Impact of developmental lead exposure on splenic factors

Abstract

Lead (Pb) is known to alter the functions of numerous organ systems, including the hematopoietic and immune systems. Pb can induce anemia and can lower host resistance to bacterial and viral infections. The anemia is due to Pb's inhibition of hemoglobin synthesis and Pb's induction of membrane changes, leading to early erythrocyte senescence. Pb also increases B-cell activation/proliferation and skews T-cell help (Th) toward Th2 subset generation. The specific mechanisms for many of the Pb effects are, as yet, not completely understood. Therefore, we performed gene expression analysis, via microarray, on RNA from the spleens of developmentally Pb-exposed mice, in order to gain further insight into these Pb effects. Splenic RNA microarray analysis indicated strong up-regulation of genes coding for proteolytic enzymes, lipases, amylase, and RNaseA. The data also showed that Pb affected the expression of many genes associated with innate immunity. Analysis of the microarray results via GeneSifter software indicated that Pb increased apoptosis, B-cell differentiation, and Th2 development. Direct up-regulation by Pb of expression of the gene encoding the heme-regulated inhibitor (HRI) suggested that Pb can decrease erythropoiesis by blocking globin mRNA translation. Pb's high elevation of digestive/catabolizing enzymes could generate immunogenic self peptides. With Pb's potential to induce new self-peptides and to enhance the expression of caspases, cytokines, and other immunomodulators, further evaluation of Pb's involvement in autoimmune phenomena, especially Th2-mediated autoantibody production, and alteration of organ system activities is warranted.

Figure 1

Spleen tissue from Pb-exposed pnd21 mice had significantly higher levels of Pb than did tissue from non-exposed mice. Experimental mice were exposed to 0.1 mM Pb through the dam’s drinking water from gd8 to pnd21. Spleens were homogenized in 1 ml of M-PER reagent including HALT (a cocktail of protease inhibitors), and were analyzed for elemental Pb as described in the methods section. The data represent an n of 4 litters for control and 4 litters for the Pb-exposed mice; significance of the difference between the groups, indicated by the asterisk was determined by the Student’s t-test at p ≤ 0.05.

Figure 2

Real-time RT-PCR analysis confirms the microarray analysis of increased expression of chymotrypsin upon developmental Pb exposure. Relative gene expression in the spleen was determined for control (n=3) and 0.1mM Pb-exposed mouse pups (n=3) representative of separate litters. The TaqMan method was performed as described in the methods. Relative gene expression was calculated with respect to the Ct for the gene normalized to gapdh, as described in the methods. Gene IDs indicate the following: Jarid1a, jumonji; Ctrc, chymotrypsin C; and Mst1r, macrophage stimulating 1 receptor. Significance of the difference between the groups, indicated by the asterisk was determined by the Student’s t-test at p ≤ 0.05.

Figure 3

Splenic amylase activity was increased by developmental Pb-exposure. Mouse pups were exposed to 0.1mM Pb from gd8 to pnd21. Spleen homogenates shown (2 control and 2 Pb-exposed) were prepared and assayed for amylase activity as described in the methods. Each spleen homogenate result shown here was from one spleen of a mouse representative of one litter. The gel photographs display the raw data, A) Control and B) Pb-exposed. Homogenates were diluted 1:10, 1:100, and 1:1000 with 0.1M phosphate buffer (pH 6.9) before applying to the gel. C shows a sample standard curve generated with purