HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 25, 22131-22139, June 21, 2002

This Article Abstract Full Text (PDF) An addition or correction has been published All Versions of this Article: 277/25/22131    most recent M111883200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hsu, H.-Y. Articles by Wen, M.-H. Search for Related Content PubMed PubMed Citation Articles by Hsu, H.-Y. Articles by Wen, M.-H. Social Bookmarking                      What's this?

Lipopolysaccharide-mediated Reactive Oxygen Species and Signal Transduction in the Regulation of Interleukin-1 Gene Expression^*

Hsien-Yeh Hsu^§ and Meng-Hsuan Wen

From the  Faculty of Medical Technology, Institute of Biotechnology in Medicine, National Yang-Ming University, 112 Taipei, Taiwan

Received for publication, December 13, 2001, and in revised form, March 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipopolysaccharide (LPS) stimulates macrophages to release inflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor (TNF). LPS-induced TNF suppresses scavenger receptor functions in macrophages (van Lenten, B. J., and Fogelman, A. M. (1992) J. Immunol. 148, 112-116), which is regulated by TNF-mediated protein kinases (Hsu, H. Y., and Twu, Y. C. (2000) J. Biol. Chem. 275, 41035-41048). To examine the molecular mechanism for LPS induction of IL-1 in macrophages, we demonstrated that LPS quickly stimulated reactive oxygen species (ROS), and 3 h later induced prointerleukin-1 (pro-IL-1, precursor of IL-1) production and IL-1 secretion. LPS stimulated pro-IL-1 message/protein between 3 and 10 h; however, there was a 40% reduction of pro-IL-1 in preincubation of the antioxidant, N-acetylcysteine (NAC). Moreover, NAC moderated LPS-induced IL-1 secretion partially via interleukin 1-converting enzyme. The maximal activity of LPS-induced ERK, JNK, and p38 was 12- (30 min), 5- (30 min), and 16-fold (15 min), respectively. In contrast, NAC reduced ERK activity to 60% and decreased p38 activity to the basal level, but JNK activity was induced 2-fold. Furthermore, the pharmacological antagonists LY294002, SB203580, curcumin, calphostin C, and PD98059 revealed the diverse roles of LPS-mediated protein kinases in pro-IL-1. On the other hand, NAC and diphenyleneiodonium chloride partially inhibited LPS-induced Rac activity and protein-tyrosine kinase (PTK), indicating that LPS-mediated ROS and NADPH oxidase correspond to Rac activation and IL-1 expression. Our findings establish for the first time that LPS-mediated PTK/phosphatidylinositol 3-kinase/Rac/p38 pathways play a more important role than pathways of PTK/PKC/MEK/ERK and of PTK/phosphatidylinositol 3-kinase/Rac/JNK in the regulation of pro-IL-1/IL-1. The findings also further elucidate the critical role of LPS-mediated ROS in signal transduction pathways. Our results suggest that understanding LPS-transduced signals in IL-1 induction upon the antibacterial action of macrophages should provide a therapeutic strategy for aberrant inflammatory responses leading to severe cellular injury or concurrent multiorgan septic damage.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipopolysaccharide (LPS¹ or endotoxin) is a potent activator in the immune system. It induces a variety of immune responses to severe infection (3-5), such as local inflammation, antibody production, and septic shock. In addition, LPS is the major mediator of the high morbidity and mortality rates characteristic of Gram-negative bacteria, i.e. endotoxic shock (3, 5). The architecture of LPS consists of three separated "building blocks" as follows: lipid A, an inner core region, and the O-specific side chain. These separate building blocks have widely different compositions and structures that are reflected in their biological activities (6). In early immune response for LPS, macrophages, which are the major cellular targets for LPS action, play a central role in host defense with physical and immune responses against bacterial infection (4). It is thought that LPS-induced signal transmission requires binding to specific cellular receptors, including toll-like receptors (5, 7-9). Whereas bacterial products are binding to toll-like receptors on macrophages, the cells are activated and stimulate a wide spectrum of host defensive systems. This requires the activation of multiple signaling molecules in transduction pathways, for example protein-tyrosine kinase (PTK), LPS receptor-associated serine/threonine kinase, Ras, Raf-1, IB kinase, MEK, mitogen-activated protein kinases (MAPKs) (4, 10-16), etc. These molecules may converge or diverge and often have "cross-talk" properties, which make signaling networks complicated and with mutual influence. Subsequently, the signals further transduce to downstream pathways and activate numerous transcription factors, including AP-1, NF-B (13), and ATF-2 (17). This in turn triggers a large amount of genes encoded for inflammatory mediators and cytokines. These elicited cytokines are believed to be responsible for cell proliferation, differentiation, immunoregulation, and cytotoxicity for bacteria and bacterial products (6).

It is known that LPS has multiple and different effects on macrophages, such as the regulation of macrophage functions (1, 2) and the induction of inflammatory cytokines such as IL-1, IL-6, and TNF (12, 15, 18, 19). Although IL-1 is a potent inflammatory cytokine with various biological activities regulating host defense and immune responses (20), there has been less investigation of LPS induction of IL-1. In the process of IL-1 maturation, a precursor molecule referred to as prointerleukin-1 (pro-IL-1) is produced in the cytosol of macrophages. Originally, pro-IL-1 is a 31-34-kDa non-active form of cytokine, until it is cleavaged via an enzymatic procession into a 17-kDa mature functional form by IL-1-converting enzyme (ICE) or caspase 1 (21-24). After the ICE cleavage, the active IL-1 is released and exhibits its diverse biological functions.

As growth factors or cytokines, LPS induces MAPKs, including extracellular signal-regulated kinase (ERK), c-JUN NH₂-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38). These play key roles in the LPS-mediated signal transduction between extracellular membrane stimulation and the cytoplasmic response and nuclear activity of the activation of the gene (11, 17). Specifically, ERK activation involves cytokine induction and regulation during responses to bacterial products (25-27). In addition to responding to numerous physiological and stress stimuli (28-31), JNK is considered to play roles in regulating the expression of various stress-induced proteins and inflammatory cytokines (12, 31). p38 is activated in response to stress signals such as LPS, osmotic stress, and pro-inflammatory cytokines (14, 27, 32, 33). Previous studies have shown that the p38 pathway is critical for LPS-stimulated cytokines release (25, 33), including IL-1 and TNF induction in monocytes (17). Moreover, previous findings (34, 35) indicated that p38 activity could be regulated via Rho GTPase (Rac1) and PAK.

Reactive oxygen species (ROS) regulate multiple cellular functions such as DNA synthesis (36), transcription factor activation (37), gene expression (38), and proliferation (39). The induction of H₂O₂ can affect various gene expressions in macrophages ((40, 41) and this work). There are several indications that H₂O₂ may act as a cellular secondary messenger (37, 42). H₂O₂ is considered to activate NF-B (37), which regulates the expression of multiple immune and inflammatory molecules. Therefore, for the cell immune system, H₂O₂ displays a critical role in the host defense mechanism. Alternatively, the activation of ROS including H₂O₂ acts as a significant and adverse participant in abnormal inflammatory diseases. One of the important sources of ROS in phagocytes, including macrophage, is NADPH oxidase activation during phagocytosis (43). NADPH oxidase, which could be inhibited by diphenyleneiodonium chloride (DPI), is a membrane-associated complex, which consists of a minimum of four proteins, i.e. membrane-bound cytochrome b₅₅₈, p47^phox, p67^phox, and Rac2 in humans or Rac1 in rodents (43). Among these components, Rac is the most critical for a functional NADPH oxidase whose activity is regulated by small GTP-binding proteins. In response to phagocytic stimuli, i.e. lipids, soluble peptides, and opsonized particulates, NADPH oxidase moves electrons from NADPH to reduce O₂ to O. These superoxide free radicals are rapidly converted to H₂O₂ and O₂ by cytosolic superoxide dismutase (44, 45) and then sequentially to other products. Nevertheless, both the mechanism for LPS induction ROS and the role of induced ROS in cytokine expression of macrophages are still unclear.

To understand how ligation of LPS induces inflammatory cytokine IL-1 gene expression in macrophages, we studied the molecular mechanism for LPS-mediated signal transduction pathways for various protein kinases in the regulation of IL-1 gene expression. Here we demonstrate for the first time that LPS regulation of pro-IL-1/IL-1 expression in macrophages J774A.1 is partially mediated by NADPH oxidase-derived ROS, as well as by accompanying the PTK/PI 3-kinase/Rac1/p38 pathway, but less by the PKC/MEK/ERK pathway and the PTK/PI 3-kinase/Rac1/JNK pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Cultures

Murine macrophage J774A.1 cell (J774A.1) was obtained from the ATCC (Manassas, VA), propagated in RPMI 1640 medium supplemented with 10% heated-inactivated fetal bovine serum (HyClone, Logan, UT) and 2 mM L-glutamine (Invitrogen), and cultured in 37 °C, 5% CO₂ incubator, unless otherwise indicated.

Materials

LPS (from Escherichia coli 0111:B4), sodium orthovanadate, phenylmethylsulfonyl fluoride, bovine serum albumin (fraction V), DPI, N-acetylcysteine (NAC), and curcumin were purchased from Sigma. Calcium Phosphate Transfection® reagent was purchased from Invitrogen. Immobilon® polyvinylidene difluoride membrane was obtained from Millipore Inc. (Bedford, MA). Non-radioactive Western blot Chemiluminescence Reagent, Renaissance®, was purchased from PerkinElmer Life Sciences. RE_ZOl®C&T was from PROtech Technology Co. (Taipei, Taiwan). GeneAmp® RNA PCR kit for RT-PCR amplification was purchased from PerkinElmer Life Sciences.

Growth Factors and Antibodies-- Anti-IL-1, 3ZD monoclonal antibody (a gift from the National Institutes of Health, Bethesda, to Dr. H.-Y. Hsu), anti-rabbit IgG-HRP, anti-mouse IgG-HRP, and protein A/G plus-agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phosphotyrosine, clone 4G10 (mouse monoclonal IgG2b), was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The CaspACE® Assay System, Fluorometric, was purchased from Promega Co. (Madison, WI).

Kinase Assay Kits-- The p44/42 MAPK assay kit, stress-activated protein kinase/JNK assay kit, and p38 MAPK assay kit were purchased from Cell Signaling Technology (Beverly, MA). The Rac activation assay kit was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).

Protein Kinase Inhibitors-- PD98059 was from Cell Signaling Technology; calphostin C and herbimycin A were from Calbiochem-Novabiochem; LY294002 and SB203580 were from Sigma.

Oligonucleotides-- Primers for pro-IL-1/IL-1 and glyceraldehyde phosphate dehydrogenase were synthesized from local MD Bio. Inc. (Taipei, Taiwan). The dominant negative JNK construct (DN-JNK) was a gift from Dr. Michael Karin (University of California, San Diego) (46). The dominant negative Rac1 construct (DN-Rac1) was a gift from Dr. S. Bagrodia (Cornell University, Ithaca, NY) (47).

