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In vitro immune toxicity of depleted uranium: effects on murine macrophages, CD[4.sup.+] T cells, and gene expression profiles.

Depleted uranium (DU) is a by-product of the uranium enrichment process and shares chemical properties with natural and enriched uranium. To investigate the toxic effects of environmental DU exposure on the immune system, we examined the influences of DU (in the form of uranyl nitrate) on viability and immune function as well as cytokine gene expression in murine peritoneal macrophages and splenic CD[4.sup.+] T cells. Macrophages and CD[4.sup.+] T cells were exposed to various concentrations of DU, and cell death via apoptosis and necrosis was analyzed using annexin-V/propidium iodide assay. DU cytotoxicity in both cell types was concentration dependent, with macrophage apoptosis and necrosis occurring within 24 hr at 100 [micro]M DU exposure, whereas CD[4.sup.+] T cells underwent cell death at 500 [micro]M DU exposure. Nuncytotoxic concentrations for macrophages and CD[4.sup.+] T cells were determined as 50 and 100 [micro]M, respectively. Lymphoproliferation analysis indicated that macrophage accessory cell function was altered with 200 [micro]M DU after exposure times as short as 2 hr. Microarray and real-time reverse-transcriptase polymerase chain reaction analyses revealed that DU alters gene expression patterns in both cell types. The most differentially expressed genes were related to signal transduction, such as c-jug NF-[kappa] Bp65, neurotrophic factors (e.g., Mdk), chemokine and chemokine receptors (e.g., TECKI CCL25), and interleukins such as IL-10 and IL-5, indicating a possible involvement of DU in cancer development, autoimmune diseases, and T helper 2 polarization ofT cells. The results are a first step in identifying molecular targets for the toxicity of DU and the elucidation of the molecular mechanisms for the immune modulation ability of DU. Key words: apoptosis, CD[4.sup.+] T cell, cytokine gene expression, depleted uranium, macrophage function, necrosis. Environ Health Perspect 114:85-91 (2006). doi: 10.1289/ehp.8085 available via http://dx.doi.org/ [Online 17 August 2005]

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Depleted uranium (DU) is a by-product of the enrichment process of natural uranium (Priest 2001). The release of uranium into the environment presents a threat to human and ecologic health in many parts of world (Hass et al. 1998; Murray et al. 2002). DU shares chemical properties with natural or enriched uranium, but the major hazard rendered by DU results from its heavy metal toxicity rather than from radiologic toxicity (Fisenne and Welford 1986; Priest 2001). The adverse health effects of DU compounds are partially dependent on its chemical form. Uranium compounds in +2 to +4 valence states are essentially insoluble. However, in vivo soluble uranium is always hexavalent, regardless of the oxidation state of uranium compound taken up (Edison 1994). It is this form (+6) that is of toxicologic importance. Because of their high affinity for phosphate, carboxyl, and hydroxyl groups, uranyl compounds readily combine with proteins and nucleotides to form stable complexes (Moss 1985).

Serum uranium forms a variety of non-diffusible complexes such as uranium-albumin compounds and diffusible ones such as ionic uranyl hydrogen carbonate complex (Moss 1985). Although the most characteristic response to DU exposure either short or long term is renal dysfunction (Domingo 1995; Leggett 1989; Zamora et al. 1998), uranium is also localized within the central nervous system, testes, lymph nodes, and spleen, suggesting the potential for uranium to cause health problems at these sites (Domingo 2001; Pellmar et al. 1999; Wrenn et al. 1985). Uranium-induced pathological changes in the testes and thyroid glands have been documented (Malenchenko et al. 1978).

In vitro studies have examined the effects of DU on a variety of cell types. For example, Chinese hamster ovary cells exposed to DU exhibit lower cell viability, depressed cell cycle kinetics, and increased sister chromatid exchanges, micronuclei, and chromosomal aberrations after DU exposure (Lin et al. 1993). Kidney cells release lactate dehydrogenase upon uranium exposure (Furuya et al. 1997), whereas human osteoblast cells are transformed to a neoplastic phenotype after in vitro DU exposure (Miller et al. 1998).

More important to this investigation, some studies indicated that immune cells are also involved in DU toxicity. Macrophages can actively internalize the uranium, with the subsequent occurrence of cell apoptosis (Kalinich and McClain 2001; Kalinich et al. 2002). Other evidence suggests the involvement of cytokine gene expression in DU toxicity, and the changes of some of these genes are associated with immune responses. For example, recent studies demonstrated that DU induces abnormal expression and release of tumor necrosis factor (TNF) and interleukin (IL)-6 in macrophages (Gazin et al. 2004: Zhou et al. 1998).

During the Gulf War, tons of DU weapons were fired, and DU shrapnel was permanently embedded in the bodies of many soldiers (sometimes removing shrapnel is fatal). In addition inhalation of DU combustion particles on the battlefield is also a major source of exposure to high concentrations of DU. It was hypothesized that Gulf War syndrome may be explained as a systemic shift in cytokine balance from a T helper (Th) 1 profile toward a Th2 profile because the syndrome is clinically similar to autoimmune diseases (Rook and Zumla 1997; Skowera et al. 2004). In this study we hypothesized that DU exposure may compromise the immune system function by inducing immune cell apoptosis and modulating immune cell cytokine gene expression, which may be predictive of DU immunotoxicity. This hypothesis is consistent with the findings of Li et al. (2001), Pallardy et al. (1999), and Rodenburg et al. (2000), which showed that cell death through apoptosis or necrosis may cause serious adverse effects such as immunosuppression or lead to an altered immune response. More specifically, because of the macrophage's phagocytosis activity and ubiquitous presence throughout the body, it is also important to assess the effect DU may have on macrophage function as accessory cells to T-lymphocyte activation/proliferation. Cytokine gene expression profiling of DU-exposed immune cells should contribute to the understanding of the molecular mechanisms of DU toxic effects on the immune system. To test the above hypotheses, we exposed macrophages and primary CD[4.sup.+] T cells to DU (in the form of uranyl nitrate) and examined for evidence of apoptosis and altered macrophage function in promoting lymphocyte proliferation. Macrophages and T cells were also exposed to DU at noncytotoxic concentrations, and the effect of DU-modulated cytokine gene expression was examined. The results of these experiments suggest a possible role for DU in carcinogenesis and autoimmune diseases.

