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Shigella dysenteriae serotype 1 and Shiga toxin (Stx)-producing. Escherichia coli cause bloody ... signaling for cytokine expression as well as cell proliferation.
Shiga toxin 1-induced cytokine production is mediated by MAP kinase pathways and translation initiation factor eIF4E in the macrophage-like THP-1 cell line Rama P. Cherla, Sang-Yun Lee, Pieter L. Mees, and Vernon L. Tesh1 Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, Texas

Abstract: Upon binding to the glycolipid receptor globotriaosylceramide, Shiga toxins (Stxs) undergo retrograde transport to reach ribosomes, cleave 28S rRNA, and inhibit protein synthesis. Stxs induce the ribotoxic stress response and cytokine and chemokine expression in some cell types. Signaling mechanisms necessary for cytokine expression in the face of toxin-mediated protein synthesis inhibition are not well characterized. Stxs may regulate cytokine expression via multiple mechanisms involving increased gene transcription, mRNA transcript stabilization, and/or increased translation initiation efficiency. We show that treatment of differentiated THP-1 cells with purified Stx1 resulted in prolonged activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) cascades, and lipopolysaccharides (LPS) rapidly triggered transient activation of JNK and p38 and prolonged activation of extracellular signal-regulated kinase cascades. Simultaneous treatment with Stx1 ⴙ LPS mediated prolonged p38 MAPK activation. Stx1 increased eukaryotic translation initiation factor 4E (eIF4E) activation by 4.3-fold within 4 – 6 h, and LPS or Stx1 ⴙ LPS treatment increased eIF4E activation by 7.8- and 11-fold, respectively, within 1 h. eIF4E activation required Stx1 enzymatic activity and was mediated by anisomycin, another ribotoxic stress inducer. A combination of MAPK inhibitors or a MAPK-interacting kinase 1 (Mnk1)-specific inhibitor blocked eIF4E activation by all stimulants. Mnk1 inhibition blocked the transient increase in total protein synthesis detected in Stx1-treated cells but failed to block long-term protein synthesis inhibition. The MAPK inhibitors or Mnk1 inhibitor blocked soluble interleukin (IL)-1␤ and IL-8 production or release by 73–96%. These data suggest that Stxs may regulate cytokine expression in part through activation of MAPK cascades, activation of Mnk1, and phosphorylation of eIF4E. J. Leukoc. Biol. 79: 397– 407; 2006. Key Words: interleukin-1␤ sponse



interleukin-8 䡠 ribotoxic stress re-

0741-5400/06/0079-397 © Society for Leukocyte Biology

INTRODUCTION Shigella dysenteriae serotype 1 and Shiga toxin (Stx)-producing Escherichia coli cause bloody diarrhea, which may progress to life-threatening sequelae, such as the hemolytic uremic syndrome (HUS) and central nervous system complications [1, 2]. Stxs comprise a family of related protein toxins, which share an AB5 structure [3, 4]. Pentameric Stx B subunits bind to the neutral glycolipid globotriaosylceramide (Gb3) on the surface of susceptible cells [5]. Once internalized, the toxins are transported in a retrograde manner to the endoplasmic reticulum and nuclear membrane [6]. The toxin A subunit mediates protein synthesis inhibition by depurination of a single adenine residue located near the 3⬘ end of the 28S rRNA of the 60S ribosomal subunit [7, 8]. Studies in nonhuman primates suggested that Stxs damage colonic capillaries [9, 10]. The toxins and other bacterial products such as lipopolysaccharides (LPS) may then enter the bloodstream to activate prothrombotic and proinflammatory cascades localized to glomerular and brain microvascular endothelial cells [11]. In response to microbes and microbial products, macrophages are known to be powerful producers of proinflammatory cytokines and chemokines. We and others have shown that human monocytes and monocytic cell lines respond to Stxs in vitro by secreting tumor necrosis factor ␣ (TNF-␣), interleukin (IL)-1␤, IL-6, and a number of CC and CXC chemokines [12–15]. The localized production of TNF-␣ and IL-1␤ may contribute to pathogenesis by upregulating the expression of Gb3 on target endothelial cells, thereby sensitizing the cells to the action of Stxs [16 –18]. Chemokines may be essential for the infiltration of monocytes and neutrophils into sites of vascular damage [15, 19]. The precise signaling pathways activated by Stxs to elicit cytokine production are not known. However, mitogen-activated protein kinases (MAPKs) are involved in intracellular signaling for cytokine expression as well as cell proliferation and/or apoptosis [20 –22]. MAPK cascades are categorized into three major pathways: c-Jun N-terminal kinases (JNK), p38 MAPKs, and extracellular signal-regulated kinases (ERK), and

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Correspondence: Department of Medical Microbiology and Immunology, 407 Reynolds Medical Building, Texas A&M University System Health Science Center, College Station, TX 77843-1114. E-mail: [email protected] Received June 13, 2005; revised October 6, 2005; accepted October 10, 2005; doi: 10.1189/jlb.0605313.

