ToxSci Advance Access originally published online on December 12, 2006
Toxicological Sciences 2007 96(1):92-100; doi:10.1093/toxsci/kfl182
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Posttranscriptional Inhibition of Interferon-Gamma Production by Lead
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* Department of Occupational Health, Catholic University of Daegu, Kyongsan-si, Kyongbuk, Korea 712-702
Laboratory of Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, New York State Department of Health, Albany, New York 12201
1 To whom correspondence should be addressed at Laboratory of Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, New York State Department of Health, PO Box 509, Albany, NY 12201-0509. Fax: (518)-474-1412. E-mail: david.lawrence{at}wadsworth.org.
Received September 29, 2006; accepted November 1, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lead (Pb) is known to preferentially suppress the activation and development of type-1 CD4+ helper T cell (Th1) responses, whereas it enhances the development of type-2 CD4+ helper T cell (Th2) responses. The inhibition of interferon-gamma (IFN
) production has been demonstrated in vitro with a Th1 clone and DO11.10 ovalbumin-transgenic (OVA-tg) CD4+ T cells, and in vivo with wild-type and OVA-tg BALB/c mice; however, the mechanisms responsible for the Pb-induced downregulation of IFN have not been reported. Here, we assessed the modulation of IFN production at the mRNA and protein levels. Pb did not significantly affect IFN mRNA expression by a Th1 clone or activated splenocytes, as measured by reverse transcriptase–polymerase chain reaction (RT-PCR), ribonuclease protection, and real-time RT-PCR. However, Pb did significantly lower the amount of IFN protein in supernatants and cell lysates of antigen-activated T cells in comparison to stimulated controls, suggesting that the lower amounts of IFN released into culture supernatants were not due to a blockage of secretion that gave rise to a cytoplasmic accumulation of IFN. Pb inhibition also was not prevented by addition of zinc or iron. Pb did not enhance protein degradation of IFN, in that lactacystin, an effective blocker of proteosomal proteolysis, did not prevent loss of IFN; additionally, Pb did not accelerate loss of IFN after cycloheximide treatment. Pb did, however, significantly suppress IFN biosynthesis, as investigated using 35S-incorporation in pulse/chase experiments, although it did not suppress total protein synthesis, indicating that Pb selectively inhibits IFN biosynthesis. Thus, Pb appears to selectively interfere with the translation of certain proteins, such as IFN. IL-12 blocked Pb's preferential promotion of Th2 cells, but absence of STAT6 did not prevent the Pb skewing. Thus, Pb may modulate unique regulatory pathways. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interferon-gamma (IFN
) is a major cytokine that regulates cell-mediated immune responses for the clearance of infectious pathogens, and it is also reported to have antitumor activity (Beatty and Paterson, 2001
; Shtrichman and Samuel, 2001). IFN is produced by natural killer (NK) cells, type-1 CD4+ and CD8+ T cells, and macrophages (Sandra and Belardelli, 1998; Young and Hardy, 1995). The elicited immunologic events associated with IFN include activation of macrophages, differentiation of progenitor helper T cells toward type-1 helper T cell (Th1) cells, and enhancement of major histocompatibility complex (MHC) molecule expression. Furthermore, IFN is known to direct immunoglobulin isotype switching to IgG2a (Snapper and Paul, 1987).
Among a variety of environmental toxicants, the heavy metals lead (Pb) and mercury are known to have detrimental effects on the immune system. Both metals have been demonstrated to preferentially suppress Th1 responses but to enhance type-2 helper T cell (Th2) responses (Heo et al., 1996, 1997, 1998; van Vliet et al., 1993), leading to suppression of host defenses against intracellular microbial infections, such as Listeria monocytogenes infection (Kishikawa et al., 1997). The heavy metal–induced skewing to a Th2 response in vivo could be critical in immunopathologic disorders caused by predominantly Th2-mediated immune responses; respiratory allergic diseases including asthma and rhinitis are notable among these disorders (Heo et al., 2001; Willis-Karp et al., 1998). In vivo Pb exposure has been shown to promote IgE production, which is considered a type-2 allergic immune response (Heo et al., 1996; Snyder et al., 2000). Since cell-mediated immunity is dependent to a large extent on the cytokine IFN, it is important that we understand the means by which Pb interferes with cell-mediated immunity via inhibition of IFN production. Thus, an analysis of Pb effects on IFN synthesis was undertaken. Although reports have documented downregulation of IFN levels in plasma (Heo et al., 1996), sera (Kishikawa et al., 1997), and splenocytes (Miller et al., 1998) of rodents exposed to Pb, and in a Th1 clone stimulated in the presence of Pb (Heo et al., 1996, 1998), the subcellular mechanism underlying the Pb-mediated inhibition of IFN production has not been delineated.
This study was designed to elucidate the intracellular basis of Pb-driven downregulation of IFN production. To this end, we examined Pb effects on expression of IFN mRNA, secretion of IFN protein, proteosomal processing, and kinetics of IFN protein biosynthesis. We found that in vitro IFN biosynthesis is suppressed by Pb exposure of Th1 cells, indicating that Pb's inhibitory role is at the translational stage of IFN biosynthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents.
Stock solutions of 10mM PbCl2 (Fisher Scientific, Pittsburgh, PA) in physiological saline were sterile filtered prior to use. Rabbit IgG (RGG), chicken egg ovalbumin (OVA), and dibutyryl cyclic-AMP (dbcAMP) (Sigma, St Louis, MO) were prepared in physiological saline and sterile filtered prior to addition to cultures. Culture medium was RPMI 1640 supplemented with nonessential amino acids (1mM), sodium pyruvate (1mM), sodium bicarbonate (1%) from Biowhittaker (Walkersville, MD), glutamine (2mM; Sigma), 2-mercaptoethanol (50µM; Fluka, Ronkonkoma, NY), gentamicin (25 µg/ml; Elkins-Sinn, Cherry Hill, NJ), penicillin-streptomycin-neomycin mixture (1%; Gibco, Grand Island, NY), and 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT). Anti-CD3 and anti-CD28 were purchased from BD Pharmingen (San Diego, CA). Murine recombinant IL-12 (MRB 02294-2) was a gift from the Genetics Institute (Cambridge, MA), and anti-IL-4 mAb (11B.11) was obtained from the National Institutes of Health (Bethesda, MD).
Animals and cells.
Male BALB/cByJ or DO11.10 OVA-transgenic (OVA-tg) mice (5–8 weeks old) were obtained from the Wadsworth Center animal production unit. OVA-tg BALB/c mice with a deficiency of STAT4 or STAT6 (Grusby, 1997) were provided by Dr Michael Grusby (Harvard School of Public Health). All mice were virus-free based on serology. All of our animal breeding and experimental procedures were approved by the Wadsworth Center's Institutional Animal Care and Use Committee. Splenic cells from the OVA-tg mice are approximately 50% CD4+ T cells bearing the OVA-specific receptor, which is reactive with monoclonal antibody KJ1-26. The in vitro cultures of the DO11.10 spleen cells from wild-type, STAT4-deficient, or STAT6-deficient BALB/c mice were evaluated for development of Th1 and Th2 activities and measurement of IL-4 and IFN production, as described previously (Heo et al., 1998). The RGG-specific D1.6 Th1 clone (Kurt-Jones et al., 1987) was maintained and utilized as previously described (Heo et al., 1998). To determine IFN mRNA levels from OVA-tg CD4+ T cells by reverse transcriptase–polymerase chain reaction (RT-PCR), we induced naïve splenic T cells from DO11.10 OVA-tg mice to undergo in vitro antigen-dependent differentiation, as described in our previous report (Heo et al., 1998). Briefly, splenocytes (2 x 106/ml) from unimmunized OVA-tg mice were stimulated with OVA (0.5 mg/ml) in the presence of various additives: PbCl2 (25µM), dbcAMP (100µM), rIL-12 (5 ng/ml), or anti-IL-4 mAbs (10 µg/ml). T cells were expanded at 72 h under the same culture conditions as the primary stimulation and were harvested on day 6. After washing of the cells 3 times with PBS, 2 x 105 cells per well were restimulated with OVA and irradiated BALB/c mouse splenocytes, in the absence of the experimental additives. Cells were collected following 24-h restimulation and were used for evaluation of IFN mRNA or protein levels. BALB/c spleen cells (1 x 106/ml) were stimulated with anti-CD3 (1 µg/ml), anti-CD3/CD28 (1 µg/ml each), or Phorbol myristate acetate (PMA) (5 ng/ml) plus ionomycin (1 µg/ml) for 2–48 h.