Materials and Methods

Chemicals. Uranyl nitrate [U[O.sub.2][(N[O.sub.3]).sub.2].6[H.sub.2]O], with a specific activity of approximately 0.2 [micro]Ci/mg, and sodium nitrate (NaN[O.sub.3]) were purchased from Mallinckrodt Specialty Chemicals Co. (Phillipsburg, NJ) and both were dissolved in water. Lipopolysacchride and concanavalin A (ConA) were from Sigma (St. Louis, MO) and were dissolved in DMSO. [[alpha]-[sup.33]P]-Deoxyadenosine 5'-triphosphate was purchased from ICN Radiochemicals (Costa Mesa, CA).

Animals. BALB/c and DO11.10 T-cell receptor (TCR)--transgenic mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and bred and housed under pathogen-free conditions in the animal care facility at the University of Tennessee, Knoxville, according to the animal protocol procedures approved by the Committee on the Care of Laboratory Animal Resources. Mice 6-8 weeks of age were used for cell preparation.

Cell preparations. Collected peritoneal elicited macrophages were collected and pooled from three to four Balb/c mice injected intraperitoneally with thioglycollate (TG) broth (3% wt/vol; 1 mL/mouse; Difco Laboratories, Livonia, MI) 4 days before cell collection. Calls were plated onto 25 [cm.sup.2] Coming cell culture flasks or polystyrene six-well flat-bottom microtiter plates and were incubated at 37[degrees]C, 5% C[O.sub.2]/95% air, and 95% humidity for 4 hr to allow the macrophages to adhere to the surfaces. The surfaces were washed twice with warm PBS to remove all nonadherent cells, and the macrophage layer was cultured overnight in complete RPMI-1640 (cRPMI-1640) containing 10% low-endotoxin, heat-deactivated fetal bovine serum (Sigma, Copenhagen, Denmark), [10.sup.-5] M 2-mercaptoethanol, L-glutamine (20 mM), and penicillin and streptomycin (100 U/mL each). The resulting macrophage purity was > 95%, determined by CD11b staining analysis. Cells were then washed twice, stained with trypan blue exclusive dye, and counted. The cells were exposed to uranyl nitrate at various concentrations in 25-[cm.sup.2] flasks (for RNA isolation) or six-well plates (for flow cytometry analysis).

Splenic CD[4.sup.+] T cells were negatively isolated using the magnetic activated cell sorting method according to the manufacturer's protocols (Miltenyi Biotec, Auburn, CA). In brief, pooled splenic cells from three to four DO11.10 mice were stained with a cocktail of biotin-conjugated monoclonal antibodies against CD8a(Ly-2) (rat IgG2a), CD11b (Mac-1) (rat IgG2b), CD45R (B220) (rat IgG2a), DX5 (rat IgM), and Ter-119 (rat IgG2b). The mixture was then incubated with Anti-Biotin MicroBeads (Miltenyi Biotec) and the cell suspension was passed through an LS magnetic separation column (Miltenyi Biotec). The major cell composition of elute is CD[4.sup.+] T cells (> 95%). After washing, the cell density was adjusted to 1 X [10.sup.6] cells/mL cRPMI-1640 media and the DU exposure was performed in anti-CD3--coated 96-well plates, followed by RNA isolation or flow cytometry analysis. Fresh cells from new living animals were purified each time the assays were repeated; three individual experiments were performed.

Cell staining and flow cytometry analysis of cell death. For both macrophages and primary CD[4.sup.+] T cells, cell death analysis using flow cytometry was performed in triplicate. We handled and treated cells according to the protocol provided with the Annexin-V--fluorescein (A-V--FITC) Apoptosis Detection Kit (Sigma, Copenhagen, Denmark). Cells (1 X [10.sup.6] cells/mL) were exposed to uranyl nitrate at 0-200 [micro]M (macrophages) or 0-500 [micro]M (CD[4.sup.+] T cells). The cells were harvested and washed once with PBS and resuspended in 1X binding buffer. A 500-[micro]L aliquot of the cell suspension was stained with 5 [micro]L of A-V-FITC and 10 [micro]L of propidium iodide (PI) in a 12 X 75 mm test tube for 10 min at room temperature, protected from light. We then analyzed cells using the flow cytometry FACScan (BD Biosciences, San Jose, CA) by counting 30,000 events. The data files were saved automatically by CellQuest software (BD Biosciences), and WinMDI (version 2.8; The Scripps Institute, Flow Cytometry Core Facility, La Jolla, CA), was used to perform quadrant analysis. A two-tailed t-test was performed to determine the significant difference between treatment and control experiments using Excel (Microsoft, Redmond, WA).

Lymphoproliferation assay. We used a T-lymphocyte proliferation assay to estimate the macrophage function as accessory cells. The exposure follows the model described by Krocova et al. (2000): elicited peritoneal macrophages were placed into the wells of 96-well microtiter plates (Corning Inc., Coming, NY), allowed to adhere for 4 hr, and then exposed to cRPMI-1640 media containing DU (10, 50, 100, 200, 500, and 1,000 [micro]M) or an equal amount of medium with no DU added. After 2 hr incubation, the cells were washed, and then the purified CD[4.sup.+] pension (1 X [10.sup.6] cells/mL with 5 [micro]g/mL ConA) was added to each well containing treated adherent macrophages. Simultaneously, we set up non--T-cell controls by replacing the T cells with same amount of culture medium. The plate was incubated at 37[degrees]C, 5% C[O.sub.2]/95% air, and 95% humidity for 48 hr. Then, we added 10 [micro]L of MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] solution (5 mg/mL) to each well, and allowed the plate to incubate for an additional 4 hr. At the end of the incubation, we added 100 [micro]L of acidic isopropanol (0.04 M HCI in absolute isopropanol) and mixed to dissolve the converted dye formazan. The absorbance data were recorded by a spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT) at 562 nm.

Mouse cytokine cDNA microarray analysis. The Panorama mouse cytokine gene array (Sigma, St. Louis, MO) consisting of 514 different cytokine-related cDNAs printed onto charged nylon membranes was used to analyze gene expression profile. A detailed description and a list of genes included on the array can be found on the Sigma-Genosys website (Sigma 2004). Briefly, we exposed macrophages and CD[4.sup.+] T cells to 50 and 100 [micro]M DU, respectively tively, for 24 hr; after treatment, we extracted total RNA from each sample using Trizol reagent and treated the RNA with RNase-free DNase I (Gibco-BRL Life Technologies Inc., Grand Island, NY). Using mouse cytokine gene cDNA-labeling primers (Sigma-Genosys, St. Louis, MO), 2 [micro]g RNA were reverse transcribed to generate a [[alpha]-[sup.33]P]-dATP--labeled cDNA probe. We removed unincorporated nucleotides from the probe using NucTrap probe purification columns (Stratagene, La Jolla, CA). EDTA was added to bring the final concentration to 10 mM, and the probe was heat denatured at 95[degrees]C for 5 min. Arrays were hybridized with probes in ULTRArray hybridization buffer (Ambion, Inc., Austin, TX) overnight at 55[degrees]C. The arrays were then washed extensively at 50[degrees]C under both low-and high-stringency conditions [2X saline sodium citrate (SSC), 0.5% SDS for low-stringency wash solution, 0.5X SSC, 0.5% SDS for high-stringency wash solution] for 2 X 30 min. The membranes were air dried, sealed in a dear plastic bag, and exposed to low-energy storage phosphoimage screens (Kodak, Rochester, NY). The images were scanned at 50-[micro]m resolution on a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The image files were analyzed using ArrayVision software (version 6.0; Imaging Research, St. Catharines, Ontario, Canada), and the numerical output was exported in Microsoft Excel format to ArrayStat (version 1.0; Imaging Research Inc.) for statistical analysis. Microarray data obtained here are available at Gene Expression Omnibus (GEO 2005; accession no. GSE2333).

Real-time reverse-transcriptase PCR analysis. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was used to verify gene expression of microarray analysis. The assay was performed in triplicate in a DNA Engine Opticon system (MJ Research Inc., Waltham, MA) using SYBR Green I as the detection format (Qiagen, Inc, Valencia, CA). First, we converted total RNA to first-strand cDNA using reverse transcription, and then performed real-time RT-PCR analysis using the SYBR Green PCR kit (Qiagen). The PCR primers are listed in Table 1. After PCR, we performed melting curve analysis and visualized the PCR products using gel electrophoresis to assess the specificity of PCR amplification reactions. For both the reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] and test gene, we constructed standard curves and determined the slope to calculate the PCR efficiency according to Pfaffl (2001; Pfaffl et al. 2002). We calculated differences in gene expression between treatment and control using PCR efficiencies and threshold cycle numbers (Ct values), which were normalized against GAPDH. The formulation used for calculating the ratio of gene expression between control and treatment groups is described by Pfaffl (2001; Pfaffl et al. 2002) as

[1]

ratio (S:C) = [E.sub.target gene] [DELTA]Ct-target gene (control-sample)

+ [E.sub.reference gene] [DELTA]Ct-reference gene (control-sample)

where ratio(S:C) is the expression ratio of DU-treated sample over control; [E.sub.target] gene is the PCR efficiency of target gene; [E.sub.reference gene] is the PCR efficiency of reference gene; [DELTA]Ct-target gene (control--sample) is the difference of target gene Ct values between control (C) and DU-treated samples (7); and [DELTA]Ct-reference gene (control--sample) is the difference of reference gene Ct values between control and DU-treated samples.

Statistics. Data were expressed as the mean [+ or -] SD, and a Student t-test was used to compare the differences between treatment and control groups, with the significance level set at p < 0.05. For microarray data analysis, blots were normalized to the mean values of the entire array with background subtraction. Three sets of data were generated from three biologic replicate experiments; if there were only one valid observation for a gene within a condition, that datum was disregarded for analysis. The outliers were detected by examining standardized residuals automatically using ArrayStat software. A curve-fit random error estimate method was employed for a proportional model with offset.

The data were transformed logarithmically, and a Z-test for two independent conditions was performed according to Benjamini and Hochberg (1995). Differentially expressed genes were identified on the basis of the significance level (p < 0.05 or effective p < 0.05/ number of analyzed genes).

Results

Macrophage cell death. We determined induction of apoptosis in macrophages using flow cytometry analysis of cells labeled with A-V-FITC. A-V protein can bind, by a calcium-dependent process, to phosphatidylserine (PS) presented on the surface of cells undergoing apoptosis (Bertho et al. 2000). PS is normally sequestered on the inner leaflet of the plasma membrane. However, during apoptosis, membrane phospholipid asymmetry is lost and PS is exposed to the outer leaflet, where it can interact with A-V. Cells that stain positively for A-V and negatively for PI are considered cells in an early stage of apoptosis, whereas those that stain positively for both indicators either are in late apoptotic stage or are undergoing necrotic cell death.

The treatment of macrophages with DU as uranyl nitrate resulted in apoptotic cell death. As shown in Figure 1A, treatment with 20 and 50 [micro]M DU for 24 hr did not cause an apparent increase in A-V and PI staining. However, 100 and 200 [micro]M DU treatment led to a significant increase of both A-V and PI staining (p < 0.05), with the percentages of A-V binding at 12.3 and 30.5%, respectively, and the percentage of PI positive cells at 12.4 and 49.2%, respectively. The results indicated that both cell apoptosis and necrosis increase with an increase in concentration. DU at 50 [micro]M was determined as noncytotoxic to macrophages.

Using light microscopy and atomic force microscopy, we investigated the morphologic changes in macrophages treated with 100 [micro]M DU for 24 hr (Figure 2). We treated the macrophages with 0 or 100 [micro]M DU for 24 hr and fixed the cells by soaking them in 2% formaldehyde (prepared in propanol), followed by incubation in 0.1% Triton-X 100 for 15 min. We then air dried the cells at room temperature in preparation for imaging by atomic force microscopy. Alternatively, cells after DU exposure were directly imaged by light microscopy. When adherent macrophages undergo necrosis or cellular disintegration, adherent cells will disassociate and float in the medium. Figure 2 shows that the apoptotic cells are still adherent to the surface and show the rough shape of the cell membrane and no apparent nuclear structure (Figure 2D). In the atomic force microscopy images, the darkness represents the height of sample surface over the vessel surface. Note that in Figure 2C, which is a normal cell, the thicker area (brightest) is the nucleus of the cell, but in Figure 2D, there is no such area, which is indicative of loss of nuclear structure. In Figure 2D, the small particle-like areas indicate apoptotic bodies.

