Posttranscriptional Inhibition of Interferon-Gamma Production by Lead

TOXICOLOGICAL SCIENCES 96(1), 92–100 (2007) doi:10.1093/toxsci/kfl182 Advance Access publication December 12, 2006

Posttranscriptional Inhibition of Interferon-Gamma Production by Lead Yong Heo,* Tapan K. Mondal,† Donghong Gao,† Jane Kasten-Jolly,† Hiroko Kishikawa,† and David A. Lawrence†,1 *Department of Occupational Health, Catholic University of Daegu, Kyongsan-si, Kyongbuk, Korea 712-702; and †Laboratory of Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, New York State Department of Health, Albany, New York 12201 Received September 29, 2006; accepted November 1, 2006

Interferon-gamma (IFNc) 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). 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 122010509. Fax: (518)-474-1412. E-mail: [email protected].


The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: [email protected]

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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 (IFNg) 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 IFNg have not been reported. Here, we assessed the modulation of IFNg production at the mRNA and protein levels. Pb did not significantly affect IFNg 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 IFNg protein in supernatants and cell lysates of antigen-activated T cells in comparison to stimulated controls, suggesting that the lower amounts of IFNg released into culture supernatants were not due to a blockage of secretion that gave rise to a cytoplasmic accumulation of IFNg. Pb inhibition also was not prevented by addition of zinc or iron. Pb did not enhance protein degradation of IFNg, in that lactacystin, an effective blocker of proteosomal proteolysis, did not prevent loss of IFNg; additionally, Pb did not accelerate loss of IFNg after cycloheximide treatment. Pb did, however, significantly suppress IFNg biosynthesis, as investigated using 35S-incorporation in pulse/chase experiments, although it did not suppress total protein synthesis, indicating that Pb selectively inhibits IFNg biosynthesis. Thus, Pb appears to selectively interfere with the translation of certain proteins, such as IFNg. 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.

IFNc is produced by natural killer (NK) cells, type-1 CD4þ and CD8þ T cells, and macrophages (Sandra and Belardelli, 1998; Young and Hardy, 1995). The elicited immunologic events associated with IFNc 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, IFNc 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 IFNc, it is important that we understand the means by which Pb interferes with cell-mediated immunity via inhibition of IFNc production. Thus, an analysis of Pb effects on IFNc synthesis was undertaken. Although reports have documented downregulation of IFNc 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 IFNc production has not been delineated. This study was designed to elucidate the intracellular basis of Pb-driven downregulation of IFNc production. To this end, we examined Pb effects on expression of IFNc mRNA, secretion of IFNc protein, proteosomal processing, and kinetics

LEAD INHIBITS IFNc PRODUCTION

of IFNc protein biosynthesis. We found that in vitro IFNc biosynthesis is suppressed by Pb exposure of Th1 cells, indicating that Pb’s inhibitory role is at the translational stage of IFNc biosynthesis.

MATERIALS AND METHODS

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% CD4þ 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 STAT6deficient BALB/c mice were evaluated for development of Th1 and Th2 activities and measurement of IL-4 and IFNc 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 IFNc mRNA levels from OVA-tg CD4þ T cells by reverse transcriptase– polymerase chain reaction (RT-PCR), we induced naı¨ve 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 3 106/ml) from unimmunized OVA-tg mice were stimulated with OVA (0.5 mg/ ml) in the presence of various additives: PbCl2 (25lM), dbcAMP (100lM), rIL12 (5 ng/ml), or anti-IL-4 mAbs (10 lg/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 3 105 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 IFNc mRNA or protein levels. BALB/c spleen cells (1 3 106/ml) were stimulated with anti-CD3 (1 lg/ml), antiCD3/CD28 (1 lg/ml each), or Phorbol myristate acetate (PMA) (5 ng/ml) plus ionomycin (1 lg/ml) for 2–48 h. Quantification of IFNc transcripts by RT-PCR. RT-PCR was used to detect the IFNc mRNA (Kishikawa et al., 1997). Resting D1.6 Th1 cells (105) were stimulated with 200 lg RGG presented by 1.5 Gy-irradiated syngeneic BALB/c mouse splenocytes (5 3 106 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 90C, the PCR reaction was performed according to the supplier’s

instructions, to amplify reverse-transcribed cDNA using primer templates of mouse IFNc and internal control b2-microglobulin (b2-M). Primers were synthesized at the Wadsworth Center’s Molecular Genetics Core, with the following sequences: IFNc sense, 5#-TTACTGCCACGGCACAGTCATTGAA-3# and IFNc anti-sense, 5#-TCGGATGAGCTCATT GAATGCTTGG-3#; b2-M sense, 5#-ATGGCTCGCTC-GGTGACCCTAG-3# and b2-M antisense, 5#-TCATGATGCTTGATCACATGTTCTG-3#. A volume of 20 ll 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 IFNc. 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 IFNc (Gilman, 1993; Walker et al., 1999). The resting D1.6 Th1 cells (5 3 105) were stimulated with 300 lg RGG and APCs (10 3 106) for 4, 8, 12, or 21 h. Total RNA extracted with RNAzol was used to evaluate IFNc mRNA levels in the cells by the RiboQuant Multi-Probe RNase Protection Assay System (PharMingen, San Diego, CA). Briefly, 2 lg 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 IFNc. A two-step process was employed for mRNA quantification. First, cDNA was prepared from 1lg of total RNA using a first-strand cDNA synthesis kit from Roche Applied Science (Indianapolis, IN). Following the synthesis reaction, an aliquot of 200 ll PCR grade water was added to each tube. The second step involved a separate amplification for the IFNc 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 IFNc protein levels in the culture supernatants and the cell lysates. Th1 cells (5 3 105) were cultured with RGG and APCs for 12 or 18 h in the presence or absence of 25lM PbCl2. PbCl2 at this concentration has been reported to significantly inhibit IFNc 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 IFNc 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 IFNc. Inhibition of proteasomal proteolytic activity. To test the possibility of Pb-induced proteasomal degradation of IFNc protein, we pretreated the Th1 cells (0.5 3 105) 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 IFNc protein levels were determined by ELISA. Analysis of IFNc degradation. BALB/c spleen cells (2 3 106) were stimulated with PMA (5 ng/ml) þ ionomycin (1 lg/ml) ± PbCl2 (25lM) for 24 h. Cycloheximide (25 lg/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%

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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 (50lM; Fluka, Ronkonkoma, NY), gentamicin (25 lg/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).

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NP40), and the culture was harvested and frozen until all culture lysates were ready for ELISA.

In vitro effect of iron or zinc. Resting D1.6 Th1 cells (0.5 3 105) were stimulated with RGG antigen and APCs (2.5 3 106) for 36 h. Zinc chloride (25lM, Sigma) or ferrous chloride (25lM, Sigma) ± lead chloride (25lM) was added into the culture. At the end of the culture period, culture supernatants were collected, and IFNc 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.

RESULTS

Pb Does Not Modulate mRNA Expression by Th1 Cells or BALB/c Spleen Cells To investigate the intracellular mechanism of Pb-driven downregulation of IFNc production from Th1 cells, we examined whether Pb (25lM) could suppress expression of IFNc mRNA expression by antigen stimulation of a RGG-specific Th1 clone or OVA-specific CD4þ 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). IFNc mRNA transcripts were quantified in relative densitometric units compared to b2-M mRNA transcripts for each culture condition (Fig. 1A). As expected, IL-12 enhanced IFNc 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 IFNc mRNA. Addition of Pb or dbcAMP, a cell-permeable cAMP analog, during Th1 clone activation has been reported (Heo et al., 1998) to inhibit IFNc production, but neither Pb nor dbcAMP showed any effect on

FIG. 1. Pb does not modulate IFNc mRNA expression. RT-PCR analysis is shown in (A) for the RGG-specific D1.6 Th1 clone or OVA-tg (DO11.10) T cells stimulated in vitro with the appropriate antigen and APCs for 24 h in the presence of antigen (lane 1) or antigen plus dibutyryl cAMP (lane 2), rIL-12 (lane 3), dbcAMP plus IL-12 (lane 4), dbcAMP plus IL-12 added 6 h after culture initiation (lane 5), IL-12 plus dbcAMP added 6 h after culture initiation (lane 6), PbCl2 (lane 7), PbCl2 plus IL-12 (lane 8), PbCl2 plus IL-12 added 6 h after culture initiation (lane 9), or IL-12 plus PbCl2 added 6 h after culture initiation (lane 10). Similar RT-PCR results were obtained at 12 h. PbCl2 was always at a final concentration of 25lM. For detection of IFNc mRNA by RPA (B), the D1.6 Th1 cells were stimulated with RGG for 12 h in the presence of additives as shown; the last lane contained no D1.6 T cells. Normalized photostimulated luminencence (PSL) values for the RPA products were obtained by dividing each IFNc PSL value by the value for L32 or GADPH, housekeeping gene mRNAs (C). The results were essentially the same in two independent separate experiments.