Quantification of IFN transcripts by RT-PCR.
RT-PCR was used to detect the IFN mRNA (Kishikawa et al., 1997). Resting D1.6 Th1 cells (105) were stimulated with 200 µg RGG presented by 1.5 Gy-irradiated syngeneic BALB/c mouse splenocytes (5 x 106 antigen-presenting cells [APCs]) for 12 or 24 h. At the end of stimulation, total RNA was extracted using the RNA extraction buffer RNAzol (Biotex Laboratories, Houston, TX). A GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, CT) was used for RT-PCR. Total cellular RNA (125 ng) was used for cDNA synthesis by reverse transcription with Moloney murine leukemia virus reverse transcriptase and random hexanucleotides. After termination of the cDNA reaction by heating for 5 min at 90°C, the PCR reaction was performed according to the supplier's instructions, to amplify reverse-transcribed cDNA using primer templates of mouse IFN and internal control ß2-microglobulin (ß2-M). Primers were synthesized at the Wadsworth Center's Molecular Genetics Core, with the following sequences: IFN sense, 5'-TTACTGCCACGGCACAGTCATTGAA-3' and IFN anti-sense, 5'-TCGGATGAGCTCATT GAATGCTTGG-3'; ß2-M sense, 5'-ATGGCTCGCTC-GGTGACCCTAG-3' and ß2-M antisense, 5'-TCATGATGCTTGATCACATGTTCTG-3'. A volume of 20 µl of each PCR product was electrophoresed in 1.1% agarose gel in Tris acetate/EDTA buffer, and the gels were stained with ethidium bromide for visualization of amplified cDNA. Densitometry analysis was performed with an IPlab gel densitometer (Signal Analytics Corporation, Vienna, VA).
Ribonuclease protection assay for IFN.
Ribonuclease protection assay (RPA) is recognized as a fairly sensitive and specific method for the detection and quantification of low-abundance cytokine mRNAs, including that for IFN (Gilman, 1993; Walker et al., 1999). The resting D1.6 Th1 cells (5 x 105) were stimulated with 300 µg RGG and APCs (10 x 106) for 4, 8, 12, or 21 h. Total RNA extracted with RNAzol was used to evaluate IFN mRNA levels in the cells by the RiboQuant Multi-Probe RNase Protection Assay System (PharMingen, San Diego, CA). Briefly, 2 µg of total cellular RNA were hybridized in solution with the [32P]-labeled anti-sense RNA probe set, mCK-2b. Following the hybridization, free probe and other single-stranded RNAs were digested with RNases. The remaining RNase-protected probes were purified, resolved on 5% denaturing polyacrylamide gels, and quantified by Fujix Bas 2000 phosphoimager (Fuji Bio-Imaging, Japan).
Quantitative real-time RT-PCR for IFN.
A two-step process was employed for mRNA quantification. First, cDNA was prepared from 1µg of total RNA using a first-strand cDNA synthesis kit from Roche Applied Science (Indianapolis, IN). Following the synthesis reaction, an aliquot of 200 µl PCR grade water was added to each tube. The second step involved a separate amplification for the IFN gene sequence using primer kits from Search-LC (Heidelberg, Germany). Amplifications were carried out in duplicate in a Roche LightCycler instrument under conditions specified by Search-LC. Standard curves were generated for each run using a standard of known copy number (CN) supplied by Search-LC. Quantitation results were recorded as CN per milliliter, and results were normalized to the CN obtained for GAPDH from the same cDNA synthesis sample.
Quantification of IFN protein levels in the culture supernatants and the cell lysates.
Th1 cells (5 x 105) were cultured with RGG and APCs for 12 or 18 h in the presence or absence of 25µM PbCl2. PbCl2 at this concentration has been reported to significantly inhibit IFN production (Heo et al., 1996, 1998). After termination of the cultivation, culture supernatants were collected, and the cells were lysed with a lysis buffer (20mM Tris, 100mM NaCl, 2mM EDTA, 1% NP40, 0.002% leupeptin and aprotinin, 1mM phenylmethyl sulfonyl fluoride). To measure IFN protein level in the samples, we performed a sandwich ELISA (Heo et al., 1996, 1998) using the mAb pair of R4-6A2 and XMG1.2 (PharMingen). The plates (Immulon 4; Dynatech, Chantilly, VA) were read with an ELISA reader (Bio-Tek CERES UV 900C, Winooski, VT) at 405 nm; the reader automatically calculated the concentration of cytokines from the linear portion of the standard curves. The lower limit of detection was 50 pg/ml for IFN.
Inhibition of proteasomal proteolytic activity.
To test the possibility of Pb-induced proteasomal degradation of IFN protein, we pretreated the Th1 cells (0.5 x 105) with various concentrations of lactacystin (Bimol, Plymouth Meeting, PA), a potent and selective irreversible proteasome inhibitor (Fenteany, 1995), for 30 min, followed by 36-h culture in the presence of APCs and antigen. At the end of the culture, supernatants were collected, and the IFN protein levels were determined by ELISA.
Analysis of IFN degradation.
BALB/c spleen cells (2 x 106) were stimulated with PMA (5 ng/ml) + ionomycin (1 µg/ml) ± PbCl2 (25µM) for 24 h. Cycloheximide (25 µg/ml; Sigma) was then added to the cultures (time 0). After 0, 2, 4, 6, 24, 48, or 72 h, NP40 was added to duplicate cultures (final 1% NP40), and the culture was harvested and frozen until all culture lysates were ready for ELISA.
Metabolic radiolabeling and immunoprecipitations.
The resting Th1 cells (5 x 105) were stimulated with RGG and APC in the presence or absence of Pb for 18 h in methionine- and cysteine-free RPMI 1640 medium containing 10% dialyzed fetal bovine serum. The cells were biosynthetically labeled with 20 µCi of [35S]-Met/Cys mix (NEG-072 EXPRE 35S [35S] Protein Labeling Mix; NEN Life Science, Boston, MA) for 6 h at 6-h intervals from the beginning of the stimulation. After each 6-h pulse labeling, the cells were chased by addition of an excess of nonradiolabeled methionine and cysteine at final concentrations of 0.1mM and 0.4mM, respectively (Bonifacino, 1993). At the end of stimulation, culture supernatants were collected. The supernatants were incubated with control agaroses (rat serum-agarose and goat IgG-agarose, Sigma) on ice for 1 h, to reduce nonspecific background adsorption to agarose or immunoglobulins; the control agaroses were then removed by centrifugation at 200 x g for 10 min. Next, the samples were immunoprecipitated with rat anti-mouse IFN mAb (PharMingen) and goat anti-rat IgG-agarose (Sigma). The immunoprecipitates were collected through centrifugation at 12,000 x g for 5 s and then washed 4 times in the buffer recommended by Sigma. For autoradiography, the samples were subjected to electrophoresis on 10% SDS-polyacrylamide gels, and the bands in autoradiography were visualized by a Fujix Bas 2000 phosphoimager.
In vitro effect of iron or zinc.
Resting D1.6 Th1 cells (0.5 x 105) were stimulated with RGG antigen and APCs (2.5 x 106) for 36 h. Zinc chloride (25µM, Sigma) or ferrous chloride (25µM, Sigma) ± lead chloride (25µM) was added into the culture. At the end of the culture period, culture supernatants were collected, and IFN protein levels were determined by ELISA.
Statistical analyses.
Data were initially evaluated for normal distribution. Statistical significances among groups were tested using Sigmaplot (SPSS, Chicago, IL) by single-factor ANOVA and Dunnett t-test or Kruskal-Wallis ANOVA and Dunn test, depending on normality of data. The significances were further confirmed by Student t-test or Mann-Whitney test. Differences were considered significant when p was less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pb Does Not Modulate mRNA Expression by Th1 Cells or BALB/c Spleen Cells
To investigate the intracellular mechanism of Pb-driven downregulation of IFN
production from Th1 cells, we examined whether Pb (25µM) could suppress expression of IFN mRNA expression by antigen stimulation of a RGG-specific Th1 clone or OVA-specific CD4+ T cells from DO11.10 transgenic BALB/c mice. First, RT-PCR was performed with total RNAs extracted from the D1.6 Th1 cells or OVA-tg T cells stimulated for 12 or 24 h with antigen (RGG for D1.6; OVA for DO11.10). IFN mRNA transcripts were quantified in relative densitometric units compared to ß2-M mRNA transcripts for each culture condition (Fig. 1A). As expected, IL-12 enhanced IFN mRNA expression (Fig. 1A), which is a finding consistent with previous reports (Nakahira et al., 2002; Okamura et al., 1998). Surprisingly, however, neither Pb nor dbcAMP lowered expression of IFN mRNA. Addition of Pb or dbcAMP, a cell-permeable cAMP analog, during Th1 clone activation has been reported (Heo et al., 1998) to inhibit IFN production, but neither Pb nor dbcAMP showed any effect on mRNA levels in our study. Results similar to those obtained at 24 h were obtained at 12 h (data not shown).
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We further evaluated the effect of Pb (25µM) on IFN
mRNA expression using RPA (Figs. 1B and 1C). Again, reduced levels of IFN mRNA were not observed with RGG-stimulated D1.6 Th1 cells in the presence of Pb, whereas addition of rIL-12 significantly upregulated the IFN mRNA level. Results similar to those obtained at 12 h were observed with other stimulation periods (4, 8, and 21 h; data not shown). Thus, these results imply that suppression of IFN production by Th1 cells exposed to Pb is not attributable to decreased IFN gene transcription.
To further assess any inhibitory effects of Pb at early time points after activation, we assayed by real-time RT-PCR the IFN mRNA levels of anti-CD3/anti-CD28–stimulated whole-spleen cell cultures. In this case, the IFN could be generated by CD4+ or CD8+ T cells as well as NK cells. However, at 2–8 h after activation, there was no significant inhibition by Pb (Fig. 2).
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Western Analysis of IFN
Production by BALB/c Spleen CellsTo confirm and extend previous assessment of the inhibition of IFN
by Pb, we cultured spleen cells with various stimulants and assayed after 48 h for the presence of IFN in the culture supernatants. As previously reported for ELISA evaluation, PMA/ionomycin induced more IFN than did anti-CD3/CD28, and anti-CD3/CD28 was more effective than anti-CD3 alone. But, for all the stimulants, the presence of Pb prevented detection of IFN (Fig. 3).
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Pb Inhibits the Intracellular and Extracellular Concentrations of IFN
ProteinNext, we questioned whether the Pb-driven downregulation of IFN
production from Th1 cells was due to inhibition of IFN secretion. For this purpose, IFN protein levels in the activated D1.6 Th1 cell culture supernatants were compared with the levels in the cell lysates (Fig. 4). IFN production was significantly lowered, both in the culture supernatants and in the cell lysates, after the addition of PbCl2, compared to the antigen control (control values: 5.9 ± 0.3 and 9.2 ± 0.9 ng/ml for the supernatants collected 12 and 18 h after stimulation, and 107 ± 20 and 387 ± 144 pg/ml for the lysates at 12 and 18 h, respectively). On the basis of this observation, it appeared that the Pb-induced suppression of IFN production was not mediated through interference with IFN secretion.
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Pb-Enhanced Proteosomal Degradation of Intracellular IFN
Is Not Responsible for the Lower Levels of IFNSince Pb had no significant modulatory effect on IFN
transcription or extracellular export (Figs. 1, 2, and 4), we investigated whether the Pb-induced inhibition of IFN production was dependent on proteosomal proteolysis. To evaluate the possibility of Pb-induced potentiation of proteosome activities, we compared the level of IFN production by D1.6 Th1 cells that had been pretreated with various concentrations of lactacystin and then stimulated with antigen in the presence or absence of Pb. Lactacystin is the most selective proteosome inhibitor known (Fenteany et al., 1995); it can block cytoplasmic degradation of denatured or aberrant proteins by proteosomes. If Pb-driven enhancement of proteosome activities is responsible for the downregulation of IFN production, lactacystin pretreatment should result in similar IFN levels between the Pb-exposed and the unexposed Th1 cells. IFN production from the Pb-exposed Th1 cells remained suppressed in comparison with the antigen control at 10nM and at 100nM lactacystin (Fig. 5). Cytotoxicity was demonstrated with 1mM lactacystin, based on propidium iodide uptake as assayed by flow cytometry (data not shown); lactacystin significantly lowered the IFN production from the Th1 cells, whether or not Pb was added. The results indicated that cytoplasmic degradation of IFN protein is not triggered by Pb treatment of Th1 cells and is not responsible for the Pb-induced inhibition of IFN production.