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each MAPK contains multiple isoforms. Studies using a rat fibroblast cell line and human monocytic and intestinal epithelial cell lines have shown that anisomycin, ricin, ␣-sarcin, and Stxs, protein synthesis inhibitors that share the property of acting on the 28S rRNA, activate the JNK and p38 MAPK cascades [23–25]. In earlier studies measuring MAPK activation in differentiated, macrophage-like THP-1 cells, we noted that a p38 MAPK inhibitor partially blocked soluble TNF-␣ production induced by Stx1 [24]. We also noted that in contrast to LPS, which mediated a rapid increase in cytokine expression, Stx1 induced the prolonged expression or release of cytokines, a phenomenon which is in part a result of the stabilization of cytokine and chemokine mRNA transcripts [15, 26]. The prolonged activation of MAPK cascades by Stxs may be critical for transcript stabilization and prolonged expression of cytokines. Finally, it has recently been shown that the transient activation of MAPKs promotes signaling for cell survival, and the prolonged activation of MAPKs may be important for triggering apoptosis [27, 28]. We showed that after a 12-h exposure to Stx1 and LPS, ⬃40% of differentiated THP-1 cells were induced to undergo apoptosis [29], suggesting that MAPK cascades may be activated for prolonged times in toxin-treated cells. Therefore, we have examined the capacity of Stx1 to induce the prolonged activation of all three MAPK cascades and studied their roles in Stx1 and/or LPS-induced cytokine expression. Stxs may regulate macrophage cytokine expression at transcriptional and post-transcriptional stages. Treatment of THP-1 cells with Stx1 resulted in the activation and nuclear translocation of nuclear factor-␬B and activation of activator protein-1 [30]. Activation of the transcription factors was associated with increased levels of TNF-␣ and IL-1␤ mRNA transcripts in toxintreated cells. Stxs also affect cytokine and chemokine expression at a post-transcriptional stage by altering mRNA decay rates [15, 26, 31]. In eukaryotic cells, however, translation initiation may be the rate-limiting step of protein expression. Initiation involves the modulation of protein-protein and protein-RNA interactions and is under control of diverse signal transduction pathways [32]. LPS was shown to increase the translation rate of endogenous or transiently transfected TNF mRNA by two- to threefold in the murine monocytic RAW264.7 cell line [33]. LPS activates translation initiation through protein kinase cascades, which ultimately alter the phosphorylation status of eukaryotic initiation factor 4E (eIF4E) and its binding protein eIF4E-BP or phosphorylated heatand acid-stable protein (PHAS)-1 [34, 35]. Recently, Colpoys et al. [36] showed that Stxs activate eIF4E in the human intestinal epithelial cell line Hct-8. Therefore, we hypothesized that in addition to stabilizing mRNAs, Stxs may also affect the translation of cytokine mRNAs through modulation of signaling pathways to further increase translation initiation. We examined the capacity of Stx1 to phosphorylate eIF4E in THP-1 cells and the requirement of eIF4E activation in increased protein synthesis activity and the production of cytokines. The requirement for Stx1 enzymatic activity in eIF4E phosphorylation was examined using purified Stx1 B subunits and Stx1 holotoxins containing sitedirected mutations in the toxin-active site. Stx1 and anisomycin, protein synthesis inhibitors that activate the ribotoxic stress response, were shown to trigger eIF4E activation in THP-1 cells. 398

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MATERIALS AND METHODS Reagents JNK/stress-activated protein kinase inhibitor SP600125, p38 MAPK inhibitor SB203580, and ERK1/2 inhibitor PD98059 were purchased from Calbiochem (San Diego, CA). MAPK-interacting kinase 1 (Mnk1) inhibitor CGP57380 was a gift from Novartis Pharma AG (Basel, Switzerland). Monoclonal phosphospecific JNK1/2 (Thr183/Tyr185) antibody and rabbit anti-human phosphospecific p38 (Thr180/Tyr182) and ERK1/2 (Thr202/Tyr204) antibodies were obtained from Cell Signaling Technology (Beverly, MA). JNK1/2, p38, and ERK1/2 protein antibodies were purchased from Santa Cruz Biotechnology, Inc. (CA). Phospho-specific eIF4E (Ser209) and eIF4E antibodies were obtained from Cell Signaling. All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Toxin preparations Purified Stx1 was prepared from cell lysates obtained from E. coli DH5␣ harboring plasmid pCKS112, which contains the stx1 operon under control of a thermoinducible promoter [37]. Stx1 was purified from cell lysates by sequential ion exchange and chromatofocusing chromatography. Purity of toxins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining and by Western blot analysis. Prepared toxins contained ⬍0.1 ng endotoxin per ml determined by Limulus amoebocyte lysate assay (Associates of Cape Cod, Inc., East Falmouth, MA). Purified Stx1-B subunits were the kind gift of Dr. Cheleste Thorpe (New England Medical Center-Tufts University, Boston, MA). Purified Stx1 holotoxin containing an A subunit double mutation (E167Q; R170L) lacking N-glycosidase activity [38] was the kind gift of Dr. Yoshifumi Takeda (Jissen Women’s University, Tokyo, Japan). Purified LPS derived from the enterohemorrhagic E. coli serotype O111 was purchased from Sigma Chemical Co.

Cell lines The human myelogenous leukemia cell line THP-1 [39] was obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI1640 medium (Gibco-BRL, Grand Island, NY) supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml), and 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT). Cells were maintained at 37°C in 5% CO2 in a humidified incubator. THP-1 cells were differentiated to the macrophagelike state using medium containing phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) for 48 h. The medium was then replaced with medium lacking PMA for 3 days, changing the medium every day. Cells were challenged with Stx1 and/or LPS for the indicated time periods, and cell extracts were prepared using the procedure below.

Preparation of cellular lysates THP-1 cells (5⫻106) were washed once in cold Dulbecco’s phosphate-buffered saline and suspended in RPMI with 0.5% FBS prior to stimulation. Cells were serum-starved for 18 h to reduce endogenous kinase activity. Cells were stimulated with Stx1 (400 ng/ml) or LPS (200 ng/ml) or both stimulants in medium containing 0.5% FBS for various time periods as indicated in the figures. Cells were harvested and lysed at 4°C in modified radioimmunoprecipitation assay buffer [1.0% Nonidet P-40, 1.0% Na-deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.25 mM Na-pyrophosphate, 2 mM each sodium vanadate and sodium fluoride, 10 ␮g/ml aprotinin, 1.0 ␮g/ml leupeptin and pepstatin, and 200 mM phenylmethylsulfonyl fluoride]. Extracts were collected and cleared by centrifugation at 15,000 g for 10 min. Cleared extracts were stored at – 80°C until further use for Western blot analysis as described below.

Western blot analysis Cell extracts prepared from stimulated THP-1 cells were used for determination of protein content using the Micro BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins (60 – 80 ␮g protein per gel lane) were separated by SDS-PAGE using 8% and 12% acrylamide gels and transferred to nitrocellulose membranes, which were blocked with 5% milk prepared in Tris-buffered saline (TBS)/Tween 20 [200 mM Tris (pH 7.6), 1.38 M NaCl, containing 0.1% Tween 20] and incubated overnight at 4°C with various

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primary antibodies specific for JNK, p38, and ERK MAPKs or eIF4E in 5% bovine serum albumin made in TBS with 0.1% Tween 20. Membranes were then incubated with the corresponding secondary antibodies (rabbit/mouse immunoglobulin G coupled with horseradish peroxidase) for 2 h at room temperature. Bands were visualized using the Western Lightning chemiluminescence system (NEN-Perkin Elmer, Boston, MA). The intensities of protein bands captured on autoradiography film were quantitated using Bio-Rad Imager quantification software (Bio-Rad, Hercules, CA). Fold induction was calculated, as stimulated protein band intensity values divided by unstimulated control protein band intensity values after normalizing for loading controls. Data shown are from at least three independent experiments.

Measurement of protein synthesis Protein synthesis was measured by the incorporation of [3H]-leucine into trichloroacetic acid-insoluble material. THP-1 cells were cultured in 24-well plates, incubated in the presence or absence of Stx1 (400 ng/ml), with or without pretreatment with Mnk1 inhibitor CGP57380 (50 ␮M), or incubated with the ribotoxic stress inducer anisomycin (1.0 ␮g/ml) for various timepoints. Thirty minutes before each time-point, [3H]-leucine (4.0 ␮Ci) was added into the culture medium containing 0.05 g/L leucine (RPMI 1640) and incubated for 30 min at 37°C. Cells were solubilized, and incorporation of [3H]-leucine into nascent polypeptides was stopped by incubation with 0.1 M KOH for 10 min at 37°C. Proteins were precipitated by the addition of 400 ␮l 20% trichloroacetic acid (TCA), incubation on ice for 15 min, and centrifugation at 6000 g for 5 min. Supernatants were discarded, and the precipitates were washed with 95% ethanol two times and allowed to air dry. Precipitates were solubilized in 250 ␮l 0.5M KOH and transferred into scintillation vials containing 10 ml scintillant. Radioactivity was measured using a scintillation counter (Beckman LS8000, Beckman Instruments Inc., Fullerton, CA).

Analysis of IL-1␤ and IL-8 production THP-1 cells (2⫻106 cells/ml) were treated with inhibitors specific for JNK1/2 (SP600125; 50 ␮M), p38 (SB203580; 20 ␮M), and ERK1/2 (PD98059; 50 ␮M) MAPKs alone, in combinations of two inhibitors, or all three together or treated with the Mnk1-specific inhibitor (CGP57380; 50 ␮M) for 1 h prior to challenge with Stx1 and/or LPS. The cells were incubated at 37°C in a humidified 5% CO2 incubator for 24 h. Cell-free supernatants were collected and used for quantification of cytokine production with human IL-1␤ and IL-8 Quantikine sandwich enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN). Supernatants (200 ␮L in the case of IL-8 or 50 ␮l in the case of IL-1␤) were added to duplicate wells on the ELISA plates. Following the manufacturer’s protocol, absorbance at 450 nm (A450) and A570 was measured using an ELISA plate reader (Dynatech MR5000, Dynatech Laboratories, Chantilly, VA.) Each assay was repeated three times, and mean cytokine values ⫾ SEM were represented as pg/ml (IL-1␤) or ng/ml (IL-8).

Statistical analysis Statistical analyses of kinetics data (fold-induction from three independent experiments) of JNK, p38, and ERK1/2 MAPK and eIF4E activation were performed using two-way ANOVA with the Duncan multiple-range test for post-hoc comparisons (SAS Institute, Cary, NC). For MAPK and Mnk1 inhibitor studies and protein synthesis inhibition assays, the results were compiled from three independent experiments, and t-test was used to calculate the significance of inhibition from the Stx1, LPS, and Stx1 ⫹ LPS-treated cells.

RESULTS Stx1 induces prolonged activation of JNK and p38, but not ERK, and the activation of all three MAPKs is enhanced in the presence of LPS Stx1 induces the rapid activation of JNK and p38 MAPKs [14, 24], but long-term activation of MAPK cascades may be important for signaling for prolonged expression of cytokines and activation of apoptotic signaling. To study the activation kinet-

ics of MAPKs, cells were treated with Stx1, LPS, or both for 16 h, and cell lysates were prepared for Western blot analyses. As shown in Figure 1, A and B, Stx1 was a modest activator of JNK1/2. Optimal activation was reached within 4 – 6 h of toxin stimulation, and levels of phospho-JNK remained elevated five- to ninefold over the entire 16-h time-course of the experiments. LPS rapidly activated JNK1/2, and a 16-fold increase for JNK2 and a 14.5-fold increase for JNK1 occurred within 1 h of treatment (Fig. 1, A and C). Levels of phosphoJNK activated by LPS at 1 h were significantly different (P⬍0.05) compared with activation induced by Stx1 alone. Levels of LPS-induced phospho-JNK gradually declined over time. Maximal JNK activation (22-fold for JNK2 and 20-fold for JNK1) occurred when cells were treated with Stx1 ⫹ LPS, although the levels of phospho-JNK detected in the blots decreased rapidly after 1 h of treatment (Fig. 1, A and D). Stx1 ⫹ LPS treatment showed a significantly higher induction (P⬍0.001) of JNK compared with treatment with Stx1 or LPS alone. A similar pattern of activation was observed for p38 MAPK (Fig. 2). Treatment with Stx1 alone compared with LPS alone activated p38 MAPK with slower kinetics; peak values, ⬃9.5fold increased above controls, were reached at 6 h and 12 h of LPS and Stx1 stimulation, respectively. Differences between Stx1 and LPS-mediated p38 activation at these peak values were statistically significant (P⬍0.05). With the exception of the 1-h time-point, treatment of THP-1 cells with Stx1 ⫹ LPS compared with treatment with either stimulant alone resulted in significantly higher (P⬍0.001) p38 MAPK activation. A major difference noted between JNK and p38 activation triggered by Stx1 ⫹ LPS was that phospho-JNK levels declined rapidly after 1 h, whereas p38 MAPK levels remained elevated over the course of the experiments. Stx1 was not a potent inducer of ERK1/2 activation, mediating only two- to 3.6-fold increases over control values (Fig. 3, A and B). Phospho-ERK values declined to near basal levels beginning 8 h after toxin stimulation. LPS was a more potent inducer of ERK1 and ERK2, with peak values of 6.4and 5.8-fold, respectively (Fig. 3, A and C). Phospho-ERK levels remained elevated up to 16 h after LPS treatment. Treatment with both stimulants together induced optimal ERK1/2 activation (13-fold for ERK1 and 19-fold for ERK2), which was elevated significantly (P⬍0.001) compared with treatment with Stx1 or LPS alone (Fig. 3, A and D). Levels of activated ERK declined after 6 h of stimulation. We failed to detect significant changes in MAPK phosphorylation status in unstimulated control cells maintained for 24 h (data not shown).

Stx1 activates translation initiation factor eIF4E in macrophage-like THP-1 cells, and activation is enhanced by the presence of LPS eIF4E activation may be a key regulatory step in the translation initiation process and requires signaling through MAPK pathways [40]. We hypothesized that in addition to altering gene expression and stabilizing cytokine mRNA transcripts in macCherla et al. Stx1 mediates MAPK and eIF4E activation

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Fig. 1. Activation of JNK1/2 (p54/p46) after stimulation of THP-1 cells with Stx1, LPS, or Stx1 ⫹ LPS. (A) Differentiated THP-1 cells (5⫻106 cells/well) were treated with purified Stx1 (400 ng/ml), LPS (200 ng/ml), or both for the indicated time periods. Cell lysates were prepared, and protein was quantified according to the procedure in Materials and Methods. Equal quantities of proteins were subjected to SDS-PAGE, and separated proteins were transferred to nitrocellulose membranes. Western blotting was performed using a phospho-JNK1/2specific antibody (WB: Phospho JNK). Membranes were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of JNK1/2 [WB: JNK (loading control)]. Blots shown are representative of three independent experiments. Depiction of the kinetics of JNK1/2 activation in response to treatment with (B) Stx1, (C) LPS, or (D) Stx1 ⫹ LPS. The densities of bands from at least three independent experiments were quantified, and fold-increase ⫾ SEM was compared with control cells represented as line graphs. *, Time-points at which Stx1 ⫹ LPS-induced JNK1/2 activation significantly differed from cells treated with Stx1 or LPS alone (P⬍0.005). **, Time-point at which LPSinduced JNK1/2 activation was significantly different from Stx1-treated cells. C, Zerohour control values.

rophage-like THP-1 cells, Stxs may also regulate cytokine expression by modulation of eIF4E phosphorylation status. eIF4E activation was monitored by Western blotting of cell lysates prepared from Stx1, LPS, or Stx1 ⫹ LPS-treated cells using a phospho-eIF4E-specific antibody. As shown in Figure 4, Stx1 induced eIF4E phosphorylation with a 4.3-fold increase in activation compared with control cells, occurring 4 – 6 h after toxin stimulation (P⬍0.05). LPS rapidly activated eIF4E, with a 7.8-fold increase in eIF4E phosphorylation above basal levels detected 1 h after stimulation. This level was significantly different from activation induced by Stx1 at 1 h (P⬍0.01). We have previously shown that maximal levels of TNF-␣ transcripts and soluble protein were induced in THP-1 cells treated with Stx1 ⫹ LPS [26, 41]. In the presence of both stimulants, optimal levels of eIF4E phosphorylation were detected, peaking at an 11-fold increase over control cells at 1 h, and activation was maintained over 4 h of toxin stimulation. These values were significantly different (P⬍0.01) 400

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compared with treatment with Stx1 or LPS alone. After 6 h, eIF4E phosphorylation returned to control values for all stimulants (data not shown). We failed to detect significant differences in eIF4E activation in unstimulated control cells over a 24-h time period (data not shown).

eIF4E activation requires Stx1 enzymatic activity and the ribotoxic stress response Purified Stx1 B subunits do not induce the increased production or secretion of TNF-␣, IL-1␤, or IL-8 from differentiated THP-1 cells in vitro [26, 29, 41]. Smith et al. [25] showed that heat-inactivated Stx1 and a single-site Stx1 A subunit mutant failed to activate p38 and JNK in human intestinal epithelial cells. If MAPK activation were required for eIF4E activation, contributing to the increased expression of cytokines, we hypothesized that Stxl B subunits and Stx1 holotoxin molecules containing a double mutation in the active site of the A subunit (E167Q; R170L) would be unable to induce activation of http://www.jleukbio.org

Fig. 2. Activation of p38 MAPK in THP-1 cells treated with Stx1, LPS, or Stx1 ⫹ LPS. (A) Differentiated THP-1 cells were treated with Stx1, LPS, or both for the indicated time periods. Cell lysates were subjected to Western blotting using a phospho-p38 MAPK-specific antibody (WB: P-p38). Blots were stripped and reprobed with antibodies recognizing activated and nonactivated forms of p38 MAPK [WB: p38 (loading control)]. Blots shown are representative of three independent experiments. MW, Molecular weight. (B) Depiction of the kinetics of p38 MAPK activation in response to treatment with Stx1 (䉬), LPS (f), or Stx1 ⫹ LPS (Œ). The densities of bands from at least three independent experiments were quantified, and fold-increase ⫾ SEM was compared with control cells. *, Time-points at which Stx1 ⫹ LPS-induced p38 MAPK activation differed significantly from cells treated with Stx1 or LPS alone (P⬍0.005). #, Time-points at which LPS-induced p38 MAPK activation differed significantly from Stx1-treated cells. C, Zero-hour control values.

eIF4E. As shown in Figure 5, A and B, neither Stx1 B subunits alone nor the Stx1A– (E167Q; R170L) double mutant were capable of inducing eIF4E phosphorylation above basal activation levels. Stxs and anisomycin have been shown to

elicit the ribotoxic stress response as defined by the rapid activation of JNK and p38 MAPKs [23–25], and we show here (Figs. 4 and 5C) that both protein synthesis inhibitors activate eIF4E in THP-1 cells. These data suggest that the ribotoxic

Fig. 3. Activation of ERK1/2 (p44/ p42) after stimulation of THP-1 cells with Stx1, LPS, or Stx1 ⫹ LPS. (A) Differentiated THP-1 cells were treated with Stx1, LPS, or both for the indicated time periods. Cell lysates were subjected to Western blotting using a phospho-ERK1/2-specific antibody (WB: PErk 1/2). Membranes were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of ERK1/2 [WB: Erk 1/2 (loading control)]. Blots shown are representative of three independent experiments. Depiction of the kinetics of ERK1/2 activation in response to treatment with (B) Stx1, (C) LPS, or (D) Stx1 ⫹ LPS. The densities of bands from at least three independent experiments were quantified, and fold-increase ⫾ SEM was compared with control cells represented as line graphs. *, Time-points at which Stx1 ⫹ LPS-induced ERK1/2 activation significantly differed from cells treated with Stx1 or LPS alone (P⬍0.005). C, Zerohour control values.

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Fig. 4. Phosphorylation of eIF4E in THP-1 cells treated with Stx1, LPS, or Stx1 ⫹ LPS. (A) Differentiated THP-1 cells (5⫻106) were treated with purified Stx1 (400 ng/ml), LPS (200 ng/ml), or Stx1 ⫹ LPS for the indicated time periods. Cells were lysed, and soluble proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes as described in Materials and Methods. Membranes were probed with antibodies specific for the phosphorylated form of eIF4E (WB: P-eIF4E). Blots were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of eIF4E [WB: eIF4E (loading control)]. Blots shown are representative of three independent experiments. (B) Graphical representation of the kinetics of eIF4E phosphorylation in cells treated with Stx1 (f), LPS (Œ), or both (䉬). Fold increases ⫾ SEM were calculated from densities of phospho-specific bands compared with basal values for each time-point from at least three independent experiments. *, Time-points that significantly differed (P⬍0.05) between THP-1 cells treated with Stx1 ⫹ LPS versus treatment with Stx1 or LPS alone. **, Time-point that significantly differed between treatment with Stx1 versus LPS alone. #, Time-point that significantly differed between THP-1 cells treated with Stx1 versus unstimulated control cells. C, Unstimulated control values.

stress response may activate eIF4E through the stress-activated protein kinase cascades.

Inhibitors of MAPKs and Mnk1 block eIF4E phosphorylation induced by Stx1 ERK and p38 MAPKs activate the downstream kinases Mnk1/2, which in turn, phosphorylate eIF4E at serine 209. Phosphorylation of eIF4E may increase the preinitiation complex affinity for 5⬘-methylated mRNAs and facilitate translation initiation, although this finding is controversial [42– 44]. We explored whether eIF4E phosphorylation induced by Stx1

was MAPK- or Mnk1-dependent by pretreating macrophagelike THP-1 cells with a Mnk1-specific inhibitor (Fig. 6A) or a combination of inhibitors specific for JNK, p38, or ERK MAPKs (Fig. 6B) for 1 h prior to treatment with Stx1 for 4 or 6 h. We had previously determined that maximal or nearmaximal levels of eIF4E activation by Stx1 occurred after 4 – 6 h of toxin treatment. eIF4E phosphorylation was analyzed by Western blotting. The Mnk1 inhibitor (20 – 40 ␮M) and MAPK inhibitors were effective at blocking Stx1-induced eIF4E phosphorylation, and the Mnk1 inhibitor reduced phosphorylation to ⬎95% of the levels expressed by Stx1-treated cells at 6 h of

Fig. 5. Effect of Stx1 B subunits, enzymatic mutant holotoxin Stx1A– (E167Q; R170L), and the ribotoxic stress inducer anisomycin on eIF4E phosphorylation. Differentiated THP-1 cells (5⫻106 cells/well) were treated with (A) purified Stx1 B subunits (800 ng/ml), (B) enzymatic mutant holotoxin Stx1A– (E167Q; R170L; 400 ng/ml), or (C) anisomycin (1.0 ␮g/ml) for the indicated time periods. Cell lysates were prepared, equal concentrations of proteins were separated by SDS-PAGE, and the proteins were transferred to nitrocellulose membranes, which were probed with antibodies recognizing activated eIF4E (WB: P-eIF4E). The same membranes were stripped and probed with eIF4E antibody recognizing activated and nonactivated forms of eIF4E to check for equal protein loading (WB: eIF4E). Data shown are representative blots from at least three independent experiments. C, Zero-hour control.

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Fig. 6. Effect of Mnk1 and MAPK inhibitors on eIF4E activation. Differentiated THP-1 cells were treated with (A) the Mnk1 inhibitor CGP57380 (20 and 40 ␮M) or (B) a combination of the JNK1/2 inhibitor SP600125 (50 ␮M), p38 MAPK inhibitor SB203580 (20 ␮M), and ERK1/2 inhibitor PD98059 (50 ␮M) for 1 h prior to stimulation with Stx1 (400 ng/ml) for 4 or 6 h. Cell lysates were prepared, and soluble proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were probed with antibodies recognizing the activated form of eIF4E (WB: P-eIF4E). Blots were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of eIF4E for equal protein loading (WB: eIF4E). Dosedependent inhibition of Stx1-induced eIF4E phosphorylation by the Mnk1 inhibitor is shown in A, lanes 4 and 5 (20 ␮M and 40 ␮M CGP57380, respectively). Pretreatment of cells with vehicle alone [dimethyl sulfoxide (DMSO)] prior to Stx1 exposure failed to alter eIF4E activation (A, lanes 2 and 3; B, lane 2). Treatment of cells with the inhibitors alone did not alter eIF4E activation (data not shown). (C) Western blotting was performed on cell lysates derived from THP-1 cells pretreated with the Mnk1 inhibitor (40 and 60 ␮M) for 1 h before exposure to LPS (200 ng/ml) or Stx1 ⫹ LPS (400 and 200 ng/ml, respectively) for 1 or 2 h (WB: P-eIF4E). Blots were stripped and reprobed to check for equal protein loading (WB: eIF4E). Dose-dependent inhibition of eIF4E phosphorylation induced by LPS or Stx1 ⫹ LPS by the Mnk1 inhibitor is shown in C, lanes 4 and 5 (40 ␮M and 60 ␮M CGP57380, respectively). Pretreatment of cells with vehicle alone (DMSO) prior to LPS or Stx1 ⫹ LPS treatment failed to alter eIF4E activation (C, lanes 2 and 3). Data shown are representative blots from at least three independent experiments. C, Zero-hour control.

toxin stimulation, and the combination of MAPK inhibitors completely blocked eIF4E activation. We also tested whether eIF4E activation, induced by LPS or Stx1 ⫹ LPS, was blocked by the Mnk1 inhibitor (Fig. 6C). eIF4E activation was analyzed by Western blotting after 1–2 h of stimulation, as we showed maximal eIF4E phosphorylation levels in response to treatment with LPS or Stx1 ⫹ LPS occurring within this time-frame. eIF4E activation induced by LPS or Stx1 ⫹ LPS was inhibited by the Mnk1 inhibitor at a 40- to 60-␮M concentration range. These observations suggest that eIF4E phosphorylation induced by LPS or Stx1 ⫹ LPS is also dependent on Mnk1 kinase activity.

Mnk1 inhibition blocks the transient increase in protein synthesis elicited by Stx1 but not basal levels of protein synthesis Stx1 appears to rapidly trigger the ribotoxic stress response, acting through MAPK signaling pathways to activate Mnk1, which in turn, enhances eIF4E phosphorylation. Thus, THP-1 cells may manifest a transiently increased translational initiation capability following intoxication. Therefore, we examined the effect of Mnk1 inhibition on total protein synthetic activity in THP-1 cells treated with Stx1. After 2 h, treatment of cells with the ribotoxic stress inducers Stx1 or anisomycin resulted in a 25–30% increase in total protein synthesis, a level that was significantly elevated (P⬍0.05) compared with control cells (Fig. 7). Protein synthetic activity declined to basal levels after 4 h and further declined over the remaining time-

Fig. 7 . Effect of Mnk1 inhibition on total protein synthesis. Differentiated THP-1 cells (1.0⫻106 cells/well) were treated with Stx1 (400 ng/ml), with or without pretreatment with the Mnk1 inhibitor CGP57380 (CGP; 50 ␮M) for 1 h or were treated with the Mnk1 inhibitor alone or with anisomycin (1.0 ␮g/ml) for the indicated time periods. [3H]-Leucine incorporation into TCA-insoluble material was measured by the procedure described in Materials and Methods. The data are expressed as percentage [3H]-leucine incorporation in comparison with basal protein synthesis (untreated cells). The data are derived from triplicate values at each time-point from two independent experiments. *, A significant difference (P⬍0.05) between the values for cells treated with Stx1 alone versus Stx1 ⫹ CGP57380-treated cells.

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TABLE 1.

Effect of Pretreatment with JNK (SP600125), p38 (SB203580), and ERK (PD98059) MAPK Inhibitors or Mnk1 Inhibitor (CGP57380) on the Production of Soluble IL-1␤ in Stx1 and/or LPS-Treated THP-1 Cells Stimulus

Inhibitor treatment Medium ⫹ DMSO JNK ⫹ p38 JNK ⫹ ERK p38 ⫹ ERK JNK ⫹ p38 ⫹ ERK Mnk1

None 11.4 ⫾ 1.2 47.5 ⫾ 13.1 20.3 ⫾ 6.5 17.0 ⫾ 4.3 13.1 ⫾ 0.8 14.9 ⫾ 2.4 a

Stx1

LPS

Stx1 ⫹ LPS

35.0 ⫾ 2.1 47.7 ⫾ 5.0 (0%)b 21.5 ⫾ 1.0 (39%) 15.9 ⫾ 5.8 (55%) 9.5 ⫾ 1.0 (73%)* 9.0 ⫾ 3.1 (74%)*

636.7 ⫾ 17.2 458.7 ⫾ 18.2 (28%) 431.0 ⫾ 28.0 (32%) 228.0 ⫾ 31.1 (64%) 132.0 ⫾ 72.7 (79%)* 78.3 ⫾ 14.4 (88%)*

897.0 ⫾ 5.9 437.7 ⫾ 110 (51%) 390.0 ⫾ 51.5 (56%) 554.7 ⫾ 29.2 (38%) 84.3 ⫾ 4.1 (91%)* 105.0 ⫾ 9.5 (88%)*

All values are expressed as pg/ml ⫾ SEM. b Numbers in parentheses represent percentage inhibition of IL-1␤ production compared with medium ⫹ vehicle-control cells. * Significant differences (P ⬍ 0.05) between values of inhibitor-treated versus untreated cells. a

points. Pretreatment of cells with the Mnk1 inhibitor prior to Stx1 (Fig. 7) or anisomycin (data not shown) stimulation blocked the rapid transient increase in protein synthesis with basal levels of synthesis maintained over the first 4 h of the experiments. However, Mnk1 inhibition did not block the decline in total protein synthetic capacity beginning 6 h after intoxication. The Mnk1 inhibitor itself showed no effect on basal protein synthetic activity over the time course of the experiments. These data suggest that Mnk1-induced eIF4E phosphorylation may play a role in rapidly enhanced protein synthesis but not in basal protein synthetic activity.

MAPK and Mnk1 inhibitors reduce soluble IL-1␤ and IL-8 production elicited by Stx1, LPS, or Stx1 ⫹ LPS To examine the role of MAPKs and Mnk1 in cytokine production, we measured soluble IL-1␤ and IL-8 levels in cell supernatants collected from THP-1 cells pretreated with combinations of MAPK inhibitors or the Mnk1 inhibitor for 1 h prior to stimulation with Stx1, LPS, or both. As we have previously shown, LPS is a more effective inducer of cytokines compared with Stx1. Treatment of cells with Stx1 and LPS resulted in slightly increased IL-1␤ and IL-8 expression. Treatment of cells with a combination of JNK ⫹ p38 inhibitors produced unexpected results. IL-1␤ expression was slightly increased above unstimulated levels by treatment with the inhibitors alone. Pretreatment of cells with JNK ⫹ p38 inhibitors prior to treatment with Stx1 resulted in a 36% increase in IL-1␤ production, and IL-1␤ expression induced by LPS was slightly TABLE 2.

decreased (28%); expression induced by Stx1 ⫹ LPS was significantly decreased (51%) by inhibition of JNK and p38 MAPKs compared with treatment with stimulants alone (Table 1). Treatment with JNK ⫹ ERK inhibitors reduced Stx1-, LPS-, and Stx1 ⫹ LPS-induced IL-1␤ expression by 39%, 32%, and 56%, respectively, and treatment of cells with p38 ⫹ ERK inhibitors reduced IL-1␤ production by 55%, 64%, and 38%, respectively. Pretreatment of cells with all three inhibitors significantly reduced IL-1␤ expression in response to all stimulants. Pretreatment of cells with the Mnk1 inhibitor reduced Stx1-, LPS-, and Stx1 ⫹ LPS-induced IL-1␤ expression by 74%, 88%, and 88%, respectively. As was the case with IL-1␤ expression, treatment of THP-1 cells with the combination of JNK ⫹ p38 inhibitors alone elicited soluble IL-8 production (Table 2). However, subsequent treatment with Stx1 reduced IL-8 expression compared with levels induced by Stx1 alone or JNK ⫹ p38 inhibitors alone. Pretreatment of THP-1 cells with JNK ⫹ ERK or p38 ⫹ ERK inhibitors reduced Stx1-induced IL-8 expression, and p38 ⫹ ERK inhibitors were more effective. Pretreatment of cells with all three inhibitors or treatment with the Mnk1 inhibitor significantly reduced Stx1-induced IL-8 expression. All combinations of MAPK inhibitors and the Mnk1 inhibitor significantly reduced IL-8 expression elicited by LPS or Stx1 ⫹ LPS.

