross talk exists between the LPS and TNF-α signaling pathways. Such cross talk may occur at the level of upstream MAP kinase activation or at the level of downstream transcription factor activation. Deletion of the TNF-α receptor has been shown to sensitize macrophages to the effects of LPS (30), possibly because of alterations in the activation of NF-κB family members. Our findings indicate that LPS and TNF-α exhibit distinct kinetics with regard to p50 and p65 activation. These differential patterns of NF-κB family member activation suggest that TNF-α acts through a parallel host defense mechanism in response to bacterial antigens, which functions independently of TLR-4.
TNF-α signaling occurs via two classes of cell surface receptors, TNFR1 and TNFR2, and is mediated via the tumor necrosis receptor-associated factor (TRAF) adapter protein family. Receptor events trigger the activation of multiple downstream kinases, including JNK, phosphoinositide 3-kinase, NF-κB-inducing kinase, and p38 MAP kinase (7, 32). Our data demonstrate that the effect of TNF-α on LTC4 synthase gene expression and activity in eosinophils and monocytes/macrophages is mediated via the MEK/ERK pathway. Although the involvement of MEK/ERK in the TNF-α signaling pathway has not been as well described as that of JNK, our findings are consistent with previous work supporting a role of the MEK/ERK pathway in TNF-α signaling via the NF-κB-associated regulation of genes such as IL-6 (32). Moreover, our previous work (27) indicates that MEK/ERK inhibition does not block the downregulatory effect of LPS on LTC4 synthase, further supporting distinct mechanisms of action of LPS and TNF-α. In contrast to prior studies that suggest that TNF-α induces the expression of iNOS (10) and COX-2 (33), our data indicate that the JNK, COX, or iNOS pathways do not play a role in TNF-α signaling in our system. Although TNF-α signaling has been reported to be mediated by a number of pathways, the activation of the transcription factor NF-κB may be a common end point in this signaling cascade.
Both LPS and TNF-α are known to induce NF-κB activation in monocytes/macrophages (8). LPS-induced activation of p50 and p65 is the most well-defined dimer in the NF-κB signaling pathway. The pattern of NF-κB induction is typically transient, occurring within 1–4 h of LPS exposure (29, 30). Our data are consistent with this finding, as we demonstrate transient LPS induction of both p50 and p65 in THP-1 cells. A similar transient induction of p65 is observed in BMDM, with normalization by 48 h. In contrast, TNF-α induces a unique pattern of sustained p50 and p65 activation in THP-1 cells and sustained p65 activation in BMDM. As we previously showed (28) that overexpression of p50 and p65 leads to downregulation of LTC4 synthase expression in THP-1 cells, our present findings indicate that TNF-α downregulates LTC4 synthase gene transcription through the persistent activation of p50 and p65. The transfection assays support this assertion, in that TNF-α decreases LTC4 synthase promoter activity via an element located within the first 620 bp of the promoter. Data from the actinomycin D studies indicate that TNF-α does not enhance LTC4 synthase mRNA degradation. These findings are consistent with a transcriptional mechanism.
We acknowledge that it is also possible that TNF-α suppresses LTC4 synthesis through alterations in expression of the upstream cytosolic (c)PLA2 or 5-LO pathway genes/enzymes (including 5-LO and FLAP). However, we previously demonstrated (22) that TNF-α upregulates FLAP gene expression in THP-1 cells. Additionally, TNF-α does not appear to significantly alter 5-LO gene expression (unpublished data). Conditioning with TNF-α does not decrease constitutive or A-23187-induced LTB4 levels in peripheral blood monocytes and THP-1 cells (data not shown). Collectively, these findings suggest that the downregulation of LTC4 synthesis by TNF-α is not mediated through suppression of other 5-LO pathway genes/enzymes. We further acknowledge that because we did not measure total arachidonic acid release, the possibility that TNF-α may decrease substrate release at the level of phospholipase cannot be completely excluded.
In summary, we demonstrate that TNF-α acts via a distinct parallel pathway to downregulate the activity and expression of the LTC4 synthase gene in mononuclear phagocytes and an eosinophilic cell line. This observation may have important implications for the modulation of synthesis of LTs and their role in host defense in conditions such as sepsis. In addition, this observation could potentially explain the role of bacterial exposure in the downregulation of LT synthesis in allergic disorders, such as asthma.
This work was supported by a VA Advanced Research Career Development Award (K. J. Serio), a University of California, San Diego Academic Senate Grant (K. J. Serio), and by National Cancer Institute Grant U01/CA-96134-01 (J. T. Mao).
We thank I-Hsien Tsu for excellent technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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