Measurement of LPS-induced Intracellular ROS

Intracellular ROS stimulated by LPS was measured by detecting the fluorescent intensity of either 2',7'-dichlorofluorescein diacetate (DCFH) or the improved analogue carboxyl-DCFH (CM-DCFH) (Molecular Probes, Inc., Eugene, OR) oxidized product, DCF (or CM-DCF), as described (48). Briefly, 0.5-1.0 × 10⁶ J774A.1 cells/ml grown in serum- and phenol red-free RPMI medium (starvation medium) for 24 h were preincubated with 2 µmol/liter CM-DCFH and NAC at 37 °C for 30 min in the dark. To these were added fresh starvation medium containing LPS for additional incubation at the indicated times. The relative fluorescent intensity of fluorophore CM-DCF, which was formed by peroxide oxidation of the non-fluorescent precursor, was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm with a fluorometer, Cytofluor 2300 (Millipore Inc., Bedford, MA). In contrast, CM-DCFH with starvation medium was used as a blank control.

Measurement of LPS-induced NO

Cells were seeded in a 1 × 10⁷/100-mm plate containing 6 ml of media. After 24 h of incubation, the media were replaced by fetal bovine serum-free medium 1 h prior to LPS treatment, and the conditioned media were collected at the indicated times. For assaying NO, 50 µl of conditioned media of each sample was mixed with 50 µl of Griess reagent I (1% sulfanilamide dissolved in 5% H₃PO₄) in a 96-well assay plate at room temperature for 10 min. This was followed by addition of Griess reagent II (0.1% (w/v) N-1-naphthylethylenediamine) at room temperature in the dark for another 10 min. The absorbance of reaction (wavelength at 550 nm) in each well was measured by an MRX microplate reader (Dynex Tech. Inc., Chantilly, VA). A standard calibration curve for NaNO₂ was constructed, and calculation of the relative absorbance unit for the NO concentration of each sample was as described (19).

RNA Isolation, RT and PCR Amplification for Detecting the Expression of Pro-IL-1/IL-1, Analysis of ICE Activity, Western Blot Analysis, Assay Activity of ERK, JNK, p38, and Enzyme-linked Immunosorbent Assay (ELISA) for Measurement of IL-1

All methods and procedures followed the previous descriptions (34).

Measurement of LPS-induced Rac Activity

The measurement of LPS-induced Rac activity followed the manufacturer's instructions from Upstate Biotechnology, Inc. Briefly, cells were starved for 18 h and then pretreated with or without NAC for 30 min prior to LPS stimulation. The whole cell lysates were collected at the indicated times and immediately incubated with PAK-1 p21-binding domain agarose (PAK-1 PBD) for immunoprecipitating active PAK-1/Rac-GTP complex. Because the Rac-GTP is able to associate with PAK-1, the amount of activated form of Rac was detected via mouse monoclonal IgG_2b anti-Rac by Western blot analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LPS-induced ROS in Murine Macrophage J774A.1 Cells-- To investigate LPS-induced ROS in J774A.1 cells, detection of fluorescent oxidative product of CM-DCFH was used to examine cell oxygen bursts (48). As shown in Fig. 1A, within several minutes LPS (1 µg/ml) stimulation of cells rapidly induced significant ROS, including more H₂O₂ production, when compared with that of LPS-untreated control samples, although there was less, but still significant, H₂O₂ generation by low doses of LPS (0.1 µg/ml) (data not shown). In contrast, pretreatment of NAC (10 mM), a potent antioxidant, quickly reduced LPS-induced ROS release (Fig. 1A) in a dose-dependent fashion (data not shown). The difference of LPS-induced ROS production in cells treated with or without NAC continued for the longer testing period of up to 240 min (Fig. 1B). NAC approximately attenuated 20% of H₂O₂ in cells at the steady state (Fig. 1, A and B). However, we could not totally rule out the involvement of other ROS including superoxide anion (O) (data not shown). Because LPS induces NO (19, 49, 50), a member of the ROS superfamily which plays multiple biological roles in macrophages, we extended our examination to measure LPS-induced NO in our system. The cells were grown at various times in media with or without LPS, and the released NO in conditioned media was measured. In the early stage of LPS stimulation, there was no detectable NO released from the cells (Fig. 1C). After incubation of LPS for 7 h, cells gradually produced NO at about 2 µM, and at 24 h the concentration of NO reached 22 µM. In contrast, in cells co-incubated with NAC, LPS-induced NO reduced to 0.8 and 6.4 µM at 7 and 24 h, respectively (Fig. 1C).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of NAC on LPS-induced release of ROS and NO production. To detect release of ROS, J774A.1 cells were preincubated with CM-DCFH (2 µM) and NAC (10 mM) for 30 min, followed by substitution with medium containing LPS (1 µg/ml) for additional incubation for the indicated times. The relative fluorescent intensity of fluorophore CM-DCF was then detected. The data expressed are one of four representative experiments. LPS-induced release of ROS over a short period between 0 and 15 min (A); over a long period between 0 and 240 min (B). C, to detect LPS-induced NO production, cells (1 × 107/6 ml of medium/100-mm plate) were stimulated with LPS (0.1 µg/ml), and supernatants were collected after LPS stimulation at the time indicated and assayed for the NO concentration by the Griess method. The data are representative of three similar experiments and expressed as mean ± S.E. ctr, control.