DISCUSSION Monocytes/macrophages are key cellular components of innate immunity. After encountering microbial pathogens or toxins,

Effect of Pretreatment with JNK (SP600125), p38 (SB203580), and ERK (PD98059) MAPK Inhibitors or Mnk 1 Inhibitor (CGP57380) on the Production of Soluble IL-8 in Stx1 and/or LPS-Treated THP-1 Cells Stimulus

Inhibitor treatment Medium ⫹ DMSO JNK ⫹ p38 JNK ⫹ ERK p38 ⫹ ERK JNK ⫹ p38 ⫹ ERK Mnk1

None

Stx1

LPS

Stx1 ⫹ LPS

0.42 ⫾ 0.04a 2.57 ⫾ 0.77 0.80 ⫾ 0.11 0.29 ⫾ 0.02 0.38 ⫾ 0.04 0.32 ⫾ 0.07

1.04 ⫾ 0.03 0.75 ⫾ 0.05 (28%)b 0.86 ⫾ 0.28 (17%) 0.30 ⫾ 0.02 (71%) 0.17 ⫾ 0.01 (84%)* 0.26 ⫾ 0.06 (75%)*

259.2 ⫾ 17.5 29.73 ⫾ 1.45 (88%) 27.94 ⫾ 1.17 (89%) 60.6 ⫾ 10.2 (77%) 26.15 ⫾ 9.33 (90%)* 38.43 ⫾ 6.49 (85%)*

304.4 ⫾ 1.45 15.86 ⫾ 24.8 (95%) 12.68 ⫾ 1.11 (96%) 154.0 ⫾ 13.8 (49%) 10.81 ⫾ 1.26 (96%)* 43.36 ⫾ 8.08 (86%)*

a All values are expressed as ng/ml ⫾ SEM. b Numbers in parentheses represent percentage inhibition of IL-8 production compared with medium ⫹ vehicle-control cells. * Significant differences (P ⬍ 0.05) between values of inhibitor-treated versus untreated cells.

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these cells produce cytokines and chemokines, which initiate inflammation, regulate leukocyte trafficking to sites of tissue damage, and activate acquired immunity. Stxs have been shown to up-regulate the expression of cytokines and chemokines from human primary blood monocytes and transformed monocytic cell lines in vitro [12–15]. Although the intracellular signaling pathways activated by Stxs to induce cytokine expression are not well characterized, it appears that the toxins may regulate cytokine expression via multiple mechanisms, including the activation of MAPK cascades leading to increased cytokine gene transcriptional activity; stabilization of cytokine mRNA transcripts; and increased translation initiation efficiency. In support of the latter mechanism, we show that Stx1 induces the phosphorylation of the eukaryotic translation initiation factor eIF4E. Maximal phosphorylation levels were detected 4 – 6 h after Stx1 treatment, and LPS-induced eIF4E phosphorylation peaked at 1–2 h poststimulation. In the presence of Stx1 and LPS, phosphorylation levels were increased an additional 6.4-fold, suggesting that Stx1 and LPS may signal through nonredundant, parallel pathways to trigger eIF4E activation. In general, the kinetics and extent of eIF4E phosphorylation corresponded with cytokine production; i.e., Stx1 was shown to induce cytokine expression from THP-1 cells with slower kinetics compared with LPS, and both stimulants together induced the highest expression of cytokine mRNA transcripts [15, 26]. Normally, eIF4E binds to cytoplasmic sequestering proteins, eIF4E-BPs (or PHAS-1), which may be phosphorylated at multiple sites, and eIF4E is phosphorylated at serine 209 in response to insulin, mitogens, or environmental stress. Phosphorylation may increase the affinity of eIF4E for the mRNA 5⬘ m7GpppN cap structure, although this finding is controversial. Thus, in response to stress, the cytosolic eIF4E/eIF4E-BP complex dissociates, and phosphoeIF4E may be incorporated into the eIF4F (initiation) complex (reviewed in ref. [45]). Stxs and LPS have been shown to phosphorylate eIF4E and eIF4E-BPs [34 –36]. Thus, the capacity of Stxs and LPS to trigger eIF4E and eIF4E-BP activation may be a key step in increasing cytokine expression by up-regulating translation initiation, even in the face of sublethal total protein synthesis inhibition mediated by Stxs. MAPK interacting kinases (Mnk)1/2 are serine/threonine kinases involved in eIF4E activation [46, 47]. Mnk1 has been shown to have low basal activity, which is enhanced by agents that activate the ERK and p38 MAPKs. Mnk2, in contrast, has high basal activity, which is not enhanced by agents that activate MAPKs, and Mnk2 is thought to be primarily responsible for maintenance of basal levels of eIF4E phosphorylation [35, 44]. We have used a low molecular weight Mnk1-specific inhibitor (CGP57380 [48]) to show that Mnk1 mediates Stx1and LPS-induced eIF4E phosphorylation. At the concentrations of the Mnk1 inhibitor used in this study, we did not detect toxic effects caused by the inhibitor alone (data not shown). Our findings are consistent with the recent report of Colpoys et al. [36], showing that Stxs induce eIF4E activation in the Hct-8 intestinal epithelial cell line. We also show that the Mnk1 activating kinases ERK1/2 and p38, as well as JNK1/2, are phosphorylated in response to Stx1. ERK1/2 transduces signals important for growth in response to serum factors and mitogens, and NIH3T3 cells overexpressing ERK1/2, show an

elevated basal level of eIF4E phosphorylation [49]. JNK1/2 and p38 MAPKs are activated by cell stressors such as ultraviolet light, DNA damage, and protein synthesis inhibition and regulate cell growth, differentiation, apoptosis, and cytokine expression [20 –22]. The kinetic evaluation of MAPK activation showed that Stx1 induced the activation of JNK and p38 MAPKs, but maximal activation required 6 and 12 h of toxin stimulation, respectively. The induction of JNK and p38 MAPK activation by LPS was rapid but not prolonged, returning to near basal levels within the time course of the experiments. Stx1 was not a vigorous inducer of ERK1/2 activation, and LPS mediated prolonged activation of ERK1/2. The simultaneous exposure of THP-1 cells to Stx1 ⫹ LPS produced the highest levels of MAPK phosphorylation, although levels of activated JNK and ERK declined over time, and levels of phospho-p38 remained elevated. We speculate that the decline in activated JNK and ERK levels in Stx1 ⫹ LPS-treated cells may be a result of apoptosis, as we have shown, using deoxyuridine triphosphate nick-end labeling (TUNEL) staining, that ⬃40% of macrophage-like THP-1 cells are TUNELpositive 12 h after treatment with Stx1 ⫹ LPS [29]. Yet, in the face of significant apoptosis, p38 activation is maintained in Stx1 ⫹ LPS-treated cells. Stx1 enzymatic activity and the ribotoxic stress response appear to be necessary to trigger eIF4E activation. Stx1 enzymatic mutant holotoxin or purified Stx1 B subunits did not activate eIF4E. These data correlate with earlier studies showing that purified B subunits do not activate MAPK cascades or trigger cytokine expression [24, 26, 41]. Stx1 and anisomycin are protein synthesis inhibitors acting on the 28S rRNA component of eukaryotic ribosomes to block the peptidyl transferase reaction and activate the ribotoxic stress response. Stx1 and anisomycin activated eIF4E in THP-1 cells. Finally, eIF4E activation appears to be important in mediating a transient increase in protein synthesis in THP-1 cells intoxicated with Stx1, as a Mnk1-specific inhibitor blocked the rapid increase in protein synthesis but did not affect the long-term inhibition of protein synthesis seen with prolonged exposure to Stx1. The consequences of prolonged activation of MAPKs by Stx1 in THP-1 cells may be correlated with the prolonged activation of cytokine genes and/or the stabilization of cytokine mRNA transcripts, resulting in increased levels of IL-1␤ and IL-8 being detected in culture supernatants. In support of this, ricin treatment of murine monocytic cells activated MAPKs, which were required for the expression of several proinflammatory mediators and transcription factors [50]. The biological consequences of transient versus prolonged activation of MAPK cascades remain to be fully characterized but may involve such fundamental processes as increased inflammation and cytokine expression and signaling for cell survival or programmed cell death. In light of these observations, we measured soluble IL-1␤ and IL-8 levels produced by Stx1, LPS, or Stx1 ⫹ LPS-treated THP-1 cells cultured in the presence of single MAPK inhibitors or in the presence of combinations of inhibitors. A partial reduction in IL-1␤ and IL-8 production was observed with individual inhibitors (data not shown) and with combinations of the inhibitors. In our hands, the p38 and ERK cascades appeared most important in Stx1-induced signaling, as inhibitors of these MAPKs were most effective at blocking Cherla et al. Stx1 mediates MAPK and eIF4E activation

405

cytokine expression. However, interpretation of the data was confounded by the fact that treatment of THP-1 cells with JNK ⫹ p38 inhibitors alone reproducibly increased IL-1␤ and IL-8 expression above basal levels expressed by unstimulated cells, suggesting that these MAPK cascades are normally involved in the negative regulation of cytokine expression. Inhibitors of all three major MAPK cascades and the Mnk1-specific inhibitor significantly reduced (73–96%) cytokine expression compared with stimulation with Stx1 and/or LPS without the inhibitors. The data presented here suggest that all three kinase cascades may contribute to increased cytokine expression. Previous work using NIH3T3 cells suggested that more than one kinase family was involved in regulating eIF4E phosphorylation. ERK1/2, activated in the presence of serum, contributed to eIF4E phosphorylation, which was inhibited by the ERK1/2 inhibitor PD98059 but not by the p38 MAPK inhibitor SB203580, whereas eIF4E activation, mediated by p38 MAPK, was inhibited by the p38 inhibitor but not by the ERK inhibitor [51]. We show here that use of inhibitors for all three kinase families, as well as the Mnk1-specific inhibitor, reduced eIF4E phosphorylation in response to Stx1 and/or LPS. The precise role of cytokines in the pathogenesis of HUS requires additional study. Serum TNF-␣ and IL-1␤ levels in HUS patients have been reported to range from 44 to 568 pg/ml and 55 to 70 pg/ml, respectively [52, 53]. These values are in the range induced by purified Stx1 but less than the values induced by Stx1 ⫹ LPS from THP-1 cells in vitro [26, 30]. However, detection of elevated serum cytokines is not a consistent finding in patients with HUS. Karpman et al. [54] detected elevated serum TNF-␣ levels (defined as ⬎25 pg/ml) in only seven of 31 HUS patients, and Murata et al. [53] reported that only six of 12 HUS patients had elevated serum TNF-␣ levels, and nine of 12 had elevated serum IL-8 levels on the first day of hospitalization. There is evidence that cytokines may be induced in a focal manner in HUS, and sites of tissue damage may correspond with sites of cytokine expression. For example, mice administered purified Stxs developed acute tubular necrosis [37]. Harel et al. [55] infused purified Stx1 into TNF-␣ promoter:chloramphenicol acetyltransferase (CAT) transgenic mice and detected CAT activity only in the kidneys. In contrast to HUS, numerous studies have shown that prolonged dysregulation of cytokine expression is a hallmark of endotoxic shock and can lead to a systemic “spillover effect” in which cytokines mediate deleterious effects. Thus, although we have shown differences in ERK activation, in the kinetics and extent of JNK, p38, and eIF4E activation, and in the amounts of soluble IL-1␤ and IL-8 produced in macrophage-like THP-1 cells treated with Stx1 versus LPS, both bacterial products appear to signal through MAPKs and eIF4E. Therefore, additional factors may be responsible for the different pathophysiological characteristics of HUS and endotoxic shock.

ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service Grant 2RO1 AI34530-10 from the National Institutes of Health. We thank Drs. Cheleste Thorpe and Yoshifumi Takeda for gifts of reagents necessary for these studies. 406

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