CD[4.sup.+] T-cell death analysis. The treatment of CD[4.sup.+] T cells with DU (as uranyl nitrate) resulted in apoptotic as well as necrotic cell death. As shown in Figure 1B, treatment with 1, 10, and 100 [micro]M DU for 24 hr did not result in significant increase of either A-V staining or PI staining, indicating that these treatments did not induce cell apoptosis or necrosis. However, there was a significant increase of apoptotic and necrotic cells after treatment with 500 [micro]M DU (p < 0.05), 64.5 and 15.3%, respectively, whereas the apoptotic and necrotic cell percentages of negative controls were 3.5 and 1.5%, respectively. As expected, the positive controls had a much higher percentage of necrotic cells, whereas the percentages of necrotic and apoptotic cells in the 1 mM NaN[O.sub.3] control group were not different from those of negative control, indicating that apoptosis and necrosis in DU treatment were not attributable to the N[O.sub.3]- ion but were due to the uranyl ion. DU at 100 [micro]M was determined as noncytotoxic to CD[4.sup.+] T cells during 24-hr exposure.

Lymphoproliferation assay. Concanavalin A (ConA) was used to activate T cells. The likely mechanism is that ConA indirectly cross-links the TCR and sends the activation signals. Activation of T cells is also dependent on the presence of non-T cells that function as accessory cells, which provide additional and essential costimulatory signals for T-cell proliferation (Coligan 1991). Exposure to DU at concentrations > 200 [micro]M significantly enhanced the functionality of macrophages, as shown by increased T-cell proliferation under the induction of ConA (Figure 3, solid bars). Lower concentrations of DU < 100 [micro]M did not change the T-cell proliferation. However, 200, 500, and 1,000 [micro]M DU treatment of macrophages significantly (p < 0.05) enhanced T-cell proliferation in a concentration-dependent manner, with the optical density measurements in MTT assay increasing from 0.56 (control) to 0.76, 0.86, and 0.87, respectively. In addition, 1 mM NaN[O.sub.3] treatment did not influence the measurement, excluding the contribution of nitrate ions (N[O.sub.3-]) to the toxic effect on macrophages. In this study, pretreatment of macrophages with various concentrations of DU for 2 hr did not cause significant loss of cells in 96-well plates, as shown by open bars in Figure 3. Therefore, it is safe to preclude the possibility that the alteration of T-cell proliferation is caused by variations in macrophage cell numbers.

DU influence on gene expression. Gene expression in macrophages and CD[4.sup.+] T cells under noncytotoxic DU exposure was analyzed by cDNA microarray. The results were from three biologic replicates. Table 2 lists 29 (6% of all analyzed genes) genes whose expressions were significantly (p < 0.05) changed in macrophages upon 50 [micro]M DU exposure. Of these 29 genes, 24 (5%) were up-regulated, and 5 (1%) were down-regulated. Although a variety of gene groups are affected, the groups with multiple affected genes include signal transduction--related, cytokine- and IL-related, and apoptosis-related groups. Other genes with altered expression such as LTBP-2 and Mdk are neurotrophic factors or involved in binding protein, respectively.

The differentially expressed genes in CD[4.sup.+] T cells under noncytotoxic (100 [micro]M) DU exposure are listed in Table 3. Although many of the same gene groups are represented, the specific genes, except for Mdh listed in Tables 2 and 3 are different. Specifically, chemokine-related genes are up-regulated in CD[4.sup.+] T cells but not in macrophages. Moreover, IL-5 is related to T-cell functionality.

Real-time RT-PCR analysis. We used real-time RT-PCR analysis as a confirmative method for the genes determined to be differentially expressed by microarray analysis and performed the analysis for selected genes in both cell types. Table 4 shows some of the RT-PCR results, along with the corresponding microarray results. RT-PCR analysis showed that under DU exposure, expression of genes such as Mdk, c-jun, and IL-10 was enhanced in macrophages, and Mdk and IL-5 in CD[4.sup.+] T cells. The ratios of all genes except for IL-10 were in accordance with those determined by microarray analysis. We performed the assay in triplicate. Generally, this quantitative RT-PCR assay confirmed the micmarray results.

Discussion

Uranium environmental contamination from mining, processing, and military industries has heightened concern of the possible environmental and health effects of DU exposure. DU can enter the body by ingestion, inhalation, contamination of wounds, and embedded shrapnel (McClain et al. 2001). At the cellular level, accumulation of DU has been observed in various macrophage cell lines (Gazin et al. 2004; Kalinich et al. 2002), and one of the first issues to address is whether DU induces macrophage death and at what level this toxic effect occurs. In this study we used flow cytometry analysis of A-V/PI binding to study apoptosis and necrosis. The results showed that apoptosis and necrosis occurred after 24 hr with DU (as uranyl nitrate) treatments [greater than or equal to] 100 [micro]M. These results are similar to those observed in a previous study that used uranyl chloride and a macrophage cell line, J774 (Kalinich et al. 2002) and were also comparable with other reported experiments using human osteoblast cells (Miller et al. 1998). Apoptosis and, to a lesser extent, necrosis occurred simultaneously after 24 hr when T cells were exposed to concentrations as high as 500 [micro]M DU. Below 500 [micro]M, apoptosis and necrosis were not observed. CD[4.sup.+] T cells are more resistant than macrophages, which may be because macrophages can actively engulf DU particles (Kalinich et al. 2002), but CD[4.sup.+] T cells do not. Compared with other heavy metals, DU is much less toxic to CD[4.sup.+] T cells than mercury, whereas lead and vanadium have approximately the same toxicity as DU (Shen et al. 2001).