LPS Induction and the Role of LPS-induced ROS in Regulation of IL-1 Gene Expression: IL-1 Secretion, Pro-IL-1 Protein Production, and IL-1/Pro-IL-1 Message Induction as Well as Activation of ICE-- To detect the effect of LPS on inflammatory cytokine and IL-1 gene expression by macrophages, we first used ELISA to quantitate mature IL-1 secretion in the conditioned medium of J774A.1 cells. As shown in Fig. 2A, compared with untreated control cells, IL-1 secretion increased with a longer LPS incubation time. For example, at 3 and 24 h, the concentration of LPS-induced IL-1 was about 5 and 22 pg/ml, respectively. In contrast, there was less IL-1 released from NAC-pretreated cells, i.e. 1 (3 h) and 12 pg/ml (24 h), indicating that LPS-stimulated ROS involves IL-1 secretion.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of LPS-induced ROS on LPS stimulation of pro-IL-1/IL-1 expression in macrophage J774A.1 cells. A, time course of LPS-induced IL-1 and inhibitory effect of NAC on IL-1 secretion. Cells (1 × 107/6 ml of medium) were stimulated with LPS (0.1 µg/ml), and the supernatants were collected after stimulation at the time indicated. To analyze the effect of NAC on LPS-induced IL-1 secretion, cells were incubated with NAC (10 mM) for 30 min prior to LPS stimulation. Samples were assayed for the IL-1 content in an IL-1-specific ELISA, as described under "Experimental Procedures." The experiments were conducted three times, and all data are expressed as mean ± S.E. B, time course and the effect of NAC on LPS-induced pro-IL-1 protein. After cells were stimulated as described above for the indicated time interval, cell lysates were analyzed by Western blotting with anti-IL-1 monoclonal antibody at a position of 34 kDa for pro-IL-1. Pro-IL-1 and -actin (internal control for protein loading) are indicated as arrows on the right-hand side, and one of three experiments is presented (n = 3). C, time course and the effect of NAC on LPS-mediated pro-IL-1/IL-1 mRNA expression. Total RNA was isolated from treated or untreated J774A.1 cells as indicated. Pro-IL-1 at 563 bp in the ethidium bromide-stained agarose gel was normalized by comparison to RT-PCR of mRNA of glyceraldehyde phosphate dehydrogenase (GAPDH), a constitutively expressed gene at 450 bp. Arrows indicate RT-PCR products of pro-IL-1 and glyceraldehyde phosphate dehydrogenase, respectively. The results shown are representative of three independent experiments. Quantification of protein and mRNA expression was carried out by PhosphorImager® of each sample using ImageQuant® software from Molecular Dynamics, which is expressed as fold increase relative to the level of cells without LPS treatment (t = 0, activity of control cells defined as 1). D, time-dependent activation of LPS-induced ICE activity in J774A.1 cells. After LPS and NAC treatments, cell extracts (90 µg of protein) were incubated in the presence of the fluorescence ICE substrate Ac-YVAD-AMC (50 µM) for 1 h at 30 °C. ICE activity was measured fluorimetrically after subtracting cleavage with excitation at 360 nm and emission at 460 nm, as described previously (34). All values shown are means of triplicate values ± S.E. ctr, control.

Next, we investigated whether the increased mature IL-1 was reflected by induction of a precursor of IL-1, pro-IL-1 protein (molecular mass of 34 kDa), and IL-1/pro-IL-1 message. A time course study of LPS-induced pro-IL-1 protein was performed by Western blot assay. For 3-h LPS stimulation, in the absence of NAC, a 21-fold increase of pro-IL-1 protein was detected as compared with the unstimulated cells, peaking at 8 h and about 24-fold relative to the control sample. After 12 h, pro-IL-1 protein decreased, gradually returning to the basal level at around 18 and 24 h (Fig. 2B). To examine further the role of LPS-stimulated ROS in pro-IL-1 expression, pro-IL-1 protein was determined by using cells preincubated with NAC followed by LPS treatment. As shown in Fig. 2B, NAC blockage of LPS-stimulated ROS decreased LPS-induced pro-IL-1 protein to about 35% of NAC-free cells between 3 and 12 h. However, after 18 h there was no difference in pro-IL-1 protein with or without NAC treatment.

Next, by using RT-PCR for 3-h LPS stimulation, pro-IL-1 mRNA increased more than 30-fold, as compared with untreated cells. Between 8 and 12 h, LPS-induced pro-IL-1 mRNA gradually reduced (Fig. 2C). After 18 h, the increased pro-IL-1 mRNA returned to the basal level. In contrast, in cells pretreated with NAC, followed by LPS stimulation between 3 and 8 h, there was generally about 70 to 40% of pro-IL-1 mRNA, as compared with NAC-free, LPS-treated samples (Fig. 2C). This indicated that NAC decreased LPS-induced pro-IL-1 mRNA. A similar regulation of pro-IL-1 mRNA detected by Northern analyses was compatible to the message detection by RT-PCR (data not shown).

Post-transcriptional regulation and processing of the pro-IL-1 protein into a mature IL-1 secretion via ICE has been well reported in various cells, including macrophages, as in our previous reports (22, 23, 34, 51). Because incubation of macrophages with LPS-induced pro-IL-1 protein simultaneously increased IL-1 secretion in a time-dependent fashion (Fig. 2, B versus A), we initially examined whether LPS affects ICE activity during IL-1 secretion. As shown in Fig. 2D, LPS increased ICE activity up to 2-fold between 3 and 12 h, peaking at a 4-fold increase by 18 h, and sequentially returning to the basal level at 24 h, as compared with the control cells. In contrast, there was no difference in ICE activity in NAC-pretreated cells under LPS stimulation, as compared with the control cells.

LPS Induces MAPKs Activity as Well as the Effect of LPS-induced ROS on MAPKs Activity-- To dissect LPS-mediated signal transduction pathways in the regulation of pro-IL-1 protein and IL-1 secretion, we systematically dissected LPS-stimulated MAPKs including ERK, JNK, and p38. Furthermore, we examined the role of LPS-induced ROS in mediating the activity of MAPKs. Incubation of cells with LPS led to phosphorylation of Elk-1, a transcriptional factor, indicating LPS activation of ERK (Fig. 3A). The experiments for time course of LPS-induced ERK activity were also conducted. As detected by Western blot analysis with an antibody that specifically recognized the activated, serine 383-phosphorylated form of Elk-1 by activated ERK (52), a 4-fold increase in ERK activity was detected at 15 min, reaching a maximum level of 12-fold at 30 min and reducing to 6-fold after 60 min (Fig. 3A). In comparison, there was almost no ERK activity in untreated control cells. To examine further whether LPS-induced ROS involves LPS mediation of ERK activation, cells were pretreated with NAC for 30 min prior to LPS incubation. Upon LPS stimulation, ERK activity in NAC-pretreated cells increased by 2- (15 min) and 7-fold (30 min), respectively, as compared with samples without LPS. The relatively lower ERK activity in NAC-treated cells compared with that of NAC-free cells indicates that ROS is involved in the LPS-induced ERK activity.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   LPS-induced ROS regulation of multiple MAPKs activation. J774A.1 cells were treated as described in Fig. 2. The activated ERK, JNK, and p38 were immunoprecipitated from cell lysates using specific antibodies. In vitro kinase assays were performed using the immunoprecipitants as kinases, as described previously (34). Recombinant Elk-1 fusion protein, c-Jun fusion protein, and ATF-2 fusion protein were used as substrates for ERK, JNK, and p38, respectively. LPS-induced ERK (A), JNK (B), and p38 (C) activities were monitored by phosphorylation of substrate. These were measured by quantitative immunoblotting with phospho-Elk-1 (Ser-383) antibody, phospho-c-Jun (Ser-63) antibody, and phospho-ATF-2 (Thr-71) antibody, respectively. Comparison and quantitative analysis of LPS-induced ERK, JNK, and p38 activities are represented in histograms (D). The values shown are the mean ± S.E. of quadruplicate determinations. All data of relative increased activity are expressed as comparison with untreated J774A.1 cells, (i.e. t = 0, activity of control cells defined as 1). This is representative of four such independent experiments.

The inflammatory stress response of J774A.1 cells to LPS prompted us to investigate the LPS-mediated JNK signaling pathway. Therefore, we examined whether LPS activates JNK in the cells. Stimulation of cells with LPS leads to activation of JNK in a time-dependent fashion, as determined by Western blot analysis via anti-phospho-c-Jun, which specifically recognizes the activated serine 63-phosphorylated form of c-Jun (28, 53, 54). Upon 15-min LPS incubation of cells, JNK activity gradually increased, with a maximum level of JNK activation at 30 min, about a 5-fold increase over that of untreated cells. Then after 60 min, it returned to the basal level (Fig. 3B). Moreover, experiments using NAC to study the effect of ROS on LPS-mediated JNK activity were conducted. Surprisingly, JNK activity of NAC-treated cells is super-induced up to 9-fold at both 15 and 30 min, as compared with the control cells (Fig. 3B). The reason for induction of higher JNK activity in NAC-treated cells than that of NAC-free cells (i.e. 9- versus 5-fold) upon LPS stimulation is unclear and requires further investigation.

To explore additional LPS-mediated signal transduction pathways, we further examined whether LPS induces p38 activity. This is another important member of the MAPK superfamily related to inflammatory responses (32, 33). Upon LPS stimulation, the p38 activity gradually increased, as detected by Western blot analysis with anti-phospho-ATF-2, an antibody that specifically recognizes the activated threonine 71-phosphorylated form of ATF-2 (55) (Fig. 3C). The result of time course study of LPS-induced p38 activity indicated that at around 15 min, the activity of p38 in LPS-treated cells was ~15 times greater than those of the control cells and gradually decreased to about 10 and 8 times approximately at 60 and 240 min, respectively (Fig. 3C). In contrast, deprivation of LPS-induced ROS by pretreatment of cells with NAC significantly reduced LPS-stimulated p38 activity to the basal level after about 30-240 min, as compared with NAC-free cells (Fig. 3C). A quantitative analysis of LPS-induced ERK, JNK, and p38 activity to compare the MAPKs activity with and without NAC is summarized in Fig. 3D.

