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Trypanocidal Drug Benznidazole Impairs Lipopolysaccharide Induction of Macrophage Nitric Oxide Synthase Gene Transcription Through Inhibition of NF-B Activation¹

Eliane Piaggio^*,, Josiane Sancéau, Silvia Revelli^*, Oscar Bottasso^2,*, Juana Wietzerbin and Esteban Serra

^* Facultad de Ciencias Médicas, Instituto de Immunología, Rosario, Argentina; Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario Consejo Nacional de Invetigaciones Cientìficas y Técnicas de Argentina, Rosario, Argentina; and Institut National de la Santé et de la Recherche Médicale Unite 365, Institut Curie, Section de Recherche, Paris, France

	Abstract

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In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN- with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-B binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-B in stimulated macrophages. NF-B is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IB. Within this setting, Western blot was also performed to show that BZL blocked IB degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-B activation after LPS plus IFN- stimulation.

	Introduction

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Benznidazole (BZL)³ is a nitroheterocyclic drug used for the treatment of Chagas’ disease, a protozoan disease caused by Trypanosoma cruzi (1). The biochemical basis by which BZL exerts its trypanocidal activity has not been fully elucidated, but evidence suggests that BZL could interfere with the synthesis of macromolecules via covalent binding or other types of interaction between nitroreduction intermediates and various cellular components, i.e., DNA, lipids, and proteins of T. cruzi (2, 3). Because BZL was also shown to affect mammalian host cells (4) and because T. cruzi mostly replicates in macrophages, we recently investigated whether BZL altered the in vitro functional activity of such cells. It was demonstrated that BZL inhibited LPS-, IFN--, and LPS- plus IFN--induced production of NO and inflammatory cytokines by RAW 264.7 macrophages (5). Such a down-regulation of NO production by BZL correlated with the inhibition of inducible NO synthase (iNOS or NOSII) mRNA accumulation, as determined by RT-PCR.

The LPS- plus IFN--dependent iNOS induction is mostly controlled by two regulatory regions present in the iNOS gene promoter, which contain binding sequences for two transcription factors, NF-B and IFN regulatory factor 1 (IRF-1). These nuclear factors were shown to be essential when NOSII gene expression is induced by IFN- and/or LPS (6, 7, 8). Deletion studies of the NOSII gene promoter showed that an IRF response element (IRF-E) which interacts with the IRF-1 protein mediates the synergistic contribution of IFN- to LPS-induced NOSII gene promoter activity (6, 8, 9). When comparing IRF-E and B binding sites, the latter was shown to be the major responsible element for NOSII gene induction by LPS (10). NF-B is a critical transcriptional activator protein which participates in the regulation of the expression of many genes encoding proteins involved in inflammation and macrophage functions (11). This nuclear factor can be activated, in monocytes and macrophages, by LPS, cytokines like TNF- and IL-1, oxidative stress, and DNA-damaging agents (12). NF-B, present in the cytoplasm of most cell types, is normally bound to a member of a family of inhibitor proteins known as IB that sequestrates NF-B in the cytoplasm. Macrophage activation by LPS leads to the phosphorylation of IB by two IB kinases (IKK-1 and IKK-2) (13). Phosphorylated IB is then selectively ubiquitinated and degraded by the 26S proteasome. Once NF-B is released, it migrates to the nucleus, where it binds to specific promoter sites activating gene transcription (see for review Refs. 14, 15). In the nucleus, NF-B participates in the regulation of a large number of genes beside NOSII, including TNF-, IL-6, and Il-1. As above mentioned, the production of these cytokines, as well as NO, was lowered when LPS- and IFN--activated RAW 264.7 macrophages were cultured in the presence of BZL. Given this background, the question arises as to whether BZL effects on the expression of iNOS and cytokines genes could involve a mechanism sharing a common nuclear factor, namely, NF-B.

To test the hypothesis that BZL could inhibit iNOS gene induction by blocking activation of NF-B, a series of experiments was now performed. Transient transfection of RAW 264.7 macrophages with plasmids containing reporter genes under the control of the complete promoter of the NOSII gene as well as the IRF-E and B regulatory elements showed that NF-B is the major target of BZL action. Furthermore, gel shift analysis by using the NF-B regulatory sequence as probe and Western blot analysis demonstrated that BZL treatment inhibited NF-B activation by blocking the degradation of IkB- following LPS plus IFN- stimulation.

	Materials and Methods

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Reagents

Reagents used for cell culture were purchased from Life Technologies (Grand Island, NY). Recombinant murine IFN-, sp. act. 2 x 10⁷ U/mg, was produced by Genentech (South San Francisco, CA), provided by Boehringer Ingelheim (Ridgefield, CT), and was shown to be endotoxin free (<10 pg/mg). LPS (from >Escherichia coli serotype 0111:B4) was purchased from Sigma (St. Louis, MO). Rabbit affinity-purified polyclonal Abs against human IB and NF-B family proteins were obtained from Santa Cruz Biotechnology Santa Cruz, CA). BZL was a gift from Roche Laboratories (Buenos Aires, Argentina). In all experiments, BZL was dissolved in 2-methoxyethanol and used at a final concentration of 1 mM, a dose already shown to give maximal inhibitory effect on NO production (5).

Cell culture

RAW 264.7 cells (murine macrophage cell line from the American Type Culture Collection, Manassas, VA) were grown in DMEM supplemented with 5% heat-inactivated FBS (shown to be endotoxin free), 2 mM L-glutamine, nonessential amino acids, 2 mM HEPES, and 2 µg/ml gentamicin at 37°C in 5% CO₂. Cells were plated at 1 x 10⁶/ml. After overnight culture, the medium was replaced by fresh medium containing the various inducers in the presence or absence of BZL. To rule out changes in cells, viability was determined by measuring lactate dehydrogenase (LDH) levels in supernatants from stimulated cells (Sigma assay). Data analysis revealed no significant differences in LDH concentrations in culture supernatants irrespective of whether or not cultures were BZL treated. In all cases, LDH values did not exceed 0.5% of the total LDH activity obtained from the corresponding cell extracts (data not shown).

Peritoneal macrophages were obtained from 60- to 90-day-old outbred Rockland mice. Cells were centrifuged and resuspended in MEM (Sigma) and cultured in 6-well plaques; 5 x 10⁶ cells/well (Nalge Nunc International, Rochester, NY) containing the same medium supplemented with 10% FBS (Life Technologies), 0.2% gentamicin (10 mg/ml; Life Technologies), 2% penicillin-streptomycin, and 2-ME. After 24 h, the culture medium was replaced, and cells were stimulated with LPS and/or IFN- in the presence or absence of BZL.

Nitrite determination

Nitrite accumulation in supernatants from cultured cells was measured using the Griess method (16). Equal amounts of macrophage culture supernatant and Griess reagent were combined and incubated for 10 min at room temperature. Absorbance was measured at 543 nm. Nitrite concentration was quantified using various NaNO₂ concentrations in culture medium as standard and data were expressed in micromolars.

Transient transfection

RAW 264.7 cells (2 x 10⁷, logarithmic growth phase) were washed three times with PBS, resuspended in 200 µl of DMEM, mixed with 20 µg of each plasmid, and electroporated at 220 V, 960 µf using a Gene Pulser (Bio-Rad, Hercules, CA). Cells were maintained at room temperature for 10 min and cells from two or three separate electroporations were pooled in DMEM supplemented with 5% FCS and distributed equally between eight culture plates. After one night at 37°C in a 5% CO₂ atmosphere, cells were stimulated for 24 h with LPS (100 ng/ml) and/or IFN- (500 U/ml) with or without BZL in fresh medium. When viral promoters -galactosidase plasmids were used, BZL was added after electroporation and cells were recovered after 24 h. Cells were then assayed for chloramphenicol acetyltransferase (CAT), -galactosidase, or luciferase activity. CAT reaction was performed as described previously (17). -Galactosidase assay (-Galactosidase Enzyme Assay System; Promega, Madison, WI) and luciferase reaction (Luciferase Assay System: Promega) were performed as indicated by the manufacturers.

The pNOSII-CAT plasmid was a generous gift from Dr. C. F. Nathan and Dr. Q.-W. Xie (Cornell University Medical College, New York, NY) (6). IRF-E pNOSII was a generous gift from Dr. J. Vilcek (Kaplan Comprehensive Cancer Center, New York University, New York, NY) (7). The p3enh-b-CONA-Luc, carrying a luciferase gene under the control of three synthetic copies of the B site from the Ig -chain gene promoter, was a generous gift from Dr. F. Arenzana-Seisdedos (Unité d’Immunologie Virale, Institut Pasteur, Paris, France) (18). PCMV, ptk, and pSV containing human CMV immediate early promoter enhancer, herpes simplex virus thymidine kinase promoter, and SV40 early promoter controlling E. coli -galactosidase gene expression were reported by MacGregor and Casley (19).

EMSA

Nuclear extracts were prepared as previously described (20). Purified oligonucleotide corresponding to the NF-B-binding element (5'-CTAGACAGAGGGGATTTCCGAGAGGT-3') was provided by Eurogentec (Seraing, Belgium). For EMSA, nuclear extracts (10 µg) were incubated with annealed radiolabeled oligonucleotide (20,000 cpm) in buffer containing 50 mM NaCl, 10 mM HEPES (pH 7.9), 5 mM Tris-HCl (pH 7.9), 1 mM DTT, 15 mM EDTA, 10% glycerol, 500 µg/ml BSA-fraction V, and 800 µg/ml denatured salmon sperm DNA in a final volume of 12.5 µl. After a 30-min incubation on ice, the nucleoprotein complexes were resolved by nondenaturing electrophoresis in a 5% polyacrylamide gel for 3 h at 20 mA in 1x TGE buffer (50 mM Tris-HCl (pH 8.8), 180 mM glycine, and 2.5 mM EDTA) in refrigerated conditions. The gels were dried and exposed overnight to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). For competition experiments, a 100-fold molar excess of unlabeled oligonucleotides was added 15 min before incubation of nuclear extracts with the labeled oligonucleotides, while antisera were mixed directly with nuclear extracts and binding buffer (without salmon sperm DNA) 1 h before adding salmon sperm DNA and radiolabeled probe.

Western blot

Cells were disrupted by three freeze-thaw cycles in the presence of protease inhibitors. Thirty micrograms of each fraction was subjected to SDS-PAGE and were transferred to polyvinylidene difluoride membranes (Immobilon-P) by electroblotting. Membranes were blocked in PBS containing 5% nonfat powdered milk and 0.1% Tween 20, washed, and incubated with primary Abs for IB and IB. After washing, membranes were revealed using a goat anti-rabbit Ig-alkaline phosphatase conjugate.

Statistical analysis

Comparisons of NO-derived metabolites in culture supernatants were performed by using the Mann-Whitney U test. The level of statistical significance was set at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
BZL inhibited NOSII induction at the transcriptional level

In a first step, we sought to analyze the effect of BZL on NO production by stimulated macrophages. RAW 264.7 macrophages were stimulated with LPS and/or IFN- in the presence or absence of BZL for assessment of nitrite levels in 24-h culture supernatants. Results obtained after three independent experiments are summarized in Table I. BZL inhibited LPS induction of NO production by more than seven times. When macrophages were stimulated with IFN- or IFN- plus LPS, BZL caused a 4- and 3-fold inhibition of NO production, respectively.

To study the effect of BZL on NOSII gene expression, we transiently transfected RAW 264.7 cells with pNOSII-CAT, a plasmid containing the bacterial CAT reporter gene (pCAT) under the control of the whole NOSII gene promoter. Transfected cells were stimulated for 24 h to measure the maximal accumulation of CAT activity. Stimulation with LPS and/or IFN- led to a 5-, 7-, or 3-fold increase of CAT expression, respectively. BZL reduced CAT activity in LPS-, IFN--, or LPS- plus IFN--stimulated transfected cells up to 50, 30, and 40%, respectively (Fig. 1A). As expected, BZL also inhibited the iNOS-dependent endogenous NO production in pNOSII-CAT-transfected RAW 264.7 cells (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Effect of BZL on NOSII gene promoter in transfected cells. RAW 264.7 cells were transfected with pNOSII-CAT (A), p3enh-kB-CONA-Luc (C), or IRF-EpNOSII plasmid (D) and induced with LPS (100 ng/ml), IFN- (500 U/ml), and LPS plus IFN- in the presence () or absence () of BZL (1 mM). CAT (A and D) or luciferase (C) activity was measured after 24 h of induction. B, RAW 264.7 cells were transfected with pCMV, ptk, and pSV plasmids containing E. coli -galactosidase gene driven by the human CMV immediate early promoter enhancer, herpes simplex virus thymidine kinase promoter, and SV40 early promoter, respectively. -Galactosidase activity was measured after 24-h incubation in the presence () or absence () of BZL (1 mM). Data correspond to a representative experiment from three independent rounds of experiments.

To rule out the possibility that BZL could block global transcription and/or translation processes, we transfected RAW 264.7 cells with plasmids containing the -galactosidase reporter gene under the control of different viral promoter regions (see Materials and Methods). As shown in Fig. 1B, in all cases, the -galactosidase activity level was similar regardless of whether cells had been treated with BZL, or not, after transfection.