Once the toxicity of DU to immune cells was determined, the issue arose as to how DU affects the function of immune cells. In the presence of ConA, in vitro T-cell activation requires accessory cells for co-stimulatory signals (Pollard and Landberg 2001). In the present study we assessed the ability of DU-exposed macrophages to function as accessory cells by measuring the CD[4.sup.+] T-cell proliferation. The results indicated that higher concentrations (200 to--1,000 [micro]M) of DU were able to alter macrophage functionality in vitro in a concentration-dependent manner, which led to significant T-cell proliferation. This response is similar but occurs at higher concentrations compared with other heavy metals such as lead, which induces lymphocyte proliferation at a concentration range of approximately 12-120 [micro]M (Krocova et al. 2000). The results in this study demonstrated that a short-term, high-concentration DU exposure was able to perturb rapidly the interaction between macrophages and T cells, and immune function.

Previous studies on the toxic effects of heavy metals, including uranium, indicated the involvement of cytokine regulation in immunomodulatory activities (Gazin et al. 2002; Krocova et al. 2000). However, these studies focused on the expression of only a few cytokine genes such as interleukins, NF-[kappa]B, or TNF-[alpha]. Global gene expression analysis in kidney tissue after DU exposure suggested that genes involved in multiple biologic functions, including signal transduction, may be altered by uranium exposure (Taulan et al. 2004). We further asked what effects DU might have on the immune system if the exposure scenario were nonlethal and long term and how it might relate to cytokine gene expression.

In this present study we used a mouse cytokine gene array, and as expected, genes related to signal transduction pathways were significantly modulated by DU exposure (Tables 2, 3). In DU-exposed macrophages the most highly expressed gene was NF-[kappa]B p65 (Table 2). Miller et al. (2004) demonstrated that DU (5-50 [micro]g/mL or 18.5-185 [micro]M) had profound influences on multiple signaling pathways in HepG2 cells; interestingly, the genes they identified also related to the NF-[micro]B pathway, as indicated in our research. The important role of NF-[kappa], B in uranium toxic effects has been reported previously by Gazin et al. (2002). Our results provide direct evidence showing that DU is able to activate NF-[kappa]B by increasing the expression of the p65 subunit. The NF-[kappa]B family of transcription factors not only are key regulators of genes involved in immune and inflammatory reactions (Li et al. 2001; Tak and Firestein 2001) but also are involved in many aspects of call growth, differentiation, and proliferation via the induction of certain growth and transcription factors (e.g., c-myc, ras, p53). The co-induction of NF-[kappa]B, MMP-13, and c-myc indicated in our microarray results is consistent with previous work by Tak and Firestein (2001).

NF-[kappa]B can mediate both inflammatory and antiinflammatory responses by regulating genes encoding either proinflammatory or antiinflammatory activities (e.g., IL-10) (Baldwin 2001; Bierhaus and Nawroth 2003; Xu and Shu 2002). In our microarray analysis, the latter was indicated by the up-regulation of IL-10 gene in macrophages upon DU exposure (Table 2). Activation of NF-[kappa]B requires degradation of I-[kappa]B (nucear factor of kappa light chain gene enhancer in B-cells inhibitor, [beta]) with the help of I-[kappa]B kinases, the activity of which depends on binding with NF-[kappa]B--inducing kinase (NIK) (then et al. 2001; Wooten 1999). The activation of NIK as shown in this study (Table 2) supports the conclusion that the NF-[kappa]B signaling pathway was adopted by macrophages under the DU exposure. DU may induce NIK activity leading to the up-regulation of NF-[kappa]B (by increasing the p65 subunit level), which further activates expression of a variety of cytokine genes, such as c-myc and MMP-13, as indicated in our array data. This hypothesis is supported by the study of Miller et al. (1998) that demonstrated DU-induced tumorigenic activity in osteoblast cells.

It is interesting to note that the expression of the neurotrophic factor Mdk was highly induced in both primary macrophages and CD[4.sup.+] T cells (Tables 2, 3) after DU exposure. Mdk gene expression is restricted to only a few types of cells such as kidney and epithelial cells (Garver et al. 1993; Hu et al. 2002); it is very unusual that this gene was regulated by DU in immune cells. To our knowledge [main references were Tully et al. (2000) and Yamada and Koizumi (2002)], the up-regulation of Mdk by heavy metal exposure has not been previously reported, which may indicate a common mechanism of DU immunotoxicity to both macrophages and T cells and may provide a biologic marker for DU exposure. Because Mdk levels often increase in the early stage of cancer progression, it has been suggested as a tumor marker (Muramatsu 2002). High induction of Mdk expression in this study presents further evidence for the possible involvement of DU in carcinogenesis, as reported by Miller et al. (1998).

A DU-induced Th1-Th2 shift has been long postulated to play a role in the development of Gulf War syndrome (Rook and Zumla 1997; Skowera et al. 2004). The complex balance between Th1 and Th2 cells can be disturbed by a variety of factors, including heavy metals; a shift to a Th2 phenotype has been correlated with the development of allergic responses and some autoimmune diseases (Harber et al. 2000; Mosmann and Sad 1996). As part of our efforts, cytokine gene expression was studied in CD4 cells to investigate the DU-induced Th2 shift hypothesis.

Our array data showed an approximately 2-fold induction of IL-5 expression in CD[4.sup.+] T cells and 1.7-fold induction of IL-10 in macrophages upon DU exposure (Tables 2, 3). We postulate that the reason changes in TNF and IL-6 were not detected, as reported by Krocova et al. (2000), is because of differences in exposure conditions and cell type (we used mouse primary peritoneal macrophages vs. rat alveolar macrophages and lung fibroblasts). However, the conclusions were similar because IL-10 and IL-5 were found to be up-regulated, and both genes are of the Th2 type. IL-5 is a signature cytokine of Th2 cells, which also produce cytokines such as IL-4 and IL-10 (Cousins et al. 2002; Mazzarella et al. 2000). IL-10 can also create a microenvironment to facilitate Th2 cell development (Malefyt et al. 1991; Mosmann and Sad 1996). Therefore, up-regulation of IL-5 and IL-10 expression in our study indicates a Th2 differentiation tendency after DU exposure. This is direct evidence, at the transcriptional level, for a DU-induced Th2 shift. Th2 domination of T-helper cell population differentiation is often found in association with strong antibody (e.g., autoimmune diseases) and allergic responses (Harber et al. 2000; Mosmann and Sad 1996). Interestingly, elevated blood IL-10 concentrations have been detected in symptomatic Gulf War veterans who were potentially exposed to DU under battlefield conditions (Skowera et al. 2004; Zhang et al. 1999). The data in our study show that DU may contribute to an increase in IL-10 levels through its action on macrophages. Additionally, the induction of IL-5 expression in CD[4.sup.+] T cells, and possibly IL-10 in macrophages suggests an important role for DU in promoting Th2 shifting.

Conclusions

In summary we have demonstrated DU-induced apoptosis and necrosis in both peritoneal macrophages and splenic CD[4.sup.+] T cells in a cell-specific and concentration-dependent manner. Short-term DU exposure (> 200 [micro]M) to macrophages interferes with the interplay between macrophages and CD[4.sup.+] T cells, resulting in an enhanced T-cell proliferation response. At lower (noncytotoxic) concentrations, DU has the potential to influence immune function by modulating cytokine gene expression mainly involved in signal transductions, interleukin production, chemokine and chemokine receptors, and neurotrophic factors. Array analyses have successfully identified differentially regulated genes implicating DU in carcinogenesis and the development of autoimmune diseases. The up-regulation of IL-5 and IL-10 genes in CD[4.sup.+] T cells and macrophages, respectively, strongly suggests a DU-induced Th2 shift during naive T-cell differentiation. Considering the substantial sequence homology between the mouse and human genome and the conserved expression patterns of orthologs reflecting common physiologic functions in these two organisms (Su et al. 2002), the alteration in immune functions and cytokine gene expression in murine immune cells demonstrated in this study identify putative molecular targets for the toxic actions of DU and suggest molecular mechanisms for the development of DU-related diseases in humans.

We acknowledge the assistance of H. Zaghouani, B. Rouse, U. Kumaraguru, J. Young, and personnel of the University of Tennessee animal facilities.

This work was supported by the Center for Environmental Biotechnology, Research Center of Excellence, by the Waste Management Research and Education Institute, and by the Office of the Vice Chancellor for Research, University of Tennessee. Partial support for this project was provided by National Science Foundation grant BES-0116610.

The authors declare they have no competing financial interests.

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Bin Wan, (1,2) James T. Fleming, (1,3) Terry W. Schultz, (1,2,4) and Gary S. Sayler (1,2,3)

(1) Center for Environmental Biotechnology, (2) Department of Ecology and Evolutionary Biology, (3) Department of Microbiology, and

(4) Department of Comparative Medicine, University of Tennessee, Knoxville, Tennessee, USA

Address correspondence to G.S. Sayler, Center for Environmental Biotechnology, University of Tennessee, Knoxville, 676 Dabney Hall, Knoxville, TN 37996-1605 USA. Telephone: (865) 974-8080. Fax: (865) 974-8086. E-mail: sayler@utk.edu

Received 2 March 2005; accepted 17 August 2005.
Table 1. Primer sequences used in quantitative
RT-PCR for differentially expressed genes

 Amplicon
Target Sequences size (bp)

IL-10 F: 5'-CAT GGG TCT TGG GAA GAG AA-3'
 R: 5'-CAT TCC CAG AGG AAT TGC AT-3' 194
Mdk F: 5'-ACC GAG GCT TCT TCC TTC TC-3'
 R: 5'-GGC TCC AAA TTC CTT CTT CC-3' 230
BMP-11 F: 5'-TTC ATG GAG CTT CGA GTC CT-3'
 R: 5'-AGC ATG TTG ATT GGG GAC AT-3' 299
c jun F: 5-TGA GAA CTT GAC TGG TTG CG-3'
 R: 5'-AAA GTC CAT CGT TCT GGT CG-3' 222
Stat-1 F: 5'-TGG TGA AAT TGC AAG AGC TG-3'
 R: 5'-TGT GTG CGT ACC CAA GAT GT-3' 119
Tlr6 F: 5'-ACA CAA TCG GTT GCA AAA CA-3'
 R: 5'-GGA AAG TCA GCT TCG TCA GG-3' 128
IL-5 F: 5'-GTC CCT ACT CAT AAA AAT CAC CA-3'
 R: 5'-GAA TAG CAT TTC CAC AGT ACC C-3' 105
GAPDH (a) F: 5'-TGA TGA CAT CAA GAA GGT GGT GAA G-3'
 R: 5'-TCC T TG GAG GCC ATG TAG GCC AT-3' 240

Abbreviations: F, forward primer; R, reverse primer.

(a) Sequences are from Lee et al. (2000).

Table 2. Differentially regulated genes in DU-exposed peritoneal
adherent macrophages as determined by array analysis.

 Accession
Gene abbreviation (a) Gene symbol (a) no. (b) Gene description

Up-regulated gene expression
NF-kB p65 Rela NM_009045 avian reticulo-
 endotheliosis
 viral (v-rel)
 oncogene homolog
 A
LTBP-2 Ltbp2 AF004874 latent TGF-beta
 binding protein-2
WNT-8b Wnt86 NM_011720 wingless related
 MMTV integration
 site 8b
Mdk Mdk NM_010784 midkine
c-jun Jun NM_010591 Jun oncogene
Map3k14 Nik--pending NM_016896 Nfkb inducing
 kinase
BDNF Bdnf NM_007540 brain derived
 neurotrophic
 factor
SOCS-1 Cish1 NM_009896 cytokine
 inducible
 SH2-containing
 protein 1
C-myc Myc NM_010849 myelocytomatosis
 oncogene
NSG1/p21 Nsg1 NM_010942 neuron specific
 gene family
 member 1
IL-10 II10 NM_010548 interleukin 10
Stat 1 Stat1 NM_009283 signal transducer
 and activator of
 transcription 1
IL-12Rb1 II12rb1 NM_008353 interleukin 12
 receptor, beta 1
CIS3 Cish3 NM_007707 cytokine
 inducible
 SH2-containing
 protein 3
BMP-9 Bmp9 AF188286 bone
 morphogenetic
 protein 9
SMAD7 Madh7 NM_008543 MAD homolog 7
 (Drosophila)
Tlr2 Tlr2 AF185284 toll-like
 receptor 2
CT-1 Ctf1 NM_007795 cardiotrophin 1
EphA3 Epha3 M68513 mouse eph-related
 receptor tyrosine
 kinase (Mek4)
MMP-13 Mmp13 NM_008607 matrix metal
 loproteinase 13
BMP-11 Gdf11 AF092734 growth/
 differentiation
 factor 11
GDNF Gdnf D49921 glial cell
 line-derived
 neurotrophic
 factor /GDNF)
Itgb7 Itgb7 M95632 integrin beta-7
 subunit
MMP-8 Mmp8 NM_008611 matrix metal
 loproteinase 8
Down-regulated gene expression
Tlr6 Tlr6 NM_011604 toll-like
 receptor 6
Cox-2 Ptgs2 NM_011198 prostaglandin-
 endoperoxide
 synthase 2
VCAM-1 Vcam1 NM_011693 vascular cell
 adhesion
 molecule 1
FL IPL Cash NM_009805 caspase homolog
SARP-1 Sdf5 NM_009144 stromal cell
 derived factor 5

Gene abbreviation (a) Gene symbol (a) Gene group

Up-regulated gene expression
NF-kB p65 Rela Signal transduction
LTBP-2 Ltbp2 Binding protein
WNT-8b Wnt86 Developmental factors
Mdk Mdk Neurotrophic group
c jun Jun Signal transduction
Map3k14 Nik--pending Apoptosis related
BDNF Bdnf Neurotrophic group
SOCS-1 Cish1 Signal transduction
C-myc Myc Signal transduction
NSG1/p21 Nsg1 Signal transduction
IL-10 II10 Interleukin
Stat 1 Stat1 Signal transduction
IL-12Rb1 II12rb1 Interleukin receptor
CIS3 Cish3 Signal transduction
BMP-9 Bmp9 TGF-beta family
SMAD7 Madh7 Signal transduction
Tlr2 Tlr2 Cell surface protein
CT-1 Ctf1 Cytokine and receptors
EphA3 Epha3 Eph family
MMP-13 Mmp13 Protease or related factor
BMP-11 Gdf11 TGF-beta family
GDNF Gdnf Neurotrophic group
Itgb7 Itgb7 Intergrin
MMP-8 Mmp8 Protease or related
 factor
Down-regulated gene expression
Tlr6 Tlr6 Cell surface protein
Cox-2 Ptgs2 Apoptosis related
VCAM-1 Vcam1 Adhesion molecule
FL IPL Cash Apoptosis related
SARP-1 Sdf5 Apoptosis related

Gene abbreviation (a) Gene symbol (a) Z-test (c)

Up-regulated gene expression
NF-kB p65 Rela 5.8 x [10.sup.8]
LTBP-2 Ltbp2 1.8 x [10.sup.6]
WNT-8b Wnt86 9.3 x [10.sup.6]
Mdk Mdk 1.5 x [10.sup.5]
c jun Jun 7.1 x [10.sup.5]
Map3k14 Nik--pending 1.5 x [10.sup.3]
BDNF Bdnf 2.4 x [10.sup.3]
SOCS-1 Cish1 2.8 x [10.sup.3]
C-myc Myc 7.2 x [10.sup.3]
NSG1/p21 Nsg1 7.8 x [10.sup.3]
IL-10 II10 8.2 x [10.sup.3]
Stat 1 Stat1 9.2 x [10.sup.3]
IL-12Rb1 II12rb1 1.1 x [10.sup.2]
CIS3 Cish3 1.1 x [10.sup.2]
BMP-9 Bmp9 1.4 x [10.sup.2]
SMAD7 Madh7 1.9 x [10.sup.2]
Tlr2 Tlr2 1.9 x [10.sup.2]
CT-1 Ctf1 1.9 x [10.sup.2]
EphA3 Epha3 2.1 x [10.sup.2]
MMP-13 Mmp13 2.2 x [10.sup.2]
BMP-11 Gdf11 3.2 x [10.sup.2]
GDNF Gdnf 3.5 x [10.sup.2]
Itgb7 Itgb7 2.9 x [10.sup.2]
MMP-8 Mmp8 4.9 x [10.sup.2]

Down-regulated gene expression
Tlr6 Tlr6 2.7 x [10.sup.2]
Cox-2 Ptgs2 2.3 x [10.sup.2]
VCAM-1 Vcam1 1.2 x [10.sup.2]
FL IPL Cash 1.1 x [10.sup.2]
SARP-1 Sdf5 7.3 x [10.sup.4]

Gene abbreviation (a) Gene symbol (a) Ratio (d) 95% Cl (e)

Up-regulated gene expression
NF-kB p65 Rela 3.9 6.3-2.4
LTBP-2 Ltbp2 3.2 5.3-2.0
WNT-8b Wnt86 3.2 5.4-1.9
Mdk Mdk 3.1 5.2-1.9
c jun Jun 3.2 5.8-1.8
Map3k14 Nik--pending 2.4 4.0-1.4
BDNF Bdnf 1.7 2.5-1.2
SOCS-1 Cish1 2.4 4.2-1.4
C-myc Myc 2.2 3.8-1.2
NSG1/p21 Nsg1 2.0 3.3-1.2
IL-10 II10 1.7 2.5-1.2
Stat 1 Stat1 2.0 3.4-1.2
IL-12Rb1 II12rb1 1.6 2.4-1.1
CIS3 Cish3 1.8 2.8-1.1
BMP-9 Bmp9 1.8 2.7-1.1
SMAD7 Madh7 1.6 2.5-1.1
Tlr2 Tlr2 1.8 2.8-1.1
CT-1 Ctf1 1.9 3.4-1.1
EphA3 Epha3 2.6 2.3-1.1
MMP-13 Mmp13 1.9 3.3-1.1
BMP-11 Gdf11 1.8 3.1-1.0
GDNF Gdnf 1.6 2.4-1.0
Itgb7 Itgb7 1.7 2.9-1.0
MMP-8 Mmp8 1.6 2.5-1.0

Down-regulated gene expression
Tlr6 Tlr6 0.6 0.9-0.4
Cox-2 Ptgs2 0.6 0.9-0.4
VCAM-1 Vcam1 0.5 0.9-0.3
FL IPL Cash 0.6 0.9-0.4
SARP-1 Sdf5 0.6 0.8-0.4

Results are from three individual experiments.

(a) From Sigma-Genosys (http://www.sigmaaldrich.com/catalog/search
/ProductDetail/GENOSYS/G2041). (b) From GenBank (http://www.ncbi.nlm.
nih.gov/Genbank/). (c) p-Values in the two-tailed Z-tests for the
comparison between control and DU treatments. (d) The ratio expression
values for the average expression values of each gene between DU and
control, that is, ratio = intensity value from DU-treated cells divided
by that from control cells. (e) Confidence intervals (Cls) determined
for ratios, p < 0.05.

Table 3. Differentially regulated genes in DU-exposed
[CD4.sup.+] T cells as determined by array analysis.

 Accession
Gene abbreviations (a) Gene symbol (a) no. (b) Gene description

Up-regulated gene expression
TECK/CCL25 Scya25 NM_009138 small inducible
 cytokine A25
Mdk Mdk NM_010784 midkine

IL-5 II5 NM_010558 interleukin 5
VEGF-A Vegf NM_009505 vascular
 endothelial
 growth factor
Down-regulated gene expression
EphA3 Epha3 M68513 eph-related
 receptor
 tyrosine kinase
 (Mek4)
CCR-4 Cmkbr4 NM_009916 chemokine
 (C-C) receptor 4
LIF Lif NM_008501 leukemia
 inhibitory
 factor
GDF-7 Gdf7 U08339 BALB/c putative
 growth factor
 GDF7 (Gdf7) gene
EBF Ebf NM_007897 early B-cell
 factor
CD27/TNFRSF7 Tnfrsf7 L24495 CD27 antigen
SLIT-3 Slit3 AF088902 SLIP protein
CX3CL1 Scyd1 NM_009142 small inducible
SMAD1 Madh1 NM_008539 MAD homolog 1
 (Drosophila)
A1 Bcl2a1a L16462 hemopoietic-
 specific early
 response protein

Gene abbreviations (a) Gene symbol (a) Gene group

Up-regulated gene expression
TECK/CCL25 Scya25 Chemokine
Mdk Mdk Neurotrophic group
IL-5 II5 Interleukin
VEGF-A Vegf Angiogenic factor
Down-regulated gene expression
EphA3 Epha3 Eph family
CCR-4 Cmkbr4 Chemokine receptor
LIF Lif Cytokine and receptors
GDF-7 Gdf7 TGF-beta superfamily
EBF Ebf Signal transduction
CD27/TNFRSF7 Tnfrsf7 TNF superfamily
SLIT-3 Slit3 Developmental factor
CX3CL1 Scyd1 Chemokine
SMAD1 Madh1 Signal transduction
A1 Bcl2a1a Apoptosis related

Gene abbreviations (a) Gene symbol (a) Z-Test (c)

Up-regulated gene expression
TECK/CCL25 Scya25 1.5 x [10.sup.-14]
Mdk Mdk 2.9 x [10.sup.-7]
IL-5 II5 9.5 x [10.sup.-4]
VEGF-A Vegf 2.5 x [10.sup.-5]
Down-regulated gene expression
EphA3 Epha3 4.0 x [10.sup.-8]
CCR-4 Cmkbr4 1.8 x [10.sup.-5]
LIF Lif 3.0 x [10.sup.-5]
GDF-7 Gdf7 2.4 x [10.sup.-4]
EBF Ebf 3.7 x [10.sup.-4]
CD27/TNFRSF7 Tnfrsf7 6.4 x [10.sup.-4]
SLIT-3 Slit3 9.2 x [10.sup.-4]
CX3CL1 Scyd1 1.2 x [10.sup.-3]
SMAD1 Madh1 1.3 x [10.sup.-3]
A1 Bcl2a1a 1.5 x [10.sup.-3]

Gene abbreviations (a) Gene symbol (a) Ratio (d) 95% Cl (e)

Up-regulated gene expression
TECK/CCL25 Scya25 4.2 6.2-2.9
Mdk Mdk 3.0 4.7-1.9
IL-5 II5 1.9 2.9-1.3
VEGF-A Vegf 1.8 2.2-1.4
Down-regulated gene expression
EphA3 Epha3 0.5 0.6-0.4
CCR-4 Cmkbr4 0.6 0.8-0.5
LIF Lif 0.6 0.8-0.4
GDF-7 Gdf7 0.5 0.7-0.3
EBF Ebf 0.5 0.7-0.4
CD27/TNFRSF7 Tnfrsf7 0.6 0.8-D.4
SLIT-3 Slit3 0.7 0.8-0.5
CX3CL1 Scyd1 0.4 0.7-0.2
SMAD1 Madh1 0.6 0.8-0.5
A1 Bcl2a1a 0.6 0.8-0.4

Results are from three individual experiments.

(a) From Sigma-Genosys (http://www.sigmaaldrich.com/catalog/search/
ProductDetail/GENOSYS/G2041). (b) From GenBank (http://www.ncbi.nim.
nih.gov/Genbank/). (C) p-Values in the two-tailed Z-tests for the
comparison between control and OU treatments. (d) The ratio expression
values for the average expression values of each gene between DU and
control, that is, ratio = intensity value from DU-treated cells
divided by that from control cells. (e) 95% Confidence intervals (Cls)
determined for ratios, p < 0.05.

Table 4. Comparison of the gene expression ratios in macrophages
determined by microarray and quantitative RT-PCR analysis.

Gene abbreviation Array ratios RT-PCR ratio (c)

Mdk (a) 3.1 3.2
C-jun (a) 3.2 1.9
BMP-11 (a) 1.8 1.6
Stat-1 (a) 2.0 2.2
IL-10 (a) 1.7 6.9
Tlr6 (a) 0.6 0.6
Mdk (b) 3.0 1.9
IL-5 (b) 1.9 3.2

(a) The gene was differentially expressed in macrophages.

(b) The gene was differentially expressed in [CD4.sup.+] T cells.

(c) Results from triplicate s (n = 3).
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Title Annotation:Research
Author:Sayler, Gary S.
Publication:Environmental Health Perspectives
Date:Jan 1, 2006
Words:9034
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