The Role of LPS-induced Protein Kinases (PK) in the Regulation of Pro-IL-1 Protein Expression-- We demonstrated above that LPS stimulates a battery of MAPK activity, comparable with other recent reports (15, 26, 27, 32, 33, 56). To elucidate further the role of various LPS-induced PK-mediated signaling pathways in the regulation of pro-IL-1 protein expression, we utilized certain specific pharmacological antagonists such as LY294002, SB203580, curcumin, calphostin C, and PD98059 that inhibit the activation of PI 3-kinase, p38, JNK, PKC, and MEK1, respectively. The dose response for specific PK inhibitors was monitored by directly assaying individual kinase activity, and the effective working concentrations of PK inhibitors were determined (data not shown) as reported previously (34). Cells were preincubated with these PK inhibitors for 1 h prior to stimulation of LPS, and pro-IL-1 protein expression in indicated samples were analyzed by Western blotting with anti-IL-1 monoclonal antibody. Pro-IL-1 protein expression was reduced to 15% by LY294002, to 20% by SB203580, to 50% by curcumin, and to 70% by either calphostin C or by PD98059, as compared with pro-IL-1 in LPS-stimulated cells (Fig. 4A).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of protein kinase inhibitors on LPS-induced pro-IL-1 expression and role of ROS in LPS-induced pro-IL-1 protein of dominant negative JNK, Rac1, or PI 3-kinase-transfected cells. A, effect of inhibitor of PKC (calphostin C), of MEK1 (PD98059), of JNK (curcumin), of p38 (SB203580), of PI 3-kinase (LY294002), and NAC on LPS-induced pro-IL-1 expression. This was followed by cells pretreated with calphostin C (12.64 nM) (sample 2), PD98059 (50 µM) (sample 3), curcumin (10 µM) (sample 4), SB203580 (1 µM) (sample 5), LY294002 (50 µM) (sample 6), for 1 h or NAC (10 mM) (sample 7) for 30 min. After this, the pretreated cells were then incubated with LPS (0.1 µg/ml) for an additional 8 h. After incubation, samples were subjected to Western blot analysis of pro-IL-1, as described in Fig. 2. The arrows on the right-hand side represent positions of pro-IL-1 and -actin (an internal control). This experiment is representative of three similar experiments. Histograms represent quantification of pro-IL-1 of each sample. All data of relative quantity are expressed as comparisons with untreated control cells (sample 1, and activity of control cells defined as 1). B, role of ROS in LPS-induced pro-IL-1 protein of dominant negative JNK, Rac1, or PI 3-kinase-transfected cells. J774A.1 was transiently transfected with various dominant negative constructs for 24 h, as described previously (2, 34), and then incubated with NAC prior to LPS stimulation. Pro-IL-1 protein expression in cells was analyzed with Western blotting as shown above in A. The values shown are the mean ± S.E. of triplicate determinations.

Moreover, experiments for transient transfection of dominant negative JNK (DN-JNK), dominant negative Rac1 (DN-Rac1), or dominant negative PI 3-kinase (DN-PI3K) into J774A.1 cells were conducted in order to investigate further the role of PI 3-kinase/Rac in LPS induction of pro-IL-1. Results of Western blot analyses show that after LPS stimulation, there was about 30% less pro-IL-1 protein in DN-JNK- or DN-Rac1-transfected cells than that of control cells, and 40% less pro-IL-1 protein in DN-PI3K-transfected cells as compared with control cells (Fig. 4B, sample 2 versus samples 4, 6, and 8). In addition, preincubation of NAC in the dominant negative transfectants synergistically reduced pro-IL-1 expression, except for DN-JNK transfectant. Specifically, with NAC, there was 30% of the pro-IL-1 protein left in mock cells, and 20% of the pro-IL-1 left in either DN-Rac1-transfected cells or DN-PI3K-transfected cells, respectively (Fig. 4B, sample 3 versus samples 7 and 9). However, there was 50% of the pro-IL-1 protein left in DN-JNK-transfected cells (Fig. 4B, sample 3 versus sample 5).

LPS-induced Rac Activity and the Role of ROS in Rac Activation-- It has been reported (57-59) that LPS induces Rac/NAPDH oxidase-dependent ROS formation. To investigate further the potential upstream signaling molecules involving in LPS-mediated transducing networks, we focused on the activity of a relevant molecule, Rac1. Upon LPS stimulation, Rac1 activity was elevated 2.5-3-fold between 5 and 60 min (Fig. 5, samples 2-4 versus 1). In contrast, blocking LPS-induced ROS via preincubation of NAC consequently reduced Rac1 activity by 40%, as compared with untreated samples (Fig. 5, samples 2-4 versus samples 6-8). Moreover, DPI, an NADPH oxidase effective inhibitor (60), significantly reduced LPS-triggered Rac1 activation by 50% as compared with the samples without DPI preincubation (Fig. 5, samples 2-4 versus samples 10-12).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   The roles of LPS-induced ROS and NADPH oxidase in differential regulation of LPS-mediated Rac1 activity. Cells were cultured in the presence or absence of antioxidant (NAC, 10 mM) or NADPH oxidase inhibitor (DPI, 25 µM), followed by LPS stimulation at the indicated times. The Rac1-GTP complex was immunoprecipitated from whole cell lysate by PAK-1 PBD as well as active Rac1. Then the immunoprecipitant was quantitatively measured, using the anti-Rac antibody as described under "Experimental Procedures" or in the manufacturer's instructions. The data of relative Rac1 activity is expressed as compared with untreated control cells (sample 1, Rac1 activity is defined as 1). The mean ± S.E. of results from three replicate determinations and a representative experiment is shown.

LPS Induces PTP as Well as the Effect of LPS-induced ROS and NADPH Oxidase on PTP in J774A.1 Cells-- To elucidate the mechanism by which LPS induces IL-1, the LPS-initiated upstream signal transduction pathway was examined. For this we examined the LPS stimulation of PTP via performance of SDS-PAGE and Western blot analysis with monoclonal anti-phosphotyrosine IgG (61). As shown in Fig. 6, incubation of J774A.1 cells with LPS induced the appearance of many phosphotyrosyl proteins as compared with cells incubated with media alone. For example, after a 15-min LPS stimulation of cells, some apparent tyrosine-phosphorylated proteins of molecular mass of 38, 42, 44, 52-75, and 100-120 kDa (Fig. 6, samples 1 versus 2) were observed. These phosphotyrosyl proteins were identified and specifically immuno-reacted with anti-p38 IgG, anti-ERK IgG, anti-JNK IgG, anti-Lyn IgG, and anti-Src IgG, respectively (data not shown). In contrast, NAC and DPI were utilized to investigate the influence of LPS-induced ROS and NADPH oxidase in the tyrosine phosphorylation of proteins. Pretreatment of the cells with either NAC or DPI for 30 min prior to LPS stimulation resulted in a decrease of LPS-induced tyrosine phosphorylation of various proteins (Fig. 6, sample 2 versus samples 4 and 6), indicating the involvement of ROS in LPS-induced PTP.