BZL down-regulated NF-B activation

We next wished to ascertain whether BZL modified the availability of NF-B DNA-binding activity in the macrophage nucleus. To pursue this, the capacity of nuclear extracts from RAW 264.7 cells (prepared after stimulation with LPS and/or IFN- in the presence or absence of BZL) to bind the NF-B site was tested. EMSA performed with nuclear extracts from LPS- and LPS- plus IFN--treated cells and B oligonucleotides allowed the detection of an inducible DNA-protein complex (Fig. 2A). This complex was specific for the NF-B-binding sequence, as shown by experiments with unlabeled oligonucleotides and supershift assays (Fig. 3). In cells stimulated in the presence of BZL, DNA-protein complex formation was impaired, with this effect being total when LPS was used alone (Fig. 2A). No inhibitory effect of BZL on DNA-protein complex formation was observed when EMSA was conducted using IRF-E oligonucleotide (data not shown), indicating that the effect of BZL was specific and restricted to NF-B.

View larger version (52K): [in this window] [in a new window]   FIGURE 2. Effect of BZL on NF-B activation. RAW 264.7 cells were stimulated with IFN- (500 U/ml), LPS (100 ng/ml), or both in the presence or absence of BZL (1 mM). Nuclear extracts were prepared after 3 h of induction. EMSAs were performed using -32P-end-labeled B binding site oligonucleotide. Control lanes correspond to nonstimulated cells. A, Assay of NF-B-binding complexes induced after different cell treatment. B, Effect of BZL on NF-B dimer composition. Supershift analysis was performed using Abs against p65, p50, or both. Competition assay was performed by including a 100-fold molar excess of nonradiolabeled NF-B oligonucleotide.

View larger version (59K): [in this window] [in a new window]   FIGURE 3. Effect of BZL on IkB degradation. RAW 264.7 (A) and mouse macrophages (B) were stimulated with 500 U/ml IFN- and 100 ng/ml LPS in the presence or absence of 1 mM BZL. Western blot analysis of soluble protein from cells recovered after different times of stimulation was performed by using polyclonal Abs to IB and IB as indicated in Materials and Methods.

BZL prevents the degradation of IB

To gain some insight into the mechanisms by which BZL inhibited the activation of NF-B, proteins obtained from stimulated macrophages, in the presence or absence of BZL, were analyzed by Western blot using Abs against IB and IB. As expected (Fig. 3A), IB diminished in the first minute after LPS plus IFN- stimulation. This decrease was abolished by treatment with BZL. By contrast, IB remained constant over the same time period. Such results suggested that BZL blocked the NF-B activation pathway by interfering with IB degradation. To ascertain whether the action of BZL over IB was not restricted to RAW 264.7 cells, an additional round of experiments were performed on mouse macrophages obtained from the peritoneal cavity. As shown in Fig. 3B, IB degradation in these cells was also diminished when adding BZL.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We has previously reported that in the presence of BZL, LPS- or LPS-plus IFN--stimulated macrophages produced significantly lower amounts of NO and proinflammatory cytokines such as IL-6 and, to a lesser extent, IL-1 and TNF- (5). In this work, we characterized, in more detail, the molecular basis of BZL effects on activated macrophages. Our study stemmed from the effect of BZL on NO production and the expression of the iNOS. In transient transfection experiments in RAW 264.7 cells with the pNOSII-CAT plasmid, BZL treatment diminished CAT activity measured 24 h following stimulation. The BZL effect was more evident when macrophages were stimulated with LPS, being lower but still significant if IFN- or LPS plus IFN- were used as stimuli. This effect could have been due neither to a BZL influence on global cell transcription or translation machinery nor to an inhibition of CAT activity.

NOSII gene expression regulation has been deeply studied in the last years. Deletion analysis of the NOSII gene promoter demonstrated the existence of two regions implied in the regulation of this gene (21). NF-B and IRF-1 are important transcription factors responsible for NOSII transcriptional activation acting at these promoter regions. Among them, NF-B is the major responsible element for the LPS-mediated iNOS stimulation in macrophages (7, 8, 10). By contrast, IFN- has a synergistic action on LPS-mediated NOSII stimulation, involving NF-B and IRF-1, even though the mechanism underlying this synergy remains unclear.

We analyzed the participation of both B and IRF-E sites in BZL-mediated inhibition of NOSII gene expression. Luciferase activity of p3enh-b-CONA-Luc-transfected cells treated with LPS and BZL was similar to that of control cells, indicating an almost 100% inhibition of BZL on the B site. Interestingly, when macrophages were stimulated with both LPS and IFN-, luciferase activity was significantly lowered by BZL, although the remaining activity was higher than in control cells. Taking into account that this plasmid does not contain any IFN--responding regulatory sequence, such a result could be reflecting an IFN--dependent pathway of NF-B activation, which is not impaired by BZL (see below). On the other side, BZL did not affect CAT activity induced by IFN- in IRF-EpNOSII-CAT-transfected cells. This selective effect of BZL on the B site was confirmed by EMSAs. LPS and LPS plus IFN- treatment induced the formation of a detectable complex between the nuclear extracts of stimulated cells and NF-B-binding probe. Gel shift of B oligonucleotide was completely inhibited in nuclear extracts obtained from LPS-stimulated cells treated with BZL. When macrophages were stimulated with LPS plus IFN-, the BZL inhibitory effect was also observed, although to a lesser extent. Further analysis of the subunit composition of the nuclear B-binding complexes was performed using Abs specifically directed to p50 and p65 proteins. Beyond the BZL effects, these Abs revealed the existence of a larger proportion of p50/p65 dimers in nuclear extracts of LPS- plus IFN--treated cells compared with those treated with LPS alone. Since p50 can bind DNA but lacks a transactivation domain that is present in p65, these results suggest an interesting explanation for the above-mentioned synergistic effect of IFN- on the B site. Thus, IFN- could modulate NF-B activity by altering p50/p65 dimer composition. A similar mechanism has been proposed for the LPS-generated desensitization phenomenon observed in macrophages in which LPS stimulation in tolerant cells induces mainly p50/p50 homodimers (22).

Finally, Western blot experiments demonstrated that BZL prevented the degradation of the NF-B inhibitor IB after LPS plus IFN- stimulation. Proteolysis of IB, the final step of the NF-B activation pathway, starts after this protein is consecutively phosphorylated and ubiquitinated. Considering that no significant alterations were observed in the cells after 24 h of BZL treatment, a BZL-linked interference with ubiquitination or ubiquitin-dependent proteolysis mechanisms seems unlikely, as it would have resulted highly deleterious for the cell. Alternatively, the BZL mechanism of action could involve the inhibition of one or more of the kinases participating in IkB phosphorylation. Because the inhibitory action of BZL on NF-B was only tested after LPS stimulation, we remain unsure on whether BZL could block NF-B up-regulation governed by other pathways.

NF-B regulates a number of inflammatory genes like proinflammatory cytokines (TNF-, IL-1, IL-2, IL-6, G-CSF), chemokines (IL-8, RANTES, macrophage-inflammatory protein 1, eotaxin), inflammatory enzymes (iNOS, cyclooxygenase 2, 5'-lipoxygenase, cytosolic phospholipase A₂), adhesion molecules (ICAM-1, VCAM-1, E-selectin), and receptors (IL-2R, TCR). During the last years, NF-B up-regulation has been associated to many disease states related to the inflammatory response like septic shock, rheumatoid arthritis, atherosclerosis, asthma, as well as to some types of cancer (see for review Ref. 13). As suggested by several authors, the possibility of developing therapeutic compounds able to specifically block NF-B may constitute a novel and alternative approach for the treatment of the above-mentioned pathologies (see for review Refs. 23, 24). Nevertheless, evidence for a drug that specifically interferes with this activation factor and is safe enough to be used in the clinical field is still lacking.

Although the possibility of BZL blocking the activation of other transcription factors cannot be completely ruled out, we have provided evidence that this compound is able to specifically inhibit NF-B activation. This demonstration, along with the fact that BZL has been used for the treatment of Chagas’ disease for 30 years, raises the potential usefulness of BZL for the management of situations accompanied by excessive inflammatory responses.

Supporting this view, we have recently demonstrated that blocking effects of BZL on NO are also operative in vivo. Thus, rats experimentally infected with T. cruzi under treatment with optimal and suboptimal BZL doses showed lower serum levels of NO and proinflammatory cytokines when compared with their untreated counterparts (25). Furthermore, work in mice with experimentally LPS-induced endotoxemia revealed that BZL not only down-regulated proinflammatory cytokine production but also protected mice from fatal outcome (data not shown)

	Acknowledgments

 
We thank Catherine Silvestri for supplying the cell line used in these studies and Ana Rosa Perez for technical help.

	Footnotes

 
¹ This work was supported by Agencia Nacional de Promocion Cientifica y Techologica (BID 802/OC-AR), Fundacion Antorchas, and Third World Academy of Sciences.

² Address correspondence and reprint requests to Dr. Oscar Bottasso, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Santa Fe 2100, 2000, Rosario, Argentina. E-mail address: bottasso{at}arnet.com.ar

³ Abbreviations used in this paper: BZL, benznidazole; NOSII, NO synthase II; iNOS, inducible NOS; IRF-1, IFN regulatory factor 1; IRF-E, IRF response element; LDH, lactate dehydrogenase; CAT, chloramphenicol acetyltransferase.

Received for publication February 22, 2001. Accepted for publication July 10, 2001.

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