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TLR4 is a target of environmentally relevant concentration of lead
Toxicology Letters 214 (2012) 301–306
Contents lists available at SciVerse ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
TLR4 is a target of environmentally relevant concentration of lead Ana L. Luna a,b , Leonor C. Acosta-Saavedra a , Marcela Martínez a , Nallely Torres-Avilés a , Rocío Gómez a , Emma S. Calderón-Aranda a,∗ a b
Departamento de Toxicología, Cinvestav, IPN, Mexico, D.F., Mexico PIBIOM, ENMH del Instituto Politécnico Nacional, Mexico, D.F., Mexico
h i g h l i g h t s
TLR4 is a target to the effect of concentrations ≤5 g/dL of Pb. Pb at concentrations up to 5 g/dL induces IL-1 and IL-6 in macrophages. The Pb differential affect macrophages stimulated with LPSs from two pathogens. Pb effect on TLR4 may be related to decrease in response to infections. The Pb effect on macrophages is dependent of affinity of LPS by the TLR4.
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Article history: Received 14 August 2012 Received in revised form 14 September 2012 Accepted 14 September 2012 Available online 21 September 2012 Keywords: Lead Macrophages TLR4 Lipopolysaccharide
a b s t r a c t Lead (Pb) alters the susceptibility to different pathogens suggesting that macrophage-mediated defense mechanisms, through activation of toll-like receptors (TLRs), may be affected by Pb. The aim of this study was to test whether activation of TLR4 is a targeted molecule to the effect of environmentally relevant Pb concentrations (0.05, 0.5 and 5 g/dL). The function of macrophages activated through TLR4 was evaluated using as TLR4 ligand lipopolysaccharides (LPSs) from two different pathogens: Escherichia coli and Salmonella typhimurium. Pb induced proliferation, increased the NO• − baseline, IL-1 and IL-6 secretion. Interestingly, Pb exposure induced differential effects on cells stimulated with the two LPS used: in macrophages stimulated with LPS from E. coli, Pb caused an early decrease in proliferation, increase NO• − production, and decrease IL-6 and TNF-␣ secretion; in macrophages stimulated with LPS from S. typhimurium, Pb decreased proliferation after 36 h, induced a biphasic effect on NO• − production, and enhance the secretion of IL-1, IL-6 and TNF-␣. Results suggest that TLR4 is a target for the Pb effect, which up to 5.0 g/dL affect immune competence against pathogens, dependent on the bacterial species. This effect may be attributable to structural differences that determine LPS affinity for TLR4. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lead (Pb) is an element ubiquitously distributed around the world that is toxic for the immune system (ATSDR, 2007). Pb at low to moderate levels (2–50 g/dL) does not produce high cytotoxicity of immune cell, but induces deregulation and shifts of functional capacity (McCabe et al., 2001). Immunomodulatory effects of Pb exposure include misbalance in T helper (Th) Th1/Th2 function, which in many conditions favors Th2, and in consequence decreases cell mediated immune response (McCabe and Lawrence, 1991), which in turn affects host resistance to some
∗ Corresponding author at: Departamento de Toxicología, Cinvestav, IPN, PO Box: 14-740, México, D.F., 07360, Mexico. Tel.: +52 55 5747 38 00x5473; fax: +52 555747 33 95. E-mail address: [email protected] (E.S. Calderón-Aranda). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.09.009
infectious diseases. Exposure to Pb (100 and 200 g for 30 days) increases the susceptibility of mice to Salmonella typhimurium (Hemphill et al., 1971) and of rats to Escherichia coli and Staphylococcus epidermis (2.0 mg/100 g, acute dose) (Cook et al., 1975). Also, it reduces the resistance to Serratia marcesens in female mice (Schlipkopter and Frieler, 1979), as well as to Listeria monocytogenes (Lawrence, 1981; Kishikawa et al., 1997). In mice exposed to Pb (10 mg/kg for 15 days) and challenged with Staphylococcus aureus an increase in the intracellular survival of this pathogen was observed. This effect correlates with a decrease in nitric oxide (NO•− ) production, a potent microbicidal produced by the activated macrophages (Bishayi and Sengupta, 2006). These negative effects of Pb on the resistance to bacterial infection suggest that Pb affects macrophage-mediated defense mechanisms through activation of toll-like receptors (TLRs). TLRs belong to PRRs (pattern recognition receptors) which are considered the primary sensors of pathogens and involved in the induction of innate immune
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responses, since PRRs recognize signature molecules of pathogens known as pathogen associated molecular patterns (PAMPs) (Akira et al., 2006). TLRs are type I membrane glycoproteins composed of an extracellular leucine rich domain (LRR), a single transmembrane domain, and an intracellular domain homologous to the interleukin (IL)-1 receptor (IL-1R) and called toll/IL-1 receptor (TIR) domain (Akira and Takeda, 2004). The extracellular domains contain repeated LRRs consisting of 19–25 tandem repeats, where each repeat has a length of 24–29 amino acids. These domains are responsible for the recognition of PAMPs from different pathogens (Gay and Gangloff, 2007). Each member of the TLR family recognizes different pathogen components; TLR4 is the most extensively studied and it recognizes molecules such as lipopolysaccharides (LPSs) from the outer membrane of Gram negative bacteria such as S. typhimurium and E. coli (Iwasaky and Medzhitov, 2004). The binding of these ligands to a single TLR chain induces the dimerization with other chain and triggers the recruitment of adaptor proteins to the intracellular TIR domains and the ensuing signaling (O’Neill and Bowie, 2007). The binding of TLR4 to its ligands induces the activation of several intracellular pathways of the immune response through the production of inflammatory cytokines – such as IL-1, IL-6 and tumor necrosis factor (TNF) TNF␣ – cell surface molecules, as well as NO•− and superoxide anion. All these components are involved in effector functions of macrophages against pathogen infection, among other processes (Kawai and Akira, 2010). The general molecular mechanisms of Pb toxicity are related to its capability of binding to cysteine, induce reactive oxygen species (ROS), and competitively bind to proteins that naturally bind calcium and zinc (Dietert and Piepenbrink, 2006). However, the precise molecular targets and the mechanisms of toxicity on macrophages, particularly those related to the susceptibility to bacterial infection, are not fully understood. Since in murine models Pb exposure modifies inflammatory cytokine profiles and decreases the efficiency to remove some pathogens – a process that involves the activation of TLRs –, the aim of this study was to test whether activation of TLR4 is a target molecule to the immunomodulatory effect of environmentally relevant Pb (0.05, 0.5 and 5 g/dL) concentrations equal or lower than the action level concentration recently established by the Center of Disease Control (CDC, 2012). For this purpose, the function of macrophages activated through TLRs using LPS from two different pathogens (E. coli and S. typhimurium) was evaluated. To carry out this evaluation the following endpoints were considered: cell proliferation, NO•− production, as well as IL-1, IL-6 and TNF-␣ secretion. Our results suggest that the effect of low Pb concentrations, as those found in the general population, produce immunomodulatory effects on macrophages which are dependent on the bacterial origin of the TLR4 ligand used for macrophage activation, indicating that TLR4 is a relevant target for Pb. 2. Materials and methods 2.1. Culture of the J774A.1 murine macrophage cell line This cell line was obtained from the American Type Culture Collection and maintained according to the paper sheets. Cultures were maintained in a Cellstar incubator with 5% CO2 at 37 ◦ C, until they reached 70% confluence. At this point cells were harvested and re-seeded in 24-well plates at a concentration of 0.5 × 105 cell/cm2 .
2.2. Treatment of J774A.1 macrophages Cultures at 70% confluence were stabilized during 16 h before starting the experimental conditions, groups consisted of: (1) control cells, neither activated nor exposed to Pb; (2) positive control, cells non-exposed but activated with the corresponding LPS (1 g/ml of LPS from S. typhimurium or 3 g/ml of LPS from E. coli); (3) cells non-activated but exposed to Pb (see below to concentration used for each assay), and (4) cells exposed to Pb 12 h before activation of macrophages with LPS
from S. typhimurium or E. coli (Fig. 1). We used Pb (C2 H3 O2 )2 ·3H2 O and considered only the Pb in this molecule for the preparation of the solutions. 2.3. Cell viability assays Considering only the metal as was described above, Pb concentrations ranging from 0.005 to 250 g/dL were tested at 12, 24 and 36 h. The cytotoxic effect was assayed by the enzymatic reduction of MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyl tetrazolium) (Carmichael et al., 1987). In the experimental conditions in which cells were pretreated with Pb and activated with LPS, only the amount of cells stimulated was considered as 100%. For cells exposed only to Pb, viability of non-treated cells was considered 100%. 2.4. Cell proliferation by [3 H]-thymidine incorporation For the different experimental conditions, cells were pulsed with 0.5 Ci/well of [3 H]-thymidine (2 Ci/mmol; Amersham Life Science) 12 h before the harvest at 12, 24 and 36 h. Cells were harvested using an automatic Skatron combi cell harvester (Skatron Inst. Lier, Norway); incorporation of [3 H]-thymidine was measured in a scintillation counter (Beckman, LS 6500). 2.5. NO• − determination NO• − was determined by the Griess method (Green et al., 1982) in the supernatant from cultures submitted to the different experimental conditions. Nitrite was used to assess NO• − and absorbance values were read at 550 nm (SpectraMax 250, Molecular Devices, Sunnyvale, CA). A calibration curve prepared with KNO3 and NO• − concentrations was used to calculate NO• − concentrations (nmol). Results of cultures non-stimulated but treated with Pb were compared with the non-stimulated, untreated control which represented baseline NO• − levels. Cultures exposed to Pb and stimulated with LPS were compared with corresponding stimulated cultures for each LPS. NO• − was also determined in presence an antioxidant (±)-6–Hydroxy–2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, at 125 mM) (Sigma–Aldrich, St. Louis, MO). Cultures were exposed to Trolox at the end of the stabilization period and 4 h before starting the activation or exposure to Pb. 2.6. Determination of IL-1ˇ, IL-6, and TNF-˛ secretion The supernatant from J744A.1 cells exposed to the different experimental conditions for 24 h, was collected and stored at −20 ◦ C until used for cytokine determination by ELISA. ELISA was performed in ELISA Maxisorb TM plate (NUNC, Rochester, NY) with DuoSet kits for IL-1, IL-6, and TNF-␣ (R&D Systems, Inc., UK). 2.7. Statistical analysis One-way ANOVA analysis and Holm–Sidak test as post hoc test were used to determine the statistical significance. Data are expressed as the mean ± SD of at least three independent experiments performed in triplicate or quadruplicate. P values <0.05 were considered statistically significant. Statistical analysis was made using SIGMA STAT 3.11 software (Systat software, Inc.).
3. Results 3.1. Cell viability Pb effect on viability was assayed at the following concentrations: 0.005, 0.05, 0.5, 5, 10, 25, 50, 125 and 250 g/dL (24 nM, 240 nM, 2.4 M, 24 M, 48 M, 124 M, 240 M, 600 M and 1.2 mM). The results on cell viability by MTT reduction are shown in Table 1. Because a severe effect on viability was not observed at any Pb concentration, and since induction of MTT reduction was produced in cultures from 0.05 to 25 g/dL of Pb, we decided to evaluate only three of the lower concentrations (0.05, 0.5 and 5 g/dL) all of which are lower or equal to the action level established by the CDC (CDC, 2012). In cells exposed solely to 0.5 g/dL of Pb, a decrease in MTT reduction was observed at 12 h, whereas at 24 h, the all concentrations showed an increase in MTT reduction, an effect that was not detected at 36 h (Table 1). In cells stimulated with LPS from E. coli, the exposure to Pb caused viability to decrease at all times tested when compared with the stimulated cells. Thus, at 12 h the effect was statistically significant for 0.5 and 5 g/dL; at 24 h it was statistically significant for all the concentrations, and at 36 h it was significantly lower in cells exposed to 0.05 and 0.5 g/dL (Fig. 2A). In cells exposed to Pb and stimulated
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Fig. 1. Treatment schemes for experimental conditions. The time course for the treatment and for the assessment of (A) exposure to lead acetate on cells non stimulated; (B) pre exposure to metal with subsequent activation of LPS (S. typhimurium or E. coli). Viability, proliferation, NO• − assays and cytokines are described in the methods section.
with LPS from S. typhimurium the outcome was: at 12 h, significant decreases in viability with 0.05 and 5 g/dL, whereas at 24 and 36 h only 5 g/dL of Pb increased viability (Fig. 2B). 3.2. Cell proliferation The exposure of non-stimulated cells to the three Pb concentrations increased cell proliferation at 12 and 24 h compared with non-exposed controls, but interestingly, at 36 h cell proliferation
was lower in cultures exposed to Pb compared with non-exposed controls (Fig. 2C). Results for non-stimulated and for LPS stimulated controls are shown in the insert of Fig. 2C. In cells stimulated with LPS from E. coli, the three Pb concentrations at 12 h exhibited a significant decrease in proliferation compared with stimulated controls; however, at 24 and 36 h proliferation was not affected at any Pb concentration, following a similar trend to stimulated controls (Fig. 2D). In cells stimulated with LPS from S. typhimurium, the exposure to Pb did not alter proliferation neither at 12 h nor
Fig. 2. Effect of Pb on viability and proliferation. Viability is cells: (A) exposed to Pb and stimulated with LPS from E. coli, (B) exposed to Pb and stimulated with LPS from S. typhimurium. Proliferation in cells: (C) exposed to Pb only, (D) exposed to Pb and stimulated with LPS from E. coli, (E) exposed to Pb and stimulated with LPS from S. typhimurium. All data are mean ± SD from four independent experiments in quadruplicate. *Significant changes as compared with the respective control (p < 0.05).
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Fig. 3. Effect of Pb on NO• − production. NO• − in cells: (A) exposed only to Pb, (B) exposed to Pb and stimulated with LPS from E. coli, (C) exposed to Pb and stimulated with LPS from S. typhimurium. Inserts show the effect of the Trolox in the respective treatment. All data are mean ± SD from four independent experiments in quadruplicate. *Significant changes as compared with the respective control (p < 0.05).
at 24 h; however, proliferation fell at all Pb concentrations at 36 h, compared with stimulated controls (Fig. 2E). 3.3. Nitric oxide In non-stimulated cells, all Pb concentrations significantly increased basal NO•− levels at 12 h; this effect was still present at 24 h with 0.5 and 5 g/dL of Pb, and only with 0.5 g/dL, the effect remained at 36 h. NO•− baseline induced by Pb decreased when an antioxidant was used (insert in Fig. 3A). In cells stimulated with LPS from E. coli, the exposure to all Pb concentrations significantly decreased NO•− at 12 h compared to the control, but at 24 h and 36 h, all concentrations increased NO•− production (Fig. 3B). The treatment with the antioxidant showed
Table 1 Effect of lead on cell viability of J774A.1 macrophages. M
g/dL
Percentage of viability (mean ± SD)a
24 nM 240 nM 2.4 M 24 M 48 M 124 M 240 M 600 M 1.2 mM
0.005 0.05 0.5 5.0 10 25 50 125 250
105 97 85 93 82 88 109 106 99
12 h ± ± ± ± ± ± ± ± ±
24 h 12 7 9* 8 3 0 16 6 7
95 127 116 138 109 114 99 90 97
± ± ± ± ± ± ± ± ±
36 h 20 15* 15* 15* 4* 7* 14 7 12
118 105 105 96 116 107 134 134 136
± ± ± ± ± ± ± ± ±
16 15 12 13 6* 12* 11* 13* 5*
a Values represent the mean ± SD of cuadruplicates from four independent experiments. * Significant changes as compared with the control at 12 h (p < 0.05). Statistical analysis was performed using a one-way ANOVA and Holm–Sidak test for post hoc analysis.
a trend to decrease NO•− production, but this effect was not statistically significant (insert in Fig. 3B). In cells stimulated with LPS from S. typhimurium, all Pb concentrations significantly increased NO•− production at 12 and 24 h, whereas a reduction at 36 h was observed in presence of any of the three Pb concentrations (Fig. 3C). The treatment with the antioxidant exerted a protective effect only for 5 g/dL of Pb at 12 h, whereas this effect was present for all Pb concentrations at 24 and 36 h (insert in Fig. 3C). 3.4. Inflammatory cytokines In cells exposed to Pb only, a trend to increase IL-1 levels was encountered. This effect was statistically different in cells treated with Pb concentrations of 0.05 and 5 g/dL (Fig. 4A). On the other hand, a decrease in IL-6 production was observed with the three concentrations assayed (Fig. 4B). No effect of Pb was found on TNF␣ (Fig. 4C). In cells stimulated with LPS from E. coli, Pb did not affect secreted levels of IL-1 (Fig. 4D), but a significant decrease in secreted IL-6 and TNF-␣ was only detected in cells exposed to 5 g/dL of Pb (Fig. 4E and F, respectively). In cells stimulated with LPS from S. typhimurium, Pb increased IL-1 secretion. This effect showed a trend that depended on Pb concentration; although a significant difference was observed in cells exposed to Pb concentrations of 0.5 and 5 g/dL (Fig. 4G). IL-6 secretion was significantly increased in cells exposed to 0.05 g/dL of Pb (Fig. 4H). Similarly, TNF-␣ was significantly increased in cells exposed to 5 g/dL of Pb (Fig. 4I). 4. Discussion The present study demonstrated that TLR4 is a target molecule to the immunomodulatory effect of low Pb concentrations, at which
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Fig. 4. Effect of Pb exposure and LPS stimulation on cytokines IL-1, IL-6 and TNF-␣. Cytokines from macrophages exposed to Pb (figures A–C), Pb and stimulated with LPS from E. coli. (figures D–F), and Pb and stimulated with LPS from S. typhimurium (figures G–I). All data are mean ± SD from four independent experiments in quadruplicate. *Significant changes, compared with the control (p < 0.05).
most of the human population is environmentally exposed. In addition, we showed a differential effect of Pb on the response of macrophages against LPS from two different pathogenic bacteria (E. coli and S. typhimurium) that bind TLR4 with different affinities (Wiese et al., 1999). The Pb concentrations studied were 0.05, 0.5, and 5 g/dL that are lower or equal to the action level (5 g/dL) established by the CDC (CDC, 2012). In fact, it has been shown that PbB ≈5 g/dL is associated with in vitro effects on cytokine production in peripheral blood mononuclear cells (PBMC) from non-exposed humans stimulated with different activator compounds (Hemdan et al., 2005). It is important to mention that most of previous in vivo and in vitro studies of the Pb effect on immune parameters have used higher Pb concentrations that lie within the range from values ≥10 g/dL to those found in exposed workers (from 21.7± 8.8 to 128.1 ± 104.7 g/dL) (Valentino et al., 2007). Results of the present study showed that at doses of up to 5 g/dL, Pb is capable of altering relevant macrophage functions both in cells only exposed to Pb and in cells activated after being exposed to Pb. Interestingly, Pb effects on macrophages were dependent on the bacterial origin of LPS used; in cells activated with LPS from E. coli, there was a decrease in MTT reduction at all times, but in those activated with LPS from S. typhimurium, the diminution in MTT reduction occurred at 12 h, but increased at 24 and 36 h. To determine whether these effects were due to cytotoxic effect or due to alteration in mitochondrial reductase activity, cell proliferation was performed. Pb exposure alone increased proliferation, except at 36 h where it was lower than the control, whereas in macrophages exposed to Pb and stimulated with LPS from E. coli, proliferation decreased at 12 h, but was not affected at 24 and 36 h; in cells exposed to Pb and stimulated with LPS from S. typhimurium,
Pb did not affect proliferation at 12 and 24 h, but caused a decrease at 36 for all Pb concentrations. Because NO•− is a bactericidal mediator of activated macrophages, we evaluated the effect of Pb on this molecule. Interestingly, in non-stimulated cells, 5 g/dL of Pb increased baseline NO•− levels, as occurred with all concentrations of Pb in cells activated with LPS from E. coli at 36 h, whereas in cells activated with LPS from S. typhimurium a biphasic trend was shown with an increase in NO•− production at 24 h and a decrease at 36 h. Previously, there have been no reports using Pb concentrations similar to the ones used in this study to show the effect of Pb on NO•− production induced by LPS. Given that Pb generates ROS (Verstraeten et al., 2008), we explored whether Pb effect on NO•− had a relationship with Pbinduced oxidative stress. As expected, NO•− production induced by each LPS alone was lower when compared to the control without antioxidant; thus the antioxidant protected against the Pb-induced NO•− in non-activated macrophages, while a partial protective effect occurred in macrophages stimulated with LPS from E. coli and to a lesser extent to those cells stimulated with LPS from S. typhimurium. These results suggest that ROS induced by Pb exposure may be partially involved in the effect on NO•− . However, the molecular mechanism remains to be established. Interestingly, the capability of Pb to inhibit NO•− production was different for each LPS to TLR4, probably due to differences in LPS affinity. Exposure to 1 M (4.8 g/dL) of Pb in bone marrow cells has been reported to increase NO•− production compared with untreated cells, upon stimulation with IFN-␥ and LPS. In addition, a high correlation among increasing transcripts of iNOS, IL-1 and IL-6 has been observed, but not of TNF-␣ transcripts (Song et al., 2001).
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In the present study, Pb affected each assayed cytokine, according to the source of LPS used. Interestingly, IL-1 secretion was induced in non-stimulated cells and in those activated with S. typhimurium, but not in cells activated with LPS from E. coli. The Pb decreased IL-6 in non-activated cells at all concentrations, as well as in cells exposed to 0.5 M of Pb and activated with LPS from E. coli. Conversely, in cells activated with LPS from S. typhimurium, all Pb concentrations originated an increase in IL-6 secretion. Pb alone did not affect TNF-␣, but 5 g/dL of Pb caused a decrease in TNF-␣ secretion in cells activated with LPS from E. coli, and an increase in those activated with LPS from S. typhimurium. TNF-␣ is a major inflammatory cytokine with systemic effects both in defense response and in severe damage (Van der Poll and Lowry, 1995). In workers exposed to Pb, it has been reported that low concentrations of this contaminant (mean of 9.7 ± 4.2 g/dL) increase TNF-␣, but at high concentrations (mean of 21.7 ± 8.8 g/dL) a decrease in this cytokine has been encountered (Valentino et al., 2007). As far as we know, this is the first study that compares the differential effect of Pb in macrophages stimulated with LPS from two different pathogens. This differential effect may be attributable to structural differences of these LPSs that determine the affinity for their common ligand, TLR4 (Wiese et al., 1999). LPS is an endotoxin that consists of a hydrophilic heteropolysaccharide covalently linked to a hydrophobic lipid portion called lipid A, which is responsible for the affinity of LPS for its receptor (Rietchel et al., 1991). The three-dimensional conformation of lipid A depends on its length, number of acyl chains, asymmetry of acyl groups and number or distribution of negative charges. It has been shown that E. coli LPS, which has a lipid A moiety of six acyl groups, exhibits an asymmetrical pattern that results in a conical shape. This conformation originates a strong binding to the LPS-binding protein, whereas a more cylindrical shaped LPS like that of Porphyromonas gingivalis (comprising five asymmetrically distributed acyl groups) confers a poor binding to the LPS-binding protein (Cunningham et al., 1999). These results provide support to the hypothesis that conical and cylindrical shapes of LPS determine different affinities to their complexes (Hirschfeld et al., 2001). Thus, the differential effects of Pb on macrophage stimulation with LPS from E. coli versus S. typhimurium could be explained by structural differences of the lipid A portion of each LPS. Lipid A of E. coli LPS has six acyl groups, whereas S. typhimurium lipid A has seven, probably resulting in a more conical shape than the E. coli LPS and as a result higher affinity for TLR4. Altogether, these results show that the effect of Pb, at low but environmentally relevant levels, produces immunomodulatory effects on macrophages, affecting differentially the immune competence against pathogens with ligands to TLR4, suggesting that the effect of Pb could be more harmful in the response of macrophages against LPS with low affinity for TLR4.The differential effect observed in this study suggests that Pb probably interferes with the binding of LPS to TLR4. However, further studies are needed to define the involved mechanism. Conflict of interest statement The authors declare they have no competing financial interests. Acknowledgements This study was funded by grants from the Mexican Council for Science and Technology (Conacyt 46297-M and 152491).
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
TLR4 is a target of environmentally relevant concentration of lead Ana L. Luna a,b , Leonor C. Acosta-Saavedra a , Marcela Martínez a , Nallely Torres-Avilés a , Rocío Gómez a , Emma S. Calderón-Aranda a,∗ a b
Departamento de Toxicología, Cinvestav, IPN, Mexico, D.F., Mexico PIBIOM, ENMH del Instituto Politécnico Nacional, Mexico, D.F., Mexico
h i g h l i g h t s
TLR4 is a target to the effect of concentrations ≤5 g/dL of Pb. Pb at concentrations up to 5 g/dL induces IL-1 and IL-6 in macrophages. The Pb differential affect macrophages stimulated with LPSs from two pathogens. Pb effect on TLR4 may be related to decrease in response to infections. The Pb effect on macrophages is dependent of affinity of LPS by the TLR4.
a r t i c l e
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Article history: Received 14 August 2012 Received in revised form 14 September 2012 Accepted 14 September 2012 Available online 21 September 2012 Keywords: Lead Macrophages TLR4 Lipopolysaccharide
a b s t r a c t Lead (Pb) alters the susceptibility to different pathogens suggesting that macrophage-mediated defense mechanisms, through activation of toll-like receptors (TLRs), may be affected by Pb. The aim of this study was to test whether activation of TLR4 is a targeted molecule to the effect of environmentally relevant Pb concentrations (0.05, 0.5 and 5 g/dL). The function of macrophages activated through TLR4 was evaluated using as TLR4 ligand lipopolysaccharides (LPSs) from two different pathogens: Escherichia coli and Salmonella typhimurium. Pb induced proliferation, increased the NO• − baseline, IL-1 and IL-6 secretion. Interestingly, Pb exposure induced differential effects on cells stimulated with the two LPS used: in macrophages stimulated with LPS from E. coli, Pb caused an early decrease in proliferation, increase NO• − production, and decrease IL-6 and TNF-␣ secretion; in macrophages stimulated with LPS from S. typhimurium, Pb decreased proliferation after 36 h, induced a biphasic effect on NO• − production, and enhance the secretion of IL-1, IL-6 and TNF-␣. Results suggest that TLR4 is a target for the Pb effect, which up to 5.0 g/dL affect immune competence against pathogens, dependent on the bacterial species. This effect may be attributable to structural differences that determine LPS affinity for TLR4. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lead (Pb) is an element ubiquitously distributed around the world that is toxic for the immune system (ATSDR, 2007). Pb at low to moderate levels (2–50 g/dL) does not produce high cytotoxicity of immune cell, but induces deregulation and shifts of functional capacity (McCabe et al., 2001). Immunomodulatory effects of Pb exposure include misbalance in T helper (Th) Th1/Th2 function, which in many conditions favors Th2, and in consequence decreases cell mediated immune response (McCabe and Lawrence, 1991), which in turn affects host resistance to some
∗ Corresponding author at: Departamento de Toxicología, Cinvestav, IPN, PO Box: 14-740, México, D.F., 07360, Mexico. Tel.: +52 55 5747 38 00x5473; fax: +52 555747 33 95. E-mail address: [email protected] (E.S. Calderón-Aranda). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.09.009
infectious diseases. Exposure to Pb (100 and 200 g for 30 days) increases the susceptibility of mice to Salmonella typhimurium (Hemphill et al., 1971) and of rats to Escherichia coli and Staphylococcus epidermis (2.0 mg/100 g, acute dose) (Cook et al., 1975). Also, it reduces the resistance to Serratia marcesens in female mice (Schlipkopter and Frieler, 1979), as well as to Listeria monocytogenes (Lawrence, 1981; Kishikawa et al., 1997). In mice exposed to Pb (10 mg/kg for 15 days) and challenged with Staphylococcus aureus an increase in the intracellular survival of this pathogen was observed. This effect correlates with a decrease in nitric oxide (NO•− ) production, a potent microbicidal produced by the activated macrophages (Bishayi and Sengupta, 2006). These negative effects of Pb on the resistance to bacterial infection suggest that Pb affects macrophage-mediated defense mechanisms through activation of toll-like receptors (TLRs). TLRs belong to PRRs (pattern recognition receptors) which are considered the primary sensors of pathogens and involved in the induction of innate immune
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responses, since PRRs recognize signature molecules of pathogens known as pathogen associated molecular patterns (PAMPs) (Akira et al., 2006). TLRs are type I membrane glycoproteins composed of an extracellular leucine rich domain (LRR), a single transmembrane domain, and an intracellular domain homologous to the interleukin (IL)-1 receptor (IL-1R) and called toll/IL-1 receptor (TIR) domain (Akira and Takeda, 2004). The extracellular domains contain repeated LRRs consisting of 19–25 tandem repeats, where each repeat has a length of 24–29 amino acids. These domains are responsible for the recognition of PAMPs from different pathogens (Gay and Gangloff, 2007). Each member of the TLR family recognizes different pathogen components; TLR4 is the most extensively studied and it recognizes molecules such as lipopolysaccharides (LPSs) from the outer membrane of Gram negative bacteria such as S. typhimurium and E. coli (Iwasaky and Medzhitov, 2004). The binding of these ligands to a single TLR chain induces the dimerization with other chain and triggers the recruitment of adaptor proteins to the intracellular TIR domains and the ensuing signaling (O’Neill and Bowie, 2007). The binding of TLR4 to its ligands induces the activation of several intracellular pathways of the immune response through the production of inflammatory cytokines – such as IL-1, IL-6 and tumor necrosis factor (TNF) TNF␣ – cell surface molecules, as well as NO•− and superoxide anion. All these components are involved in effector functions of macrophages against pathogen infection, among other processes (Kawai and Akira, 2010). The general molecular mechanisms of Pb toxicity are related to its capability of binding to cysteine, induce reactive oxygen species (ROS), and competitively bind to proteins that naturally bind calcium and zinc (Dietert and Piepenbrink, 2006). However, the precise molecular targets and the mechanisms of toxicity on macrophages, particularly those related to the susceptibility to bacterial infection, are not fully understood. Since in murine models Pb exposure modifies inflammatory cytokine profiles and decreases the efficiency to remove some pathogens – a process that involves the activation of TLRs –, the aim of this study was to test whether activation of TLR4 is a target molecule to the immunomodulatory effect of environmentally relevant Pb (0.05, 0.5 and 5 g/dL) concentrations equal or lower than the action level concentration recently established by the Center of Disease Control (CDC, 2012). For this purpose, the function of macrophages activated through TLRs using LPS from two different pathogens (E. coli and S. typhimurium) was evaluated. To carry out this evaluation the following endpoints were considered: cell proliferation, NO•− production, as well as IL-1, IL-6 and TNF-␣ secretion. Our results suggest that the effect of low Pb concentrations, as those found in the general population, produce immunomodulatory effects on macrophages which are dependent on the bacterial origin of the TLR4 ligand used for macrophage activation, indicating that TLR4 is a relevant target for Pb. 2. Materials and methods 2.1. Culture of the J774A.1 murine macrophage cell line This cell line was obtained from the American Type Culture Collection and maintained according to the paper sheets. Cultures were maintained in a Cellstar incubator with 5% CO2 at 37 ◦ C, until they reached 70% confluence. At this point cells were harvested and re-seeded in 24-well plates at a concentration of 0.5 × 105 cell/cm2 .
2.2. Treatment of J774A.1 macrophages Cultures at 70% confluence were stabilized during 16 h before starting the experimental conditions, groups consisted of: (1) control cells, neither activated nor exposed to Pb; (2) positive control, cells non-exposed but activated with the corresponding LPS (1 g/ml of LPS from S. typhimurium or 3 g/ml of LPS from E. coli); (3) cells non-activated but exposed to Pb (see below to concentration used for each assay), and (4) cells exposed to Pb 12 h before activation of macrophages with LPS
from S. typhimurium or E. coli (Fig. 1). We used Pb (C2 H3 O2 )2 ·3H2 O and considered only the Pb in this molecule for the preparation of the solutions. 2.3. Cell viability assays Considering only the metal as was described above, Pb concentrations ranging from 0.005 to 250 g/dL were tested at 12, 24 and 36 h. The cytotoxic effect was assayed by the enzymatic reduction of MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyl tetrazolium) (Carmichael et al., 1987). In the experimental conditions in which cells were pretreated with Pb and activated with LPS, only the amount of cells stimulated was considered as 100%. For cells exposed only to Pb, viability of non-treated cells was considered 100%. 2.4. Cell proliferation by [3 H]-thymidine incorporation For the different experimental conditions, cells were pulsed with 0.5 Ci/well of [3 H]-thymidine (2 Ci/mmol; Amersham Life Science) 12 h before the harvest at 12, 24 and 36 h. Cells were harvested using an automatic Skatron combi cell harvester (Skatron Inst. Lier, Norway); incorporation of [3 H]-thymidine was measured in a scintillation counter (Beckman, LS 6500). 2.5. NO• − determination NO• − was determined by the Griess method (Green et al., 1982) in the supernatant from cultures submitted to the different experimental conditions. Nitrite was used to assess NO• − and absorbance values were read at 550 nm (SpectraMax 250, Molecular Devices, Sunnyvale, CA). A calibration curve prepared with KNO3 and NO• − concentrations was used to calculate NO• − concentrations (nmol). Results of cultures non-stimulated but treated with Pb were compared with the non-stimulated, untreated control which represented baseline NO• − levels. Cultures exposed to Pb and stimulated with LPS were compared with corresponding stimulated cultures for each LPS. NO• − was also determined in presence an antioxidant (±)-6–Hydroxy–2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, at 125 mM) (Sigma–Aldrich, St. Louis, MO). Cultures were exposed to Trolox at the end of the stabilization period and 4 h before starting the activation or exposure to Pb. 2.6. Determination of IL-1ˇ, IL-6, and TNF-˛ secretion The supernatant from J744A.1 cells exposed to the different experimental conditions for 24 h, was collected and stored at −20 ◦ C until used for cytokine determination by ELISA. ELISA was performed in ELISA Maxisorb TM plate (NUNC, Rochester, NY) with DuoSet kits for IL-1, IL-6, and TNF-␣ (R&D Systems, Inc., UK). 2.7. Statistical analysis One-way ANOVA analysis and Holm–Sidak test as post hoc test were used to determine the statistical significance. Data are expressed as the mean ± SD of at least three independent experiments performed in triplicate or quadruplicate. P values <0.05 were considered statistically significant. Statistical analysis was made using SIGMA STAT 3.11 software (Systat software, Inc.).
3. Results 3.1. Cell viability Pb effect on viability was assayed at the following concentrations: 0.005, 0.05, 0.5, 5, 10, 25, 50, 125 and 250 g/dL (24 nM, 240 nM, 2.4 M, 24 M, 48 M, 124 M, 240 M, 600 M and 1.2 mM). The results on cell viability by MTT reduction are shown in Table 1. Because a severe effect on viability was not observed at any Pb concentration, and since induction of MTT reduction was produced in cultures from 0.05 to 25 g/dL of Pb, we decided to evaluate only three of the lower concentrations (0.05, 0.5 and 5 g/dL) all of which are lower or equal to the action level established by the CDC (CDC, 2012). In cells exposed solely to 0.5 g/dL of Pb, a decrease in MTT reduction was observed at 12 h, whereas at 24 h, the all concentrations showed an increase in MTT reduction, an effect that was not detected at 36 h (Table 1). In cells stimulated with LPS from E. coli, the exposure to Pb caused viability to decrease at all times tested when compared with the stimulated cells. Thus, at 12 h the effect was statistically significant for 0.5 and 5 g/dL; at 24 h it was statistically significant for all the concentrations, and at 36 h it was significantly lower in cells exposed to 0.05 and 0.5 g/dL (Fig. 2A). In cells exposed to Pb and stimulated
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Fig. 1. Treatment schemes for experimental conditions. The time course for the treatment and for the assessment of (A) exposure to lead acetate on cells non stimulated; (B) pre exposure to metal with subsequent activation of LPS (S. typhimurium or E. coli). Viability, proliferation, NO• − assays and cytokines are described in the methods section.
with LPS from S. typhimurium the outcome was: at 12 h, significant decreases in viability with 0.05 and 5 g/dL, whereas at 24 and 36 h only 5 g/dL of Pb increased viability (Fig. 2B). 3.2. Cell proliferation The exposure of non-stimulated cells to the three Pb concentrations increased cell proliferation at 12 and 24 h compared with non-exposed controls, but interestingly, at 36 h cell proliferation
was lower in cultures exposed to Pb compared with non-exposed controls (Fig. 2C). Results for non-stimulated and for LPS stimulated controls are shown in the insert of Fig. 2C. In cells stimulated with LPS from E. coli, the three Pb concentrations at 12 h exhibited a significant decrease in proliferation compared with stimulated controls; however, at 24 and 36 h proliferation was not affected at any Pb concentration, following a similar trend to stimulated controls (Fig. 2D). In cells stimulated with LPS from S. typhimurium, the exposure to Pb did not alter proliferation neither at 12 h nor
Fig. 2. Effect of Pb on viability and proliferation. Viability is cells: (A) exposed to Pb and stimulated with LPS from E. coli, (B) exposed to Pb and stimulated with LPS from S. typhimurium. Proliferation in cells: (C) exposed to Pb only, (D) exposed to Pb and stimulated with LPS from E. coli, (E) exposed to Pb and stimulated with LPS from S. typhimurium. All data are mean ± SD from four independent experiments in quadruplicate. *Significant changes as compared with the respective control (p < 0.05).
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Fig. 3. Effect of Pb on NO• − production. NO• − in cells: (A) exposed only to Pb, (B) exposed to Pb and stimulated with LPS from E. coli, (C) exposed to Pb and stimulated with LPS from S. typhimurium. Inserts show the effect of the Trolox in the respective treatment. All data are mean ± SD from four independent experiments in quadruplicate. *Significant changes as compared with the respective control (p < 0.05).
at 24 h; however, proliferation fell at all Pb concentrations at 36 h, compared with stimulated controls (Fig. 2E). 3.3. Nitric oxide In non-stimulated cells, all Pb concentrations significantly increased basal NO•− levels at 12 h; this effect was still present at 24 h with 0.5 and 5 g/dL of Pb, and only with 0.5 g/dL, the effect remained at 36 h. NO•− baseline induced by Pb decreased when an antioxidant was used (insert in Fig. 3A). In cells stimulated with LPS from E. coli, the exposure to all Pb concentrations significantly decreased NO•− at 12 h compared to the control, but at 24 h and 36 h, all concentrations increased NO•− production (Fig. 3B). The treatment with the antioxidant showed
Table 1 Effect of lead on cell viability of J774A.1 macrophages. M
g/dL
Percentage of viability (mean ± SD)a
24 nM 240 nM 2.4 M 24 M 48 M 124 M 240 M 600 M 1.2 mM
0.005 0.05 0.5 5.0 10 25 50 125 250
105 97 85 93 82 88 109 106 99
12 h ± ± ± ± ± ± ± ± ±
24 h 12 7 9* 8 3 0 16 6 7
95 127 116 138 109 114 99 90 97
± ± ± ± ± ± ± ± ±
36 h 20 15* 15* 15* 4* 7* 14 7 12
118 105 105 96 116 107 134 134 136
± ± ± ± ± ± ± ± ±
16 15 12 13 6* 12* 11* 13* 5*
a Values represent the mean ± SD of cuadruplicates from four independent experiments. * Significant changes as compared with the control at 12 h (p < 0.05). Statistical analysis was performed using a one-way ANOVA and Holm–Sidak test for post hoc analysis.
a trend to decrease NO•− production, but this effect was not statistically significant (insert in Fig. 3B). In cells stimulated with LPS from S. typhimurium, all Pb concentrations significantly increased NO•− production at 12 and 24 h, whereas a reduction at 36 h was observed in presence of any of the three Pb concentrations (Fig. 3C). The treatment with the antioxidant exerted a protective effect only for 5 g/dL of Pb at 12 h, whereas this effect was present for all Pb concentrations at 24 and 36 h (insert in Fig. 3C). 3.4. Inflammatory cytokines In cells exposed to Pb only, a trend to increase IL-1 levels was encountered. This effect was statistically different in cells treated with Pb concentrations of 0.05 and 5 g/dL (Fig. 4A). On the other hand, a decrease in IL-6 production was observed with the three concentrations assayed (Fig. 4B). No effect of Pb was found on TNF␣ (Fig. 4C). In cells stimulated with LPS from E. coli, Pb did not affect secreted levels of IL-1 (Fig. 4D), but a significant decrease in secreted IL-6 and TNF-␣ was only detected in cells exposed to 5 g/dL of Pb (Fig. 4E and F, respectively). In cells stimulated with LPS from S. typhimurium, Pb increased IL-1 secretion. This effect showed a trend that depended on Pb concentration; although a significant difference was observed in cells exposed to Pb concentrations of 0.5 and 5 g/dL (Fig. 4G). IL-6 secretion was significantly increased in cells exposed to 0.05 g/dL of Pb (Fig. 4H). Similarly, TNF-␣ was significantly increased in cells exposed to 5 g/dL of Pb (Fig. 4I). 4. Discussion The present study demonstrated that TLR4 is a target molecule to the immunomodulatory effect of low Pb concentrations, at which
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Fig. 4. Effect of Pb exposure and LPS stimulation on cytokines IL-1, IL-6 and TNF-␣. Cytokines from macrophages exposed to Pb (figures A–C), Pb and stimulated with LPS from E. coli. (figures D–F), and Pb and stimulated with LPS from S. typhimurium (figures G–I). All data are mean ± SD from four independent experiments in quadruplicate. *Significant changes, compared with the control (p < 0.05).
most of the human population is environmentally exposed. In addition, we showed a differential effect of Pb on the response of macrophages against LPS from two different pathogenic bacteria (E. coli and S. typhimurium) that bind TLR4 with different affinities (Wiese et al., 1999). The Pb concentrations studied were 0.05, 0.5, and 5 g/dL that are lower or equal to the action level (5 g/dL) established by the CDC (CDC, 2012). In fact, it has been shown that PbB ≈5 g/dL is associated with in vitro effects on cytokine production in peripheral blood mononuclear cells (PBMC) from non-exposed humans stimulated with different activator compounds (Hemdan et al., 2005). It is important to mention that most of previous in vivo and in vitro studies of the Pb effect on immune parameters have used higher Pb concentrations that lie within the range from values ≥10 g/dL to those found in exposed workers (from 21.7± 8.8 to 128.1 ± 104.7 g/dL) (Valentino et al., 2007). Results of the present study showed that at doses of up to 5 g/dL, Pb is capable of altering relevant macrophage functions both in cells only exposed to Pb and in cells activated after being exposed to Pb. Interestingly, Pb effects on macrophages were dependent on the bacterial origin of LPS used; in cells activated with LPS from E. coli, there was a decrease in MTT reduction at all times, but in those activated with LPS from S. typhimurium, the diminution in MTT reduction occurred at 12 h, but increased at 24 and 36 h. To determine whether these effects were due to cytotoxic effect or due to alteration in mitochondrial reductase activity, cell proliferation was performed. Pb exposure alone increased proliferation, except at 36 h where it was lower than the control, whereas in macrophages exposed to Pb and stimulated with LPS from E. coli, proliferation decreased at 12 h, but was not affected at 24 and 36 h; in cells exposed to Pb and stimulated with LPS from S. typhimurium,
Pb did not affect proliferation at 12 and 24 h, but caused a decrease at 36 for all Pb concentrations. Because NO•− is a bactericidal mediator of activated macrophages, we evaluated the effect of Pb on this molecule. Interestingly, in non-stimulated cells, 5 g/dL of Pb increased baseline NO•− levels, as occurred with all concentrations of Pb in cells activated with LPS from E. coli at 36 h, whereas in cells activated with LPS from S. typhimurium a biphasic trend was shown with an increase in NO•− production at 24 h and a decrease at 36 h. Previously, there have been no reports using Pb concentrations similar to the ones used in this study to show the effect of Pb on NO•− production induced by LPS. Given that Pb generates ROS (Verstraeten et al., 2008), we explored whether Pb effect on NO•− had a relationship with Pbinduced oxidative stress. As expected, NO•− production induced by each LPS alone was lower when compared to the control without antioxidant; thus the antioxidant protected against the Pb-induced NO•− in non-activated macrophages, while a partial protective effect occurred in macrophages stimulated with LPS from E. coli and to a lesser extent to those cells stimulated with LPS from S. typhimurium. These results suggest that ROS induced by Pb exposure may be partially involved in the effect on NO•− . However, the molecular mechanism remains to be established. Interestingly, the capability of Pb to inhibit NO•− production was different for each LPS to TLR4, probably due to differences in LPS affinity. Exposure to 1 M (4.8 g/dL) of Pb in bone marrow cells has been reported to increase NO•− production compared with untreated cells, upon stimulation with IFN-␥ and LPS. In addition, a high correlation among increasing transcripts of iNOS, IL-1 and IL-6 has been observed, but not of TNF-␣ transcripts (Song et al., 2001).
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In the present study, Pb affected each assayed cytokine, according to the source of LPS used. Interestingly, IL-1 secretion was induced in non-stimulated cells and in those activated with S. typhimurium, but not in cells activated with LPS from E. coli. The Pb decreased IL-6 in non-activated cells at all concentrations, as well as in cells exposed to 0.5 M of Pb and activated with LPS from E. coli. Conversely, in cells activated with LPS from S. typhimurium, all Pb concentrations originated an increase in IL-6 secretion. Pb alone did not affect TNF-␣, but 5 g/dL of Pb caused a decrease in TNF-␣ secretion in cells activated with LPS from E. coli, and an increase in those activated with LPS from S. typhimurium. TNF-␣ is a major inflammatory cytokine with systemic effects both in defense response and in severe damage (Van der Poll and Lowry, 1995). In workers exposed to Pb, it has been reported that low concentrations of this contaminant (mean of 9.7 ± 4.2 g/dL) increase TNF-␣, but at high concentrations (mean of 21.7 ± 8.8 g/dL) a decrease in this cytokine has been encountered (Valentino et al., 2007). As far as we know, this is the first study that compares the differential effect of Pb in macrophages stimulated with LPS from two different pathogens. This differential effect may be attributable to structural differences of these LPSs that determine the affinity for their common ligand, TLR4 (Wiese et al., 1999). LPS is an endotoxin that consists of a hydrophilic heteropolysaccharide covalently linked to a hydrophobic lipid portion called lipid A, which is responsible for the affinity of LPS for its receptor (Rietchel et al., 1991). The three-dimensional conformation of lipid A depends on its length, number of acyl chains, asymmetry of acyl groups and number or distribution of negative charges. It has been shown that E. coli LPS, which has a lipid A moiety of six acyl groups, exhibits an asymmetrical pattern that results in a conical shape. This conformation originates a strong binding to the LPS-binding protein, whereas a more cylindrical shaped LPS like that of Porphyromonas gingivalis (comprising five asymmetrically distributed acyl groups) confers a poor binding to the LPS-binding protein (Cunningham et al., 1999). These results provide support to the hypothesis that conical and cylindrical shapes of LPS determine different affinities to their complexes (Hirschfeld et al., 2001). Thus, the differential effects of Pb on macrophage stimulation with LPS from E. coli versus S. typhimurium could be explained by structural differences of the lipid A portion of each LPS. Lipid A of E. coli LPS has six acyl groups, whereas S. typhimurium lipid A has seven, probably resulting in a more conical shape than the E. coli LPS and as a result higher affinity for TLR4. Altogether, these results show that the effect of Pb, at low but environmentally relevant levels, produces immunomodulatory effects on macrophages, affecting differentially the immune competence against pathogens with ligands to TLR4, suggesting that the effect of Pb could be more harmful in the response of macrophages against LPS with low affinity for TLR4.The differential effect observed in this study suggests that Pb probably interferes with the binding of LPS to TLR4. However, further studies are needed to define the involved mechanism. Conflict of interest statement The authors declare they have no competing financial interests. Acknowledgements This study was funded by grants from the Mexican Council for Science and Technology (Conacyt 46297-M and 152491).
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