View larger version (105K): [in this window] [in a new window]   Fig. 6.   LPS stimulation of protein tyrosine phosphorylation in J774A.1 cells. Cells were pretreated with NAC (10 mM) or DPI (25 µM) for 30 min prior to stimulation with or without LPS (1 µg/ml). Briefly, Sample 1, un-stimulated control; sample 2, LPS 15 min; sample 3, NAC 30 min; sample 4, NAC 30 min sequentially with LPS 15 min; sample 5, DPI 30 min; sample 6, DPI 30 min sequentially with LPS 15 min. Cells were lysed and analyzed by Western blot with anti-phosphotyrosine monoclonal antibody. The detailed method was as described previously (61). The indicated bars on the left-hand side represent molecular mass (kDa), and those on the right-hand side represent some significant molecules, as discussed in text. The experiment is representative of three similar experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LPS, a potent activator for macrophages, stimulates ROS such as H₂O₂ (see Ref. 40 and this work), superoxides (see Ref. 62 and data not shown) and NO (see Ref. 49 and this work) as well as inducing a battery of signal transductions leading to gene expression (10-16, 26, 63). However, the relationship among LPS-induced ROS and mediated various PK activation and regulation of the inflammatory cytokine IL-1 is unclear. Here we analyze and define the molecular mechanisms for LPS-stimulated ROS production, signal transductions, and PK activity in regulation of IL-1 expression.

By using the CM-DCFH fluorescent method, we demonstrate that LPS immediately induced H₂O₂ in J774A.1 cells but that pretreatment of cells with NAC effectively inhibited LPS-induced H₂O₂ elevation. We also report here that LPS induced detectable NO production, which was obviously much later than both pro-IL-1/IL-1 induction (7 versus 3 h, Figs. 1 and 2) and MAPKs activation (7 versus 1 h, Figs. 1 and 3), although NO is an important reactive oxygen molecule mediating response of LPS in macrophages (49). Our current findings suggest that LPS-induced ROS, rather than NO, play a more significant role in the activation of MAPKs and regulation of pro-IL-1/IL-1 in our system (see below). However, we could not totally rule out the effects of NO or cross-talk among NO and other ROS in these reactions.

Initially, the data from studies of ICE activity, ELISA, Western blot analysis, and RT-PCR showed that LPS-triggered H₂O₂ played an important role in the induction of IL-1 gene. To investigate further the molecular mechanism for ROS effects, we found evidence that ROS participated in IL-1/pro-IL-1 expression involving multiple levels of regulation. Specifically, in the presence or absence of antioxidant NAC, our results indicate LPS-induced ROS regulation of IL-1 expression, probably at pro-IL-1 message (transcriptional), at pro-IL-1 protein production (translational), and at the processing of pro-IL-1 to mature IL-1 releasing via modification of ICE activity (post-translational). In addition to the antioxidation of NAC involving the elevation of intracellular glutathione levels (38, 40, 64, 65), NAC is able to block the activation of JNK in response to TNF (2) or interferon co-stimulation with TNF in macrophages (66). Nevertheless, the mechanism for NAC reduction of LPS-induced ICE activity related to IL-1 secretion requires further study.

LPS stimulation of MAPKs (ERK, JNK, and p38) activity and activation of transcription factors (AP-1, NF-B, and ATF-2) have been reported previously (25, 26, 67-69). In addition, activation of MEK/ERK and p38 critical for LPS-induced cytokine production by monocytes/macrophages has been documented (25, 26, 33). However, there is little discussion of the molecular mechanism for LPS-mediated MAPKs in the regulation and expression of pro-IL-1/IL-1. We demonstrate for the first time that there were differences in the activation time and in extent of activity for LPS-stimulated ERK, JNK, and p38. Moreover, the effect of LPS-induced ROS on the activity of specific MAPK was also diverse (Fig. 3). For example, NAC blockage of LPS-induced ROS resulted in a decrease in ERK and p38 activity but an increase in JNK activity as compared with untreated cells. We summarize the effect of LPS-induced ROS on the relative activity of LPS-induced MAPKs (Fig. 3D), which indicates that the existence of ROS was more effective and important to p38 activity than either ERK or JNK activity in LPS-mediated regulation of pro-IL-1/IL-1 expression. Nevertheless, when all of the three MAPK pathways were activated simultaneously, a dramatic induction of IL-1 gene expression was observed, suggesting a cooperative effect on regulation of IL-1 among these kinases (15). We further used various pharmacological antagonists to PKs. It dissected the specific role of individual PK in the regulation of pro-IL-1 expression (Fig. 4A). Additional studies of the use of dominant PK negative transfectants verified that both NAC and specific dominant negative PK synergistically reduced LPS-induced pro-IL-1 protein expression (Fig. 4B). Although stimulation of a single PK produced only a modest effect on pro-IL-1 protein, activation of each PK pathway was important for complete induction of IL-1 gene expression.

