Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Flohé, S. B.
Right arrow Articles by Kolb, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Flohé, S. B.
Right arrow Articles by Kolb, H.
(Journal of Leukocyte Biology. 2002;71:417-424.)
© 2002 by Society for Leukocyte Biology

Enhanced proinflammatory response to endotoxin after priming of macrophages with lead ions

Stefanie B. Flohé, Jutta Brüggemann, Christian Herder, Carsten Goebel and Hubert Kolb

German Diabetes Research Institute, University of Düsseldorf, Germany

Correspondence: Dr. Stefanie B. Flohé, German Diabetes Research Institute, Auf’m Hennekamp 65, 40225 Düsseldorf, Germany. E-mail: flohe{at}ddfi.uni-duesseldorf.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to lead ions strongly enhances the susceptibility of rodents to endotoxin shock and parasitical infections. Macrophages play a key role during the immune response to lipopolysaccharide (LPS) and during the defense against parasites and might be a target of lead. In the present study, bone marrow-derived macrophages (BMM{phi}) pretreated with lead chloride prior to stimulation with LPS were analyzed for their release of immune mediators. Lead-pretreated cells released up to tenfold increased amounts of tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-6, IL-12, and prostaglandin E2 (PGE2) but less IL-10 compared with controls. These effects were paralleled by enhanced mRNA levels and were dependent on the duration of lead pretreatment. Inhibition of protein kinase C or of protein synthesis during the priming phase blocked the lead-induced increase of TNF-{alpha} and IL-6 release. In conclusion, lead ions prime BMM{phi} for enhanced proinflammatory cytokine secretion in response to LPS, likely by activation of protein kinase C and subsequent synthesis of an unidentified mediator.

Key Words: heavy metals • LPS • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Humans are constantly exposed to lead that is released into air or into water by industry, car exhaust fumes, or water pipes. Especially in industrial production, workers are exposed to lead concentrations that often exceed international threshold values [1 , 2 ]. Toxic effects of lead on the central and peripheral nervous system and on heme biosynthesis are well known [3 ].

In addition, effects of lead on immune functions have been demonstrated. Exposure to low doses of lead ions drastically enhances the susceptibility of rodents for lipopolysaccharide (LPS)-induced shock. This phenomenon is associated with enhanced serum of tumor necrosis factor-{alpha} (TNF-{alpha}) levels in response to endotoxin injection [4 ]. In addition, the resistance of C57BL/6 or CBA/J mice against infection with Listeria monocytogenes is impaired after treatment with lead [5 ]. Detailed analyses of the cellular mechanisms underlying these lead-mediated effects are still missing. Because antigen-presenting cells (APC), e.g., macrophages, are a target of LPS-induced effects, they seem to be a primary target for lead.

After stimulation of macrophages with LPS, proinflammatory [e.g., TNF-{alpha}, interleukin (IL)-6, IL-1ß, IL-12] and anti-inflammatory [IL-10, prostaglandin E2 (PGE2)] mediators are secreted, each acting in an autocrine/paracrine manner, thus forming a complex regulatory network. TNF-{alpha} mainly mediates the symptoms associated with endotoxin shock. Moreover, some of the above cytokines promote T-helper (Th) cell differentiation toward Th1 (e.g., IL-12; refs. [6 , 7 ]) or Th2 (e.g., IL-10; ref. [8 ]). Thus, modulation of the LPS-induced cytokine cascade might alter the innate as well as the adaptive immune response.

In the present study, the potential effect of lead chloride on the release of cytokines and other mediators by bone marrow-derived macrophages (BMM{phi}) after stimulation with LPS was investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture medium and reagents
Very low endotoxin (VLE) RPMI-1640 (Seromed, Berlin, Germany), containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; Seromed), 2 mM L-glutamine (Seromed), 20 µg/ml gentamicin, 60 µg/ml penicillin, 50 µM 2-mercaptoethanol, and 10% heat-inactivated fetal calf serum (all from Sigma, Deisenhofen, Germany), was used as culture medium. PbCl2 (2 mM; Fluka, Buchs, Switzerland) in phosphate-buffered saline (PBS; Gibco, Karlsruhe, Germany) was used as stock solution. All solutions contained less than 0.1 EU/ml endotoxin, as stated by the manufacturer and confirmed by the Limulus amebocyte lysate QCL 1000 (BioWhittaker, Walkersville, MD) assay in our hands.

Generation and culture of BMM{phi}
Bone marrow cells were flushed from tibiae and femurs from 7- to 8-week-old female C57BL/6J Bom or nonobese diabetic (NOD) Bom mice (M&B, Ry, Denmark) with culture medium using 27-gauge needles and were depleted from erythrocytes by NH4Cl treatment. For differentiation into macrophages, 3.5 x 105 cells per ml were seeded in 150 cm2 culture flasks (Falcon, Heidelberg, Germany) in 45 ml culture medium containing 30% L929-conditioned medium [9 ]. On day 5, 30% fresh L929-conditioned medium was added. Differentiated macrophages were harvested on day 7, washed, and cultured at 106 cells in 1 ml per well (24-well plates; Falcon) for 18 h. Before onset of experiments, cells were washed twice with 37°C warm PBS.

For priming with PbCl2, macrophages were treated with 0.2–20 µM PbCl2 in medium for different periods of time. After washing twice with warm PBS, macrophages were stimulated with 10 ng/ml Escherichia coli strain 026:B6 LPS (Sigma Chemical Co., St. Louis, MO) in culture medium, and supernatants were harvested after 7 h (for determination of TNF-{alpha}, IL-6, and PGE2) and 20 h (for detection of IL-10 and IL-12).

For inhibition of protein kinase C (PKC) and of protein synthesis, cells were treated with 100 nM calphostin C (Sigma Chemical Co.) or 20 µg/ml puromycin (Sigma Chemical Co.), respectively, for 30 min before adding PbCl2 at a final concentration of 20 µM. Before stimulation with LPS, the cells were washed three times with warm PBS to remove the metal ions and the inhibitor. All culture conditions were proven to be nontoxic by determination of dehydrogenase activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

Determination of cytokines and PGE2
Commercial enzyme-linked immunosorbent assay (ELISA) kits were used for the determination of TNF-{alpha}, IL-12(p70), IL-10, and IL-6 (all OptEIA kits; Pharmingen, Heidelberg, Germany) and the EIA PGE2 kit (Cayman, Ann Arbor, MI), following the manufacturers’ instructions. The detection limit was 16 pg/ml TNF-{alpha}, IL-6, PGE2, and IL-10 and 32 pg/ml IL-12(p70).

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA of 2–3 x 106 BMM{phi} was isolated with 1 ml TriReagent (Sigma Chemical Co.) following the manufacturer’s instructions. RT-PCR was performed as described elsewhere [10 ]. Primer sequences were obtained from Clontech (Palo Alto, CA). Cycle numbers were 22 for ß-actin and 29 for TNF-{alpha} and prostaglandin H synthase type 2 (PGHS-2). To get semiquantitative results, the linearity between the amount of cDNA and signal intensity of the PCR products must be guaranteed. Therefore, serial dilutions of each cDNA were used. Furthermore, each cDNA was tested twice to show the reliability of the results. In all amplifications, template-negative controls confirmed the absence of contaminating cDNA. Amplificates were visualized in a 1.5% agarose gel containing ethidium bromide. For quantification, the fluorescence intensity of each band was determined as Boehringer light units (BLU) using a Lumi-ImagerTM (Boehringer Mannheim, Mannheim, Germany) and the specific analysis software (LumiAnalystTM 3.x). For each cDNA, a curve was generated by plotting the amount of cDNA in the respective dilution versus the corresponding BLU. Linear regression was performed with this curve, and the mRNA amount was set as the BLU value for 1 µl cDNA. The correlation between different amounts of cDNA and the corresponding fluorescence intensity ranged between 0.90 and 0.99. The relative amounts of cytokine mRNA were expressed as arbitrary units as the ratio of cytokine to the respective ß-actin mRNA.

Statistical analyses
To present the extent of variability of duplicate cultures, the difference of each value to the respective mean is shown. The paired Student’s t-test was used to compare medium- with PbCl2-treated cells after repeated measurement, according to the recommendations of GraphPad Prism 3.0 software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PbCl2 primes macrophages for enhanced secretion of TNF-{alpha} after stimulation with LPS
BMM{phi} of C57BL/6 mice were cultured for 0.5–4.5 h in the presence of 0.2–20 µM PbCl2. Controls were cultured in medium only. After extensive washing to remove extracellular lead ions, the cells were stimulated with 10 ng/ml LPS. Supernatants were harvested after 7 h, and amounts of TNF-{alpha} were determined by ELISA.

BMM{phi} that had been treated with PbCl2 before stimulation with LPS released much more TNF-{alpha} into the supernatant than controls (Fig. 1 ). This effect was dependent on the duration of PbCl2 contact and on the PbCl2 concentration used. The highest increase (more than 12-fold) was observed in supernatants of cells that had been treated with 20 µM PbCl2 for 4.5 h. This concentration was nontoxic (as verified by MTT assays) and was selected for further experiments. Lowering the concentration of PbCl2 by two orders of magnitude still enhanced the TNF-{alpha} release by nearly threefold after 4.5 h. No TNF-{alpha} could be detected in supernatants of unstimulated cells or cells that had been treated with PbCl2 only (Fig. 2 ).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. TNF-{alpha} release by BMM{phi} cultured in the presence or absence of PbCl2 before stimulation with LPS. BMM{phi} of C57BL/6 mice were cultured for 0.5, 2, or 4.5 h in the presence of 0.2, 2, or 20 µM PbCl2. Controls were cultured in medium only. After extensive washing, the cells were stimulated with 10 ng/ml LPS. Supernatants were harvested after 7 h, and amounts of TNF-{alpha} were determined by ELISA. Data are representative of two experiments and show the mean of duplicate cultures. Error bars express half of the difference between both values.

 


View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. TNF-{alpha} release by BMM{phi} after parallel stimulation with PbCl2 and LPS. BMM{phi} of C57BL/6 mice were cultured with 0, 2, or 20 µM PbCl2, together with 0, 10, or 100 ng/ml LPS. Supernatants were harvested after 7 h, and amounts of TNF-{alpha} were determined by ELISA. Data are representative of three experiments and show the mean of duplicate cultures. Error bars express half of the difference between both values.

 
To test whether PbCl2 increases the release of TNF-{alpha} when added simultaneously with LPS, BMM{phi} of C57BL/6 mice were cultured with 0–20 µM PbCl2 and 0–100 ng/ml LPS. After 7 h, the supernatants were harvested and analyzed for TNF-{alpha} by ELISA. PbCl2 (2 µM) failed to increase TNF-{alpha} production stimulated by 10 ng/ml LPS (Fig. 2) . A tendency for increased TNF-{alpha} synthesis was found in the presence of 100 ng/ml LPS. A 1.5-fold increased TNF-{alpha} release was found when 20 µM PbCl2 together with 10 or 100 ng/ml LPS was used. However, the absolute amounts of TNF-{alpha} were higher in the presence of 100 ng/ml LPS.