mRNA levels in our study. Results similar to those obtained at 24 h were obtained at 12 h (data not shown). We further evaluated the effect of Pb (25lM) on IFNc mRNA expression using RPA (Figs. 1B and 1C). Again, reduced levels of IFNc mRNA were not observed with RGGstimulated D1.6 Th1 cells in the presence of Pb, whereas addition of rIL-12 significantly upregulated the IFNc mRNA

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Metabolic radiolabeling and immunoprecipitations. The resting Th1 cells (5 3 105) 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 lCi 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 3 g for 10 min. Next, the samples were immunoprecipitated with rat anti-mouse IFNc mAb (PharMingen) and goat anti-rat IgG-agarose (Sigma). The immunoprecipitates were collected through centrifugation at 12,000 3 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% SDSpolyacrylamide gels, and the bands in autoradiography were visualized by a Fujix Bas 2000 phosphoimager.

LEAD INHIBITS IFNc PRODUCTION

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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 IFNc production by Th1 cells exposed to Pb is not attributable to decreased IFNc gene transcription. To further assess any inhibitory effects of Pb at early time points after activation, we assayed by real-time RT-PCR the IFNc mRNA levels of anti-CD3/anti-CD28–stimulated wholespleen cell cultures. In this case, the IFNc could be generated by CD4þ or CD8þ T cells as well as NK cells. However, at 2–8 h after activation, there was no significant inhibition by Pb (Fig. 2). Western Analysis of IFNc Production by BALB/c Spleen Cells

Pb Inhibits the Intracellular and Extracellular Concentrations of IFNc Protein Next, we questioned whether the Pb-driven downregulation of IFNc production from Th1 cells was due to inhibition of IFNc secretion. For this purpose, IFNc protein levels in the activated D1.6 Th1 cell culture supernatants were compared with the levels in the cell lysates (Fig. 4). IFNc 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

FIG. 2. Pb effects on IFNc mRNA expression by stimulated BALB/c splenocytes measured by real-time RT-PCR. Splenocytes were stimulated with anti-CD3/anti-CD28 ± 25lM PbCl2, and RNA was isolated at 2, 4, 6, or 8 h. RNA quantification was performed by real-time RT-PCR using SYBR Green I to measure amplification. All results for IFNc copy number were normalized to GAPDH. Error bars indicate the SEM based on three independent experiments.

FIG. 3. Western blot analysis of Pb-induced inhibition of IFNc production by mouse spleen cells. BALB/c spleen cells stimulated with anti-CD3 (lane 1), anti-CD3/CD28 (lane 3), or PMA plus ionomycin (lane 5) in the absence or presence of 25lM PbCl2 (lanes 2, 4, and 6, respectively) were assayed after incubation for 48 h. The supernatants were immunoprecipitated with monoclonal R4-6A2, SDS-PAGE fractionated, and Western blotted with biotin-XMG1.2.

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 IFNc production was not mediated through interference with IFNc secretion. Pb-Enhanced Proteosomal Degradation of Intracellular IFNc Is Not Responsible for the Lower Levels of IFNc Since Pb had no significant modulatory effect on IFNc transcription or extracellular export (Figs. 1, 2, and 4), we

FIG. 4. Secretion of IFNc synthesized inside the D1.6 Th1 cells is not inhibited by Pb. The resting D1.6 Th1 clone was stimulated with RGG for 12 h (black bar) or 18 h (gray bar) ± PbCl2 (25lM). At the end of stimulation, culture supernatants were collected, and the cell pellets were lysed. The results are expressed as percent production compared with the control culture in the absence of Pb, and values are the means (± SEM) of three independent experiments. The * (p < 0.05) or ** (p < 0.01) indicates a significant difference from the RGG control.

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To confirm and extend previous assessment of the inhibition of IFNc by Pb, we cultured spleen cells with various stimulants and assayed after 48 h for the presence of IFNc in the culture supernatants. As previously reported for ELISA evaluation, PMA/ionomycin induced more IFNc 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 IFNc (Fig. 3).

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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 IFNc production (~30% at time 0). However, with and without Pb, there was equivalent loss of IFNc over the next 72 h, and the rate of degradation (slope of the decline from 0–72 h) was also comparable. 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). Pb Effects on the Biosynthesis of IFNc Protein

To determine the overall degradation of IFNc, we assessed the entire culture (supernatant and cell lysate) for immunoreactive IFNc in the presence or absence of Pb, we added

Based on our results indicating no detectable effects of Pb on IFNc protein transcription, secretion, and proteosomal degradation, we next asked whether Pb could modify translational events for IFNc 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 antimouse IFNc 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, ´ alons et al., 1998). The autoradiograph that is shown 1993; GoA

FIG. 5. Proteosomal lysis of intracellular protein is not responsible for the lowered production of IFNc induced by Pb (25lM). D1.6 Th1 cells were treated with 10nM, 100nM, or 1lM lactacystin, followed by stimulation with the antigen, RGG. Culture supernatants collected at 36 h were used for IFNc quantification. The results are expressed as the means (± SEM) of three independent experiments. The * (p < 0.05) indicates significant difference from the antigen control.

FIG. 6. Pb does not accelerate IFNc degradation. BALB/c spleen cells (2 3 106) were stimulated with PMA (5 ng/ml) þ ionomycin (1 lg/ml) for 24 h. Cultures were then treated with 25 lg/ml cycloheximide, and cultures were harvested after additional incubation for 2, 4, 6, 24, 48, or 72 h, to quantify presence of IFNc by ELISA. To accommodate the variance of IFNc produced in each experiment, the values are reported as percentage of the IFNc concentration in the absence of Pb at the time of the addition of cycloheximide (time 0). The results are expressed as the means (± SEM) of four independent experiments.

Pb Does Not Enhance IFNc Degradation

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investigated whether the Pb-induced inhibition of IFNc production was dependent on proteosomal proteolysis. To evaluate the possibility of Pb-induced potentiation of proteosome activities, we compared the level of IFNc 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 IFNc production, lactacystin pretreatment should result in similar IFNc levels between the Pb-exposed and the unexposed Th1 cells. IFNc 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 IFNc production from the Th1 cells, whether or not Pb was added. The results indicated that cytoplasmic degradation of IFNc protein is not triggered by Pb treatment of Th1 cells and is not responsible for the Pbinduced inhibition of IFNc production.

LEAD INHIBITS IFNc PRODUCTION

(Fig. 8) is representative of results from multiple experiments. We found a suppression of IFNc 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 IFNc biosynthesis. The difficulty with this experiment was in identifying which bands were IFNc 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 IFNc 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 IFNc. Reversal of Pb’s Inhibition of IFNc Production by IL-12 As previously reported (Heo et al., 1998), IL-12 is a strong promoter of Th1 activity, and it is able to prevent the Pbinduced inhibition of IFNc production (Fig. 9). Spleen cells from OVA-tg mice were stimulated with OVA (0.5 mg) in the

FIG. 8. Pb exerts a downregulatory effect on the biosynthesis of IFNc in D1.6 Th1 clone. Resting D1.6 Th1 cells were pulsed using the [35S] Protein Labeling Mix for 6 h at 0, 6, and 12 h after initiation of stimulation with antigen and APCs in the presence or absence of 25lM PbCl2, and the chase with nonradiolabeled amino acids was followed until the end of culture (18 h stimulation). Culture supernatants were collected at the end of each 6-h pulse (A) or after pulse and chase at 18 h (B), and each supernatant was immunoprecipitated and fractionated by SDS-PAGE. Levels of biosynthetically labeled IFNc were assessed by phosphorimaging analysis. The results shown are representative of three and five independent experiments for A and B, respectively. Slight differences were obtained dependent on the antibodies used for the immunoprecipitation. The results shown utilized rabbit polyclonal antiserum from National Institutes of Health. Arrows show MW markers of 30 and 12.3 kDa.

absence (control) or presence of PbCl2 (25lM) 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 IFNc response, and IL-12 prevented the Pb-induced skewing toward a type-2 response by significantly blocking Pb’s inhibition of IFNc and inhibiting the IL-4 response.

FIG. 9. IL-12 reversal of Pb-induced inhibition of IFNc production. OVA-tg spleen cells were stimulated and expanded in vitro, as previously described (Heo et al., 1998), in the absence of any additive other than OVA (control); they were then compared with cultures containing PbCl2 (25lM) or Pb plus IL-12 (5 ng/ml). After 6 days, the culture supernatants were quantified for IFNc or IL-4 by ELISA. The data shown represent the mean ± SEM from five independent experiments; the ‘‘*’’ indicates a significant (p < 0.05) difference from the control.

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FIG. 7. Supplementation of Zn or Fe cannot compensate the Pb-induced suppression of IFNc production. The resting D1.6 Th1 clone was stimulated with RGG for 36 h in the presence or absence of 25lM PbCl2, 25lM ZnCl2, or 25lM FeCl2, or a paired combination (Pb þ Zn and Pb þ Fe). The results are expressed as percent IFNc production in the culture supernatants compared with the control culture stimulated with the antigen alone. Values are the means (± SEM) of three independent experiments. The level of IFNc production in the control culture was 2683 ± 279 pg/ml. The ‘‘*’’ (p < 0.05) or ‘‘**’’ (p < 0.01) indicates significant difference from the RGG control.

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TABLE 1 Pb Effects on IFNg and IL-4 Production by OVA-tg Splenocytes Lacking STAT4 or STAT6 STAT4
/
Cytokine IL-4 (ng/ml) IFNc (ng/ml) IFNc:IL-4

STAT6
/



Pb

þ Pb


Pb

þ Pb

1.89 ± 0.93* 1.52 ± 0.22 0.80

1.98 ± 0.87 1.61 ± 0.21 0.81

0.17 ± 0.04 41.7 ± 6.1 245

0.23 ± 0.04 32.6 ± 5.8 142*

Note. The OVA-tg response was significantly (*) skewed toward Th2 responses with the STAT6-deficient DO11.10 BALB/c mice. Results represent mean ± SE of mean of six mice assayed in six separate experiments.

Pb Effect on OVA-tg Splenocytes Lacking STAT4 or STAT6

DISCUSSION

Pb exposure is known to interfere with the development and/or activation of Th1 cells, which are the CD4þ T cells responsible, in part, for the production of IFNc. In vivo, ex vivo, and in vitro studies of the immune system of mice have shown that Pb is able to suppress IFNc 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 IFNc 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 IFNc 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 IFNc mRNA level at one early time point with BALB/c spleen cells, there was no inhibition of mRNA expression for IFNc with either a T-cell clone or a transgenic T cell line; in fact, Pb consistently enhanced IFNc gene expression. The expression of many cytokines is known to be regulated at the transcriptional and

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Since IL-12 can overcome the Pb-induced inhibition of IFNc 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 naı¨ve 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).

posttranscription levels. Inosine has been reported to modulate the levels of IL-1, TNFa, IL-12, and IFNc, 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 IFNc 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 IFNc after Pb exposure. In addition, most studies limited analysis to the presence of IFNc in the sera or culture supernatants. Thus, the lower quantities of IFNc, 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 IFNc 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 IFNc did not show any indication of increase, suggesting that degradation is not the cause for the lower amounts of IFNc. This was confirmed by evaluation of IFNc 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 IFNc 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 IFNc 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]-IFNc can detect IFNc in Western blot analysis, and they also can recognize recombinant IFNc; thus, it is not likely that the glycosylation of IFNc can account for the biosynthetic results—inhibition of IFNc 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 IFNc. 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 IFNc’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

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which are critical for host defenses against numerous pathogens. In the absence of STAT4, Pb was unable to further skew the naı¨ve 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 IFNc and enhance that of IL-4, suggesting that the Pb effects on T-cell skewing are likely independent of signaling via STAT6.

ACKNOWLEDGMENTS 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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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 IFNc. Regardless of the epitope specificities, Pb appears to inhibit the presence of IFNc. We also previously assessed by ELISA whether Pb could bind to recombinant mouse IFNc and reduce its recognition; Pb was unable to inhibit (Lynes et al., 2006). However, recombinant IFNc may have slightly different structure than the native molecule (it is not glycosylated), and the recombinant IFNc was not synthesized in the presence of Pb, which could alter its conformational epitopes. Even under the best of conditions, quantification of IFNc by Western and/or immunoprecipitation analysis is not easily achieved. IFNc can be present in multiple forms. The mouse produces a 155-amino acid IFNc polypeptide, which has a 23-amino acid leader sequence containing three cysteines. The processed, secreted IFNc 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 IFNc, 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 IFNc molecule. The translational blockage of IFNc by Pb is evidently reversible, in that the addition of IL-12 eliminates the inhibition. IL-12 is well known to enhance IFNc 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 IFNc production. However, IL-10 cannot block the activity of IL-12 (Hsieh et al., 1992). In addition to promoting signals for IFNc expression, IL-12 induces multiple molecular effects independent of IFNc (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 IFNc 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 (IFNc-promoted) cell-mediated immune responses,

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