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Pb Does Not Enhance IFN
DegradationTo determine the overall degradation of IFN
, we assessed the entire culture (supernatant and cell lysate) for immunoreactive IFN in the presence or absence of Pb, we added cycloheximide to block all protein synthesis at 24 h after stimulation with PMA and ionomycin. As shown (Fig. 6), Pb induced the expected inhibition of the IFN production (
30% at time 0). However, with and without Pb, there was equivalent loss of IFN over the next 72 h, and the rate of degradation (slope of the decline from 0–72 h) was also comparable.
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Effects of Iron and Zinc
Since iron and zinc are two metals known to be able to regulate protein synthesis at the translational level (Brumlik and Storey, 1992
; Pantopoulos, 2004; Prasad, 1998; Rogers et al., 2002), we examined whether they could either further exacerbate or lessen the inhibitory effects of Pb. Neither metal significantly altered the suppressive effects of Pb (Fig. 7).
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Pb Effects on the Biosynthesis of IFN
ProteinBased on our results indicating no detectable effects of Pb on IFN
protein transcription, secretion, and proteosomal degradation, we next asked whether Pb could modify translational events for IFN protein expression. To evaluate Pb's potential to suppress a translational step, we performed biosynthetic labeling of the D1.6 Th1 cells with [35S]-methionine/cystine, and we carried out subsequent immunoprecipitation using anti-mouse IFN mAbs. Biosynthetic labeling methods have frequently been adopted to investigate alterations in various translational steps including synthesis, secretion, processing, intracellular transport, and degradation of proteins (Bonifacino, 1993; GoÁalons et al., 1998). The autoradiograph that is shown (Fig. 8) is representative of results from multiple experiments. We found a suppression of IFN protein synthesis for the D1.6 Th1 cells exposed to Pb throughout the metabolic labeling and the chasing periods, in comparison with the Th1 cells cultured with antigen alone. There was consistent suppression of IFN biosynthesis. The difficulty with this experiment was in identifying which bands were IFN because multiple products are known to be generated due to proteolytic processing (Dijkmans et al., 1987). We used two different monoclonal antibodies and polyclonal rabbit antibodies for the immunoprecipitation to aid in the identification. The 6–12 h pulse and pulse/chase are virtually identical; the 12–18 h pulse and pulse/chase are the same since all cultures were harvested at 18 h. The Pb treatment did not significantly alter incorporation of [35S]-methionine/cysteine into total cellular proteins, as we determined by a radiolabeled protein precipitation method using trichloroacetic acid (Bonifacino, 1993). Incorporation ratios (Pb treatment vs. nontreatment) were 108 ± 21% for the 0–6 h, 114 ± 20% for the 6–12 h, and 119 ± 2% for the 12–18 h labeling period. These results indicate that IFN protein synthesis in vitro can be downregulated at an early translational stage by exposure of Th1 cells to Pb, and that the inhibition is selective for particular proteins, including IFN.
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Reversal of Pb's Inhibition of IFN
Production by IL-12As previously reported (Heo et al., 1998
), IL-12 is a strong promoter of Th1 activity, and it is able to prevent the Pb-induced inhibition of IFN production (Fig. 9). Spleen cells from OVA-tg mice were stimulated with OVA (0.5 mg) in the absence (control) or presence of PbCl2 (25µM) or PbCl2 plus recombinant mouse IL-12, for the first 7 days in culture, with reculturing on day 3. On day 7, the cells were restimulated with OVA plus newly isolated syngeneic APCs in the absence of any additional additives except antigen (OVA). Pb promoted a Th2 response by significantly suppressing the IFN response, and IL-12 prevented the Pb-induced skewing toward a type-2 response by significantly blocking Pb's inhibition of IFN and inhibiting the IL-4 response.
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Pb Effect on OVA-tg Splenocytes Lacking STAT4 or STAT6
Since IL-12 can overcome the Pb-induced inhibition of IFN
production, we assessed the effect of Pb on the OVA-tg Th1 and Th2 responses. Deficiencies of STAT4 and STAT6 have been reported to severely curtail, respectively, Th1 (Kaplan et al., 1996a) and Th2 (Kaplan et al., 1996b) development. The assay system that we used was the same as described for the assessment of the IL-12 effects. Although the Th2 response was significantly lower under the condition of the STAT6 deficiency, Pb was still able to preferentially promote a type-2 response (Table 1). As expected, the naïve OVA-tg T cells in the absence of STAT4 were highly skewed toward type-2 responses and there were no significant Pb effects (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pb exposure is known to interfere with the development and/or activation of Th1 cells, which are the CD4+ T cells responsible, in part, for the production of IFN
. In vivo, ex vivo, and in vitro studies of the immune system of mice have shown that Pb is able to suppress IFN production and to enhance Th2 cell responses (Heo et al., 1996, 1997, 1998). Additionally, it has been demonstrated that the effects of Pb on immune cells from different species are similar (mouse, Heo et al., 1998; rat, Miller et al., 1998; and human, Yucesoy et al., 1997).
Since IFN is the cytokine that plays major roles in both innate immunity and adaptive cell-mediated immunity, a better understanding of the mechanisms by which Pb inhibits its production is warranted. The results of this study suggest that Pb inhibits IFN production at a posttranscriptional stage. It is important to note that the inhibition is selective, in that protein synthesis in general was not inhibited by Pb. Although there was a slight, but nonsignificant, lowering of the IFN mRNA level at one early time point with BALB/c spleen cells, there was no inhibition of mRNA expression for IFN with either a T-cell clone or a transgenic T cell line; in fact, Pb consistently enhanced IFN gene expression. The expression of many cytokines is known to be regulated at the transcriptional and posttranscription levels. Inosine has been reported to modulate the levels of IL-1, TNF
, IL-12, and IFN, but not the level of IL-10, at the posttranscriptional stage (Hasko et al., 2000). The production of biologically active IL-12 (heterodimer of p40 and p35), the cytokine that promotes type-1 immunity and IFN production, is regulated in mice by inhibition of p35 translation (Babik et al., 1999). Numerous other chemicals, such as tetracycline (Shapira et al., 1996), chloroquine (Jeong and Jue, 1997), and spermine (Zhang et al., 1997), have been reported to posttranscriptionally regulate inflammatory cytokine production. Thus, it should not be too surprising that an environmental agent, such as Pb, with its multiple regulatory effects on numerous enzymes (Vallee and Ulmer, 1972) can modulate at the posttranscriptional level.
Previous experiments had mainly used ELISA to assay the level of IFN after Pb exposure. In addition, most studies limited analysis to the presence of IFN in the sera or culture supernatants. Thus, the lower quantities of IFN, as a result of Pb exposure, could have been due to blockage of secretion, blockage of a necessary posttranslational process such as glycosylation, or enhanced degradation. Here, these individual possibilities have been examined. The level of IFN in the cells was inhibited to the same extent as was the amount in the supernatant, thus ruling out blockage of secretion. The inhibition of proteosomal degradation was prevented with lactacystin, and the amount of IFN did not show any indication of increase, suggesting that degradation is not the cause for the lower amounts of IFN. This was confirmed by evaluation of IFN degradation in the presence of cycloheximide. The biosynthetic analyses also indicated that Pb affects synthesis rather than degradation. At each pulsing time point, biosynthesis of IFN was seen to be significantly suppressed, based on incorporation of the [35S]-Met/Cys pulse, and there was no apparent further loss of the radiolabeled IFN during the chase period. The biosynthetic results also suggested that translation is inhibited, although these results did not rule out the possibility that an inhibition of a posttranslational process also occurs. The monoclonal antibodies used to immunoprecipitate the [35S]-IFN can detect IFN in Western blot analysis, and they also can recognize recombinant IFN; thus, it is not likely that the glycosylation of IFN can account for the biosynthetic results—inhibition of IFN production.
Although the specific translational or posttranslational process inhibited by Pb remains to be determined, our biosynthetic analysis suggested that Pb inhibits the synthesis of IFN. Reliance solely on the ELISA methodology, whether a sandwich ELISA or a competitive ELISA, opens up the possibility that the linear or conformational epitope recognized by the capture or detection antibody is lost upon Pb binding. Additionally, Pb could modify a normal posttranslational event, causing a change in IFN's refolding in the endoplasmic reticulum, which modifies its tertiary structure causing a loss in its function. Unfortunately, Western blot analysis and the immunoprecipitation of [35S]-labeled protein rely on antibody binding. In attempts to rule out the possibility that Pb caused a structural change, that is, eliminated an immunoreactive epitope, as opposed to causing a blockage of translation, we employed polyclonal and monoclonal antibodies to IFN. Regardless of the epitope specificities, Pb appears to inhibit the presence of IFN. We also previously assessed by ELISA whether Pb could bind to recombinant mouse IFN and reduce its recognition; Pb was unable to inhibit (Lynes et al., 2006). However, recombinant IFN may have slightly different structure than the native molecule (it is not glycosylated), and the recombinant IFN was not synthesized in the presence of Pb, which could alter its conformational epitopes. Even under the best of conditions, quantification of IFN by Western and/or immunoprecipitation analysis is not easily achieved. IFN can be present in multiple forms. The mouse produces a 155-amino acid IFN polypeptide, which has a 23-amino acid leader sequence containing three cysteines. The processed, secreted IFN polypeptide has been shown to exist in multiple molecular weight forms due to glycosylation, aggregation, and proteolysis (Gribaudo et al., 1985; Rinderknecht and Burton, 1985). There are two general species of IFN, having either one or two carbohydrate side chains, and each species naturally aggregates to form functionally active dimers or trimers. These multimers undergo additional modifications, due to loss of one to five C-terminal amino acids, which are cleaved by exopeptidases intracellularly, or at the cell surface (Dijkmans et al., 1987). Given the CYC amino acid sequence at the cleavage site of the leader sequence and the unique cysteine as the C-terminal amino acid, it may be that Pb, with its relatively high affinity for thiols, affects the overall structure and, thus, the normal processing of the IFN molecule.
The translational blockage of IFN by Pb is evidently reversible, in that the addition of IL-12 eliminates the inhibition. IL-12 is well known to enhance IFN production and to suppress the development and activation of type-2 immunity. As previously noted, Pb is selective in its inhibition of protein synthesis, in that it does not inhibit overall protein synthesis. Additionally, previous reports have shown that Pb enhances expression of MHC class II molecules (McCabe and Lawrence, 1990) and Th2 cytokines (Heo et al., 1996). It is unknown whether Pb inhibits IL-12 production, but the type-2 cytokine IL-10 can inhibit IL-12 and IFN production. However, IL-10 cannot block the activity of IL-12 (Hsieh et al., 1992). In addition to promoting signals for IFN expression, IL-12 induces multiple molecular effects independent of IFN (Shi et al., 2004). IL-12 also causes changes in the compartmentalization of T-cell plasma membrane proteins (Salgado et al., 2003). The molecular mechanisms, by which Pb blocks IFN production and by which IL-12 is able to reverse the effect, still need to be investigated. The effects of IL-12 on Th1 cells may be either direct, overcoming the inhibition, or else indirect, inhibiting Pb's promotion of Th2 activities. In either case, it is important that IL-12 eliminates Pb's negative effects on innate and type-1 (IFN-promoted) cell-mediated immune responses, which are critical for host defenses against numerous pathogens. In the absence of STAT4, Pb was unable to further skew the naïve T cells toward an enhanced type-2 response; however, with the absence of STAT4, the response is already highly skewed toward Th2 responses. On the other hand, Pb was able to further enhance Th2 responses in the absence of STAT6. It has been reported that STAT6-mediated signaling is critical for antigen-specific Th2 development (Grusby, 1997; Kaplan et al., 1996b). Without STAT6, the responses were significantly lower than those of the wild-type (approximately threefold difference) or STAT4–/– (11-fold difference) mice. However, Pb was still able to preferentially lower the production of IFN and enhance that of IL-4, suggesting that the Pb effects on T-cell skewing are likely independent of signaling via STAT6.
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ACKNOWLEDGMENTS |
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We thank Dr Michael Grusby (Harvard School of Public Health) for the generous donation of the DO11.10 BALB/c mice with STAT4 or STAT6 deficiency. We also thank the Immunology Core facility of the Wadsworth Center for assistance with flow cytometry and phosphoimager use.
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