Priming with PbCl2 has increasing and decreasing effects on cytokine release
To assess whether the PbCl2-mediated priming effect is restricted to the proinflammatory cytokine TNF-{alpha}, further immune mediators that are released after stimulation with LPS were determined. BMM{phi} of C57BL/6 mice were cultured for 0.5–4.5 h in the presence of 20 µM PbCl2. Controls were cultured in medium only. After extensive washing, the cells were stimulated with 10 ng/ml LPS. Supernatants were harvested after 7 h and 20 h, and amounts of TNF-{alpha}, IL-6, IL-12, PGE2, and IL-10 were determined by ELISA.

In addition to TNF-{alpha} (Fig. 3 A ), the release of IL-6 and IL-12 in response to LPS was increased by priming with PbCl2. The secretion of the proinflammatory cytokine IL-6 was enhanced 6.7-fold in a time-dependent manner (Fig. 3B) . The amount of IL-12 in supernatants of controls and cells primed with PbCl2 for 0.5 or 2 h did not reach the detection limit but was clearly detectable after 4.5 h priming with PbCl2 (Fig. 3C) . In contrast, the release of the anti-inflammatory and Th2-promoting cytokine IL-10 was reduced threefold by priming with PbCl2 (Fig. 3D) . Similar to TNF-{alpha} and IL-6, this effect was time-dependent. It is interesting that 6.6-fold increased levels of PGE2 were found in supernatants of cells primed with PbCl2 for 4.5 h (Fig. 3E) . This effect was time-dependent as well.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Cytokine secretion pattern of BMM{phi} pretreated with PbCl2 or medium before stimulation with LPS. BMM{phi} of C57BL/6 mice were cultured for 0.5–4.5 h in the presence of 20 µM PbCl2. Controls were cultured in medium only. After extensive washing, the cells were stimulated with 10 ng/ml LPS. After 7 h, the levels of TNF-{alpha} (A), IL-6 (B), and PGE2 (E) were determined in the supernatants. IL-12 (C) and IL-10 (D) ELISA was performed with supernatants of 20 h cultures. Data are representative of three experiments and show the mean of duplicate cultures. Error bars express half of the difference between both values. (b.d., Below detection limit.)

 
These data are representative of three separate experiments, all showing the identical pattern of up- and down-regulation of LPS-induced mediators by priming with PbCl2. However, the extent of the priming effect of PbCl2 varied among different preparations of BMM{phi} (Table 1 ). Experiment 3 is presented in detail (Fig. 3) to allow direct comparison with the corresponding mRNA levels analyzed in parallel (see below).


View this table:
[in this window]
[in a new window]
  		Table 1. Modulation of LPS-Induced Cytokine Secretion by C57BL/6 BMM{Phi} Previously Treated with Medium or 20 µM PbCl2 for 4.5 ha

 
The PbCl2-mediated increase of TNF-{alpha} and PGE2 correlates with enhanced TNF-{alpha} and PGHS-2 mRNA levels
To test whether the enhanced release of TNF-{alpha} and PGE2 that was observed after priming with PbCl2 was associated with enhanced mRNA levels, the transcripts of TNF-{alpha} and PGHS-2 were quantified. BMM{phi} were primed with 20 µM PbCl2 or medium for 4.5 h and were washed and stimulated with 10 ng/ml LPS for 10 min, 0.5 h, 1 h, 4 h, or 7 h. Controls were treated for 4 h with medium or PbCl2 only. Total RNA was isolated and pooled from duplicate cultures. The mRNA was reverse-transcribed, and semiquantitative RT-PCR reactions were performed for ß-actin, TNF-{alpha}, and PGHS-2.

Low levels of TNF-{alpha} transcripts were detectable in control cells treated with medium or PbCl2 alone and did not increase within 10 min after LPS stimulation (Fig. 4 A ). Within 1 h after LPS stimulation, the TNF-{alpha} mRNA levels increased strongly. However, neither control cells nor cells analyzed 10 min or 1 h after LPS stimulation showed any obviously different values between treatment with medium and PbCl2. In BMM{phi} pretreated with medium, TNF-{alpha} mRNA levels were maximal 1 h after LPS stimulation and decreased continuously to a level comparable with the 0.5 h value during the following 6 h. In contrast, TNF-{alpha} mRNA levels of BMM{phi} that had been primed with PbCl2 further increased reaching its maximum 4 h after LPS stimulation. At this time point, PbCl2-treated cells contain 2.6-fold more TNF-{alpha} transcripts than BMM{phi} pretretreated with medium. This divergence was still apparent 7 h after LPS stimulation. Although the TNF-{alpha} mRNA levels in PbCl2-treated cells decreased after 4 h, these cells still contained twofold more transcripts than medium-treated cells after 7 h.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 4. Kinetics of TNF-{alpha} and PGHS-2 mRNA expression after stimulation with LPS in BMM{phi} pretreated with medium or PbCl2. BMM{phi} of C57BL/6 mice were cultured for 4.5 h in the presence or absence of 20 µM PbCl2. After extensive washing, the cells were stimulated with 10 ng/ml LPS, and total RNA was isolated after indicated time points. Control cells were treated with medium or PbCl2 only. After reverse transcription of the mRNA, RT-PCR was performed with primers for ß-actin, TNF-{alpha} (A), and PGHS-2 (B). The relative amounts of cytokine mRNA were expressed as arbitrary units (a.u.) as the ratio of cytokine to the respective ß-actin mRNA. Data represent the mean of two RT-PCR reactions. The variability of two RT-PCR reactions was <15% of the mean.

 
Transcripts of PGHS-2 were not found in control cells treated with medium or PbCl2 alone or in cells 10 min after LPS stimulation (Fig. 4B) . PGHS-2 mRNA was detected 30 min after LPS stimulation and increased during the following 6.5 h. Within the first hour after LPS stimulation, the PGHS-2 mRNA levels of PbCl2 and medium-pretreated cells did not differ. However, PGHS-2 mRNA levels increased dramatically in PbCl2-treated cells within 4 h showing 4.5-fold more transcripts than medium-pretreated cells. Seven hours after LPS stimulation, the difference between PbCl2 and medium-treated cells was even more pronounced because PGHS-2 mRNA levels were enhanced further in PbCl2-treated cells, whereas they increased only slightly in untreated cells. At this time point, sevenfold more PGHS-2 mRNA was found in PbCl2-primed cells.

Priming with PbCl2 also modulates the LPS-induced cytokine release of BMM{phi} of diabetes-prone NOD mice
We tested whether the PbCl2-mediated priming effects are restricted to C57BL/6 mice by analyzing macrophages of diabetes-prone NOD mice. NOD BMM{phi} were treated with 20 µM PbCl2 or medium for 4.5 h and were washed and stimulated with 10 ng/ml LPS. After 7 h and 20 h, supernatants were harvested and analyzed for TNF-{alpha} and IL-10. The release of TNF-{alpha} after stimulation with LPS was increased 3.5-fold by BMM{phi} primed with PbCl2 (Fig. 5 ). In parallel, priming with PbCl2 decreased the LPS-induced IL-10 release by 70% (Fig. 5) . These results are representative of three experiments showing that TNF-{alpha} levels increased 300–600% (P<0.001), and IL-10 levels decreased 50–70% (P<0.001).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. Cytokine expression of NOD BMM{phi} primed with PbCl2 before stimulation with LPS. BMM{phi} of NOD mice were cultured for 4.5 h in the presence or absence of 20 µM PbCl2. After extensive washing, the cells were stimulated with 10 ng/ml LPS. After 7 h and 20 h, the supernatants were analyzed for TNF-{alpha} and IL-10, respectively. Data are representative of three experiments and show the mean of duplicate cultures. Error bars express half of the difference between both values.

 
Inhibition of PKC and protein synthesis abolishes the priming effects mediated by PbCl2
To test whether de novo protein synthesis and/or activation of PKC are involved in the mechanism(s) underlying the PbCl2-mediated increase of cytokine production, BMM{phi} of C57BL/6 mice were primed with medium or 20 µM PbCl2 for 4.5 h each in the absence or presence of 20 µg/ml puromycin (inhibitor of protein synthesis) or 100 nM calphostin C (inhibitor of PKC). After extensive washing to remove the inhibitors, the macrophages were stimulated with 10 ng/ml LPS. Supernatants collected after 7 h were analyzed for TNF-{alpha} and IL-6.

Figure 6 shows the release of TNF-{alpha} (Fig. 6A) and IL-6 (Fig. 6B) in supernatants of BMM{phi} pretreated with PbCl2 or medium each in the absence or presence of puromycin or calphostin C. It is interesting that addition of the inhibitors during the preincubation period induced enhanced TNF-{alpha} release after subsequent LPS stimulation. However, it is clearly visible that the PbCl2-mediated increase of TNF-{alpha} release was almost completely blocked by puromycin as well as by calphostin. The enhancing effect of PbCl2 on the release of IL-6 was also susceptible to inhibition of protein synthesis and PKC, although to a lesser extent than for TNF-{alpha}.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. Impact of PKC and protein synthesis inhibition on cytokine release by BMM{phi} primed with PbCl2. BMM{phi} of C57BL/6 mice were cultured in medium or 20 µM PbCl2 each in the absence or presence of 20 µg/ml puromycin or 100 nM calphostin C for 4.5 h. After extensive washing, the cells were stimulated with 10 ng/ml LPS. After 7 h, the supernatants were analyzed for TNF-{alpha} (A) and IL-6 (B). Data are representative of two experiments and show the mean of duplicate cultures. Error bars express half of the difference between both values.

 
The addition of puromycin or calphostin C during the pretreatment period only did not reduce the release of any cytokine after subsequent stimulation with LPS. These data are representative of two separate experiments with similar results.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study examined the effect of lead on the formation of immune mediators by murine BMM{phi} after stimulation with LPS. It was found that PbCl2 primes BMM{phi} for strongly enhanced release of TNF-{alpha}, IL-6, IL-12, and PGE2 in response to LPS. In parallel, PbCl2 primes for decreased production of IL-10. The increased release of TNF-{alpha} and PGE2 is paralleled by increased expression of TNF-{alpha} and PGHS-2 mRNA. Activation of PKC and the synthesis of unknown proteins seem to be involved during priming with PbCl2.

The most prominent priming effect of PbCl2 was found for the expression of TNF-{alpha}. This proinflammatory cytokine plays a key role in the lethal outcome of endotoxin shock [11 , 12 ]. It is interesting that treatment of mice with PbCl2 enhances the mortality after administration of LPS [4 ]. The serum of these animals contains elevated levels of TNF-{alpha}. Our data suggest that this in vivo finding is caused by a strongly increased TNF-{alpha} release by macrophages primed with PbCl2 before stimulation with LPS.

The priming effect of PbCl2 is not restricted to C57BL/6 BMM{phi}. The development of type-1 diabetes in NOD mice is characterized by autoimmune destruction of the insulin-producing ß-cells and a Th1 cytokine bias in the pancreas. Recently, we could show that the immune system of NOD mice is susceptible to modulation by PbCl2 [13 ]. In this case, oral application of PbCl2 to NOD mice induced a shift of the intestinal Th1/Th2 cytokine balance toward Th1 and impaired the development of oral tolerance to the model antigen ovalbumin. It is well known that cells of the innate immune system, such as macrophages, play a key role in the development of oral tolerance that is dependent on a Th2 cytokine milieu [14 ]. The mechanisms of the above-mentioned in vivo effect of lead ions in NOD mice have not yet been clarified, because appropriate protocols for the isolation of sufficient numbers of nonstimulated intestinal macrophages are still lacking. However, our data might provide a potential explanation. Oral dosage with lead ions might prime intestinal macrophages for the release of proinflammatory mediators that then counteract the development of oral tolerance. NOD BMM{phi} release less TNF-{alpha} because of defective TNF-{alpha} gene regulation [15 ] and less IL-10 [16 ] than normal mouse strains. However, the present data show that the increase of TNF-{alpha} and the decrease of IL-10 after priming with lead ions are also present in NOD BMM{phi}. Thus, lead priming seems to be a general mechanism not associated with a unique major histocompatibility complex (MHC) or non-MHC genotype.

Further, we found that priming of macrophages with lead ions also increased the secretion of the proinflammatory cytokine IL-6 after subsequent stimulation with LPS. This was not unexpected because TNF-{alpha} induces the release of IL-6 [17 ]; thus, increased IL-6 levels as found after priming with lead might result from up-regulated TNF-{alpha} expression.

In addition, priming with lead enhances the release of IL-12, a cytokine that is synthesized by APC and that promotes the expression of IFN-{gamma} by Th and natural killer (NK) cells, thus favoring Th1 development [18 , 19 ]. Hence, the effect of lead ions is not restricted to alterations of the local cytokine milieu via TNF-{alpha} and IL-6, but furthermore, it might act systemically on Th cell-cytokine balance via IL-12. The increase of IL-12 production does not seem to be as high as observed for TNF-{alpha} or IL-6. One explanation could be the inhibitory effect of TNF-{alpha} on the release of IL-12, as described for human macrophages [20 ]. Increasing TNF-{alpha} levels after priming with lead ions might restrict further elevation of IL-12 production. However, TNF-{alpha} synergizes with IL-12 for Th1-cell development [21 ] and might potentiate the Th1 promoting effect induced by IL-12 alone.

In contrast to LPS-induced TNF-{alpha}, IL-6, and IL-12, the release of IL-10 is reduced after priming with lead. IL-10 has anti-inflammatory capacity by inhibiting TNF-{alpha} production by macrophages as well as Th2 promoting capacity by suppressing IFN-{gamma} release from Th1 cells [22 ]. Therefore, the lead-mediated decrease of IL-10 synthesis is expected to drive Th-cell development further toward Th1. TNF-{alpha} is a prerequisite for the induction of IL-10 synthesis after LPS stimulation [23 ]. However, an existing IL-10 synthesis is down-regulated by TNF-{alpha} [24 ]. Hence, the lead-induced reduction of IL-10 within 20 h might result from increased TNF-{alpha} production that was observed within the first 7 h after LPS stimulation.

It is interesting that the release of PGE2 after stimulation with LPS was also increased after priming with lead. PGE2 is synthesized by the inducible form (type 2) of the PGHS and has anti-inflammatory capacity by inhibiting TNF-{alpha} synthesis via induction of IL-10 production [25 ]. Although PGE2 levels were increased after priming with lead, we observed neither reduction of TNF-{alpha} synthesis nor increased IL-10 levels. Exogenous PGE2 has been shown to regulate TNF-{alpha} secretion and vice versa [26 27 28 ]. However, recent studies about murine [17 ] and human [29 ] macrophages showed that capture of endogenous TNF-{alpha} by specific antibodies did not alter the LPS-induced PGE2 release or PGHS-2 transcription. Thus, the LPS-induced production of PGE2 seems to be independent from TNF-{alpha}, using a separate pathway that might be a direct target for lead ions.

LPS induces several signaling cascades resulting in the synthesis of immune mediators that form a complex network of autocrine and paracrine regulation. Theoretically, every part of these pathways could be a target for lead ions. A simple explanation for the lead-mediated increase of TNF-{alpha} production is a lead-induced activation of the enzyme metalloproteinase. This enzyme enhances the release of soluble TNF-{alpha} by cleavage of membrane-bound TNF-{alpha} molecules [30 ]. However, when added in parallel to LPS, PbCl2 failed to enhance TNF-{alpha} release strongly. This observation renders it unlikely that the metalloproteinase is responsible for the lead-mediated effect.

Even after prolonged culture, TNF-{alpha} levels induced after parallel addition of PbCl2 and LPS did not exceed the levels found after pretreatment with lead ions (unpublished results). This finding excludes a delayed onset of increased TNF-{alpha} release at this culture condition, suggesting a crucial role for pretreatment with lead ions alone before subsequent induction of TNF-{alpha} synthesis by LPS. Therefore, we assume that preincubation with lead ions allows the activation or expansion of distinct components of the LPS-signaling cascade, resulting in more efficient cytokine expression after subsequent addition of LPS.

A candidate could be the activation of PKC that occurs early during LPS stimulation. Based on a study about activation of PKC by lead in osteoblastic bone cells [31 ], we hypothesized that PKC might be involved in lead-mediated priming of macrophages as presented here. This hypothesis was tested by inhibition of PKC during pretreatment of macrophages with lead. Blocking PKC activity almost completely abolished the priming activity of lead ions. It is unlikely that this effect was a result of the maintained presence of the PKC inhibitor during LPS stimulation, because inhibition of PKC during pretreatment with medium did not reduce the synthesis of the respective cytokines in response to LPS. Thus, lead ions might activate PKC and allow accelerated LPS signal transduction, resulting in enhanced levels of TNF-{alpha} and PGHS-2 mRNA. However, in this experimental system, treatment with lead ions alone failed to induce cytokine gene transcription and/or protein release. These data are in line with earlier studies from Guo et al. [32 ], who did not find any effects of lead ions on TNF-{alpha} mRNA or protein levels in human peripheral blood mononuclear cells. The finding that parallel addition of lead ions and LPS enhanced the release of TNF-{alpha} only slightly (Fig. 2 ; and ref. [32 ]) without increased mRNA levels (unpublished results; and ref. [32 ]) suggests that there also exists a translational or post-translational mechanism or that the semiquantitative method (also used by Guo et al. [32 ]) is not sensitive enough to detect only small differences of mRNA levels. In vitro and in vivo contrasting results have been demonstrated by others, suggesting a stimulatory capacity of lead ions alone on cytokine-gene transcription. A glia cell line showed enhanced levels of TNF-{alpha} mRNA in the presence of lead ions [33 ], and treatment of rats with lead ions resulted in increased TNF-{alpha} mRNA levels in the liver [34 ]. Furthermore, Lee and Battles [35 ] observed a lead ion-mediated release of PGE2 by splenic macrophages. Glia cells and splenic macrophages might react differently to lead ions as compared with BMM{phi} used by us. A more simple explanation could be a contamination of the lead-containing solution in the latter studies with LPS. Concerning the in vivo findings, it remains unclear whether lead ions alone are responsible for the induction of TNF-{alpha} gene transcription or whether further stimuli such as LPS coming from the gut are involved. Our preliminary results indicate that lead priming is not restricted to the LPS-induced cytokine response, but a modulatory capacity of these ions was also observed after stimulation of BMM{phi} with CpG-containing oligonucleotides, human heat-shock proteins, and zymosan (unpublished results).

The finding that mRNA levels of TNF-{alpha}, PGHS-2 (Fig. 4) , and IL-10 (unpublished results) of lead-treated and untreated cells do not differ considerably 1 h after LPS stimulation could account for a stabilizing effect of lead ions on mRNA as an additional explanation for increased cytokine transcripts. However, to our knowledge, no information about the effect of lead ions on cytokine mRNA stability exists so far.

The activation of PKC is a very fast event and, therefore, does not explain the correlation between the increase of the priming effect and the duration of lead exposure. Rather, the time dependence accounts for an additional mechanism of a much slower rate, such as protein synthesis. This assumption is supported by the finding that inhibition of protein synthesis during lead exposure resulted in a loss of lead-mediated enhancement of cytokine synthesis. When compared with untreated cells, addition of puromycin during preincubation with medium did not reduce cytokine synthesis after subsequent LPS stimulation. Thus, sustained inhibition of protein synthesis during LPS stimulation as cause for inhibition of the lead-mediated priming effect can be excluded. However, the nature of the protein(s) involved in lead-mediated priming remains unknown. Potential candidates could be proteins involved in cytokine mRNA stabilization [36 ], as discussed above, or cytokines that act in an autocrine way, such as IFN-{gamma}, TNF-{alpha}, or granulocyte-macrophage colony-stimulating factor (GM-CSF).

Previously, the capacity of TNF-{alpha} and GM-CSF [37 38 39 ] to prime macrophages for increased TNF-{alpha} release in response to LPS has been described. Thus, a potential mechanism of lead priming could be an induction of IFN-{gamma} or TNF-{alpha} synthesis by lead ions alone, resulting in enhanced TNF-{alpha} release by autocrine stimulation. However, we did not detect increased mRNA levels of these cytokines in macrophages after exposure to lead (unpublished results). Recent studies about priming with GM-CSF revealed that this cytokine increases the release of LPS-induced TNF-{alpha}, but it does not modulate the secretion of IL-10 and IL-12 [40 ]. This finding suggests that priming with GM-CSF and lead occurs via different mechanisms. Furthermore, the lead-mediated priming effect seems not to be based on the secretion of a soluble factor, because transfer of supernatants of cells primed with lead ions did not result in enhanced TNF-{alpha} secretion after subsequent LPS stimulation (unpublished results).

In conclusion, treatment of macrophages with lead ions selectively increases the proinflammatory and Th1-promoting cytokine response to LPS, presumably via PKC activation and synthesis of an unknown mediator. This lead-mediated priming effect might enhance the susceptibility to bacterial, parasitical, and viral infections that has been observed for lead-treated rodents [4 , 5 ] and workers exposed to lead [41 ]. Further, our findings that NOD macrophages are primed by lead ions toward a Th1-inducing phenotype support the hypothesis that lead ions as an environmental factor favor the development of type-1 diabetes in predisposed individuals. Moreover, lead ions are considered to promote the development of autoimmune disorders, because enhanced expression of MHC class-II molecules [42 ] and altered stimulation of the autologous mixed-lymphocyte reaction have been shown [43 ]. Thus, lead exposure might promote Th1-dependent immunopathological conditions.


   ACKNOWLEDGEMENTS
 
This work was supported by grant SFB503/A2 from the Deutsche Forschungsgemeinschaft and by the Bundesminister für Gesundheit and the Minister für Forschung und Wissenschaft des Landes Nordrhein-Westfalen.


   FOOTNOTES
 
Current address of Christian Herder: Blood Donor Service Hessen, Frankfurt, Germany.

Received June 18, 2001; revised September 30, 2001; accepted October 30, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nosal, R. M., Wilhelm, W. J. (1990) Lead toxicity in the shipbreaking industry: the Ontario experience Can. J. Public Health 81,259-262[Medline]
  2. Jakubowski, M., Trzcinka-Ochocka, M., Razniewska, G., Frydrych, J. (1998) Blood lead levels in industrial workers in Poland Int. J. Occup. Med. Environ. Health 11,59-67[Medline]
  3. Grandjean, P. (1978) Widening perspectives of lead toxicity. A review of health effects of lead exposure in adults Environ. Res. 17,303-321[Medline]
  4. Dentener, M. A., Greve, J. W., Maessen, J. G., Buurman, W. A. (1989) Role of tumour necrosis factor in the enhanced sensitivity of mice to endotoxin after exposure to lead Immunopharmacol. Immunotoxicol. 11,321-334[Medline]
  5. Lawrence, D. A. (1981) In vivo and in vitro effects of lead on humoral and cell-mediated immunity Infect. Immun. 31,136-143[Abstract/Free Full Text]
  6. Kennedy, M. K., Picha, K. S., Shanebeck, K. D., Anderson, D. M., Grabstein, K. H. (1994) Interleukin-12 regulates the proliferation of Th1, but not Th2 or Th0, clones Eur. J. Immunol. 24,2271-2278[Medline]
  7. Trinchieri, G., Wysocka, M., D’Andrea, A., Rengaraju, M., Aste Amezaga, M., Kubin, M., Valiante, N. M., Chehimi, J. (1992) Natural killer cell stimulatory factor (NKSF) or interleukin-12 is a key regulator of immune response and inflammation Prog. Growth Factor. Res. 4,355-368[Medline]
  8. Fiorentino, D. F., Bond, M. W., Mosmann, T. R. (1989) Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones J. Exp. Med. 170,2081-2095[Abstract/Free Full Text]
  9. Kruisbeek, A. M. (2000) Isolation and fractionation of mononuclear cell populations Caligan, J. E. Kruisbeck, A. M. Margulies, D. H. Shevach, E. M. Strober, W. eds. Current Protocols in Immunology ,246-249 New York John Wiley and Sons Inc..
  10. Flohé, S. B., Bauer, C., Flohé, S., Moll, H. (1998) Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major Eur. J. Immunol. 28,3800-3811[Medline]
  11. Beutler, B., Cerami, A. (1989) The biology of cachectin/TNF—a primary mediator of the host response Annu. Rev. Immunol. 7,625-655[Medline]
  12. Vassalli, P. (1992) The pathophysiology of tumor necrosis factors Annu. Rev. Immunol. 10,411-452[Medline]
  13. Goebel, C., Flohé, S. B., Kirchhoff, K., Herder, C., Kolb, H. (2000) Orally administered lead chloride induces bias of mucosal immunity Cytokine 12,1414-1418[Medline]
  14. Strobel, S., Mowat, A. M. (1998) Immune responses to dietary antigens: oral tolerance Immunol. Today 19,173-181[Medline]
  15. Bazzoni, F., Beutler, B. (1995) Comparative expression of TNF-alpha alleles from normal and autoimmune-prone MHC haplotypes J. Inflamm. 45,106-114[Medline]
  16. Alleva, D. G., Pavlovich, R. P., Grant, C., Kaser, S. B., Beller, D. I. (2000) Aberrant macrophage cytokine production is a conserved feature among autoimmune-prone mouse strains: elevated interleukin (IL)-12 and an imbalance in tumor necrosis factor-alpha and IL-10 define a unique cytokine profile in macrophages from young nonobese diabetic mice Diabetes 49,1106-1115[Abstract]
  17. Ghezzi, P., Sacco, S., Agnello, D., Marullo, A., Caselli, G., Bertini, R. (2000) LPS induces IL-6 in the brain and in serum largely through TNF production Cytokine 12,1205-1210[Medline]
  18. Hsieh, C. S., Macatonia, S. E., Tripp, C. S., Wolf, S. F., O’Garra, A., Murphy, K. M. (1993) Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages Science 260,547-549[Abstract/Free Full Text]
  19. Manetti, R., Parronchi, P., Giudizi, M. G., Piccinni, M. P., Maggi, E., Trinchieri, G., Romagnani, S. (1993) Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells J. Exp. Med. 177,1199-1204[Abstract/Free Full Text]
  20. Ma, X., Sun, J., Papasavvas, E., Riemann, H., Robertson, S., Marshall, J., Bailer, R. T., Moore, A., Donnelly, R. P., Trinchieri, G., Montaner, L. J. (2000) Inhibition of IL-12 production in human monocyte-derived macrophages by TNF J. Immunol. 164,1722-1729[Abstract/Free Full Text]
  21. Gazzinelli, R. T., Hieny, S., Wynn, T. A., Wolf, S., Sher, A. (1993) Interleukin 12 is required for the T-lymphocyte-independent induction of interferon gamma by an intracellular parasite and induces resistance in T-cell-deficient hosts Proc. Natl. Acad. Sci. USA 90,6115-6119[Abstract/Free Full Text]
  22. Moore, K. W., O’Garra, A., de Waal, M. R., Vieira, P., Mosmann, T. R. (1993) Interleukin-10 Annu. Rev. Immunol. 11,165-190[Medline]
  23. Wanidworanun, C., Strober, W. (1993) Predominant role of tumor necrosis factor-alpha in human monocyte IL-10 synthesis J. Immunol. 151,6853-6861[Abstract]
  24. Bogdan, C., Vodovotz, Y., Nathan, C. (1991) Macrophage deactivation by interleukin 10 J. Exp. Med. 174,1549-1555[Abstract/Free Full Text]
  25. Strassmann, G., Patil-Koota, V., Finkelman, F., Fong, M., Kambayashi, T. (1994) Evidence for the involvement of interleukin 10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2 J. Exp. Med. 180,2365-2370[Abstract/Free Full Text]
  26. Lehmmann, V., Benninghoff, B., Dröge, W. (1988) Tumor necrosis factor-induced activation of peritoneal macrophages is regulated by prostaglandin E2 and cAMP J. Immunol. 141,587-591[Abstract]
  27. Bachwich, P. R., Chensue, S. W., Larrick, J. W., Kunkel, S. L. (1986) Tumor necrosis factor stimulates interleukin-1 and prostaglandin E2 production in resting macrophages Biochem. Biophys. Res. Commun. 136,94-101[Medline]
  28. Renz, H., Gong, J. H., Schmidt, A., Nain, M., Gemsa, D. (1988) Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides J. Immunol. 141,2388-2393[Abstract]
  29. Barrios-Rodiles, M., Tiraloche, G., Chadee, K. (1999) Lipopolysaccharide modulates cyclooxygenase-2 transcriptionally and posttranscriptionally in human macrophages independently from endogenous IL-1 beta and TNF-alpha J. Immunol. 163,963-969[Abstract/Free Full Text]
  30. Gearing, A. J., Beckett, P., Christodoulou, M., Churchill, M., Clements, J. M., Crimmin, M., Davidson, A. H., Drummond, A. H., Galloway, W. A., Gilbert, R. (1995) Matrix metalloproteinases and processing of pro-TNF-alpha J. Leukoc. Biol. 57,774-777[Abstract]
  31. Schanne, F. A., Long, G. J., Rosen, J. F. (1997) Lead induced rise in intracellular free calcium is mediated through activation of protein kinase C in osteoblastic bone cells Biochim. Biophys. Acta 1360,247-254[Medline]
  32. Guo, T. L., Mudzinski, S. P., Lawrence, D. A. (1996) The heavy metal lead modulates the expression of both TNF-alpha and TNF-alpha receptors in lipopolysaccharide-activated human peripheral blood mononuclear cells J. Leukoc. Biol. 59,932-939[Abstract]
  33. Liu, M. Y., Hsieh, W. C., Yang, B. C. (2000) In vitro aberrant gene expression as the indicator of lead-induced neurotoxicity in U-373MG cells Toxicology 147,59-64[Medline]
  34. Shinozuka, H., Kubo, Y., Katyal, S. L., Coni, P., Ledda-Columbano, G. M., Columbano, A., Nakamura, T. (1994) Roles of growth factors and of tumor necrosis factor-alpha on liver cell proliferation induced in rats by lead nitrate Lab. Investig. 71,35-41[Medline]
  35. Lee, J. J., Battles, A. H. (1994) Lead toxicity via arachidonate signal transduction to growth responses in the splenic macrophage Environ. Res. 67,209-219[Medline]
  36. Guhaniyogi, J., Brewer, G. (2001) Regulation of mRNA stability in mammalian cells Gene 265,11-23[Medline]
  37. Williams, J. G., Jurkovich, G. J., Hahnel, G. B., Maier, R. V. (1992) Macrophage priming by interferon gamma: a selective process with potentially harmful effects J. Leukoc. Biol. 52,579-584[Abstract]
  38. Singh, R. K., Sodhi, A. (1991) Effect of TNF priming of murine peritoneal macrophages on their activation to a tumoricidal state Immunol. Lett. 28,127-133[Medline]
  39. Heidenreich, S., Gong, J. H., Schmidt, A., Nain, M., Gemsa, D. (1989) Macrophage activation by granulocyte/macrophage colony-stimulating factor. Priming for enhanced release of tumor necrosis factor-alpha and prostaglandin E2 J. Immunol. 143,1198-1205[Abstract]
  40. Bergamini, A., Bolacchi, F., Bongiovanni, B., Cepparulo, M., Ventura, L., Capozzi, M., Sarrecchia, C., Rocchi, G. (2000) Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms Immunology 101,254-261[Medline]
  41. Horiguchi, S., Endo, G., Kiyota, I., Teramoto, K., Shinagawa, K., Wakitani, F., Tanaka, H., Konishi, Y., Kiyota, A., Ota, A. (1992) Frequency of cold infections in workers at a lead refinery Osaka City Med. J. 38,79-81[Medline]
  42. McCabe, M. J., Jr, Dias, J. A., Lawrence, D. A. (1991) Lead influences translational or posttranslational regulation of Ia expression and increases invariant chain expression in mouse B cells J. Biochem. Toxicol. 6,269-276[Medline]
  43. Kowolenko, M., Tracy, L., Mudzinski, S., Lawrence, D. A. (1988) Effect of lead on macrophage function J. Leukoc. Biol. 43,357-364[Abstract]



This article has been cited by other articles:


Home page
 Hum Exp ToxicolHome page
M. Valentino, V. Rapisarda, L. Santarelli, M. Bracci, M. Scorcelletti, L. Di Lorenzo, F. Cassano, and L. Soleo
Effect of lead on the levels of some immunoregulatory cytokines in occupationally exposed workers
Human and Experimental Toxicology, July 1, 2007; 26(7): 551 - 556.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Flohé, S. B.
Right arrow Articles by Kolb, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Flohé, S. B.
Right arrow Articles by Kolb, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS