In vitro immune toxicity of depleted uranium: effects on murine macrophages, CD4+ T cells, and gene expression profiles

Abstract

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 CD4+ T cells. Macrophages and CD4+ 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 microM DU exposure, whereas CD4+ T cells underwent cell death at 500 microM DU exposure. Noncytotoxic concentrations for macrophages and CD4+ T cells were determined as 50 and 100 microM, respectively. Lymphoproliferation analysis indicated that macrophage accessory cell function was altered with 200 microM 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-jun, NF- kappa Bp65, neurotrophic factors (e.g., Mdk), chemokine and chemokine receptors (e.g., TECK/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 of T 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.

Figure 1

Cell apoptosis and necrosis under DU exposure. (A) Macrophages were treated with 0 (control), 20, 50, 100, or 200 μM DU for 24 hr. (B) CD4⁺ T cells were treated with 0 (negative control), 1, 10, 100, or 500 μM DU, 1 mM NaNO₃, or 1 μg/mL staurosporine (positive control) for 24 hr. Data are presented as percentage of cells in the apoptotic or necrotic state and are the means of triplicate experiments. Error bars represent SD. *Difference from negative control is statistically significant (p < 0.05).

Figure 2

Representative bright-field and atomic force photomicrographs of macrophages with or without DU exposure. The cells were cultured in medium without or with 100 μM DU (as uranyl nitrate) for 24 hr and then processed for microscopy. Bright-field images (40 × magnification) of control (A) and DU-treated cells (B); the rough membrane structure of DU-treated cells was shown in B. Atomic force microscopy images of single control (C) and DU-treated cells (D), with or without nucleus area, respectively. The apoptotic body is presented in D as smaller separated bodies. The arrows in B and D indicate cells undergoing apoptosis after 24 hr of 100 μM DU exposure. Arrow in C indicates the nucleus feature of a normal cell.

Figure 3

Effect of DU on the accessory cell function of peritoneal adherent macrophages. The effect of DU on lymphocyte (CD4⁺ T cell) proliferation was determined by MTT assay, which indirectly reflects the accessory cell function of macrophages in promoting lymphocyte proliferation. Results are expressed in optical density values read at 562 nm wavelength as mean and SD of triplicate analyses. *Difference from control is statistically significant (p < 0.05).