Various studies have shown the activation of JNK and p38 were mediated via PI 3-kinase/Rac1/PAK signaling pathway (2, 47, 70). Rac1 is important to LPS-induced pro-IL-1 in at least two aspects as follows: first the role of Rac1 in NADPH oxidase assemble/function (71), and second its role in PI 3-kinase/Rac1/PAK signaling pathway. Rac is known as an important component of functional NADPH oxidase, which is localized at the plasma membrane or specific granule vesicle membrane of phagocytes, and which produces O upon phagocytosis processing (71). Because of the DPI reduction of LPS-triggered Rac1 activation (Fig. 5B), our data imply that LPS activates phagocytosis-related NADPH oxidase, which interacts with Rac1, leading signals as the initial of ROS (Fig. 7). Moreover, data on transient transfection of DN-Rac1 indicates partially reduced LPS-mediated pro-IL-1 expression. Although preincubation of NAC led to additional decreasing of pro-IL-1 (Fig. 4B), indicating the importance of Rac1 and/or NADPH oxidase and ROS in LPS-induced pro-IL-1. Our finding of NADPH oxidase and Rac1 involving ROS production is consistent with the recent report (58) of Rac1-dependent ROS in LPS-mediated TNF secretion. Indeed, our current results clearly demonstrate that the originality of LPS-induced ROS including H₂O₂ as a secondary messenger is mainly derived from NADPH oxidase and less from LPS-damaged mitochondria in the short term (data not shown) or via other molecule(s), which mediate signal transductions and regulate pro-IL-1 protein and IL-1 secretion.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   The proposed model for LPS-mediated ROS and signal transduction pathways in the regulation of pro-IL-1/IL-1 expression.

Ligation of LPS quickly induces diverse phosphotyrosyl proteins, leading to activation of various downstream molecules (27), although there is no evidence that the receptors including toll-like receptors have a functional tyrosine kinase domain. Because alteration of PTP is one of the most important signaling events leading to cellular responses (6), and to further understand how LPS is coupled to the initiation of early signaling by inducing various PTP resulting in up-regulation of IL-1 gene expression, we initially examined and identified the LPS-induced protein tyrosine phosphorylation, including MAPKs (ERK, JNK, and p38) and the Src family (Src and Lyn). Furthermore, we investigated LPS ligation, showing the relationships among LPS-induced ROS and NADPH oxidase and observed alteration of PTP. The results of antioxidant of ROS (NAC) or NADPH oxidase inhibitor (DPI) reducing LPS-induced tyrosine kinase activation suggest that ROS indeed involves LPS-induced protein tyrosine phosphorylation.

In summary, we are the first to demonstrate that in macrophages the relationship among NADPH oxidase-derived ROS, protein tyrosine phosphorylation, and the role of ROS in the regulation of IL-1 gene expression during LPS stimulation. Our results dissect the molecular mechanism of LPS-induced ROS regulation of pro-IL-1 protein and IL-1 secretion. This regulation is at multiple levels and involves LPS-mediated PTK, PK, and MAPK activity. Although LPS-mediated cooperation of multiple MAPKs induces IL-1 gene expression, we show for the first time that the LPS-induced PTK/Rac1/PI 3-kinase/p38 pathway plays a relatively more important role than the pathways of PTK/PKC/MEK/ERK and of PTK/Rac1/PI 3-kinase/JNK in the specific cytokine regulation (Fig. 7). Thus, understanding how LPS-mediated signal transduction pathways in IL-1 induction is responsible for macrophage antibacterial action may provide improved treatments for severe inflammatory responses leading to cellular injury, multiorgan damage, and concurrent sepsis.

	ACKNOWLEDGEMENTS

We gratefully acknowledge the DN-JNK construct from Dr. M. Karin (University of California, San Diego), DN-Rac1 construct from Dr. S. Bagrodia (Cornell University, Ithaca, NY), and the anti-IL-1, 3ZD monoclonal antibody from the National Institutes of Health (Bethesda).

	FOOTNOTES

^* This work was supported by Research Grants NSC 90-2321-B-010-005 (to H.-Y. H.) from the National Science Council, Taiwan, NHRI-EX90-8937SL (to H.-Y. H.) from the National Health Research Institutes, Taiwan, and A-91-B-FA09-2-4 (to H.-Y. H.) Program for Promoting Academic Excellence of Universities from the Ministry of Education, Taiwan.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.

^§ To whom reprint requests and correspondence should be addressed: Faculty of Medical Technology, Institute of Biotechnology in Medicine, National Yang-Ming University, 155 Li-Nong St., Shih-Pai, Taipei, Taiwan. Tel.: 11-886-2-2826-7252; Fax: 11-886-2-2826-4092, E-mail: hyhsu@ym.edu.tw.

Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M111883200

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; ROS, reactive oxygen species; IL-1, interleukin-1; pro-IL-1, prointerleukin-1; NAC, N-acetyl-cysteine; DPI, diphenyleneiodonium chloride; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-JUN NH₂-terminal protein kinase; p38, p38 mitogen activated protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PAK, p21-activated kinase; PTK, protein-tyrosine kinase; PTP, protein tyrosine phosphorylation; ICE, interleukin 1-converting enzyme; TNF, tumor necrosis factor; CM-DCFH, carboxyl-2',7'-dichlorofluorescein diacetate; DN, dominant negative; ELISA, enzyme-Linked immunosorbent assay; PK, protein kinases; PKC, protein kinase C; RT, reverse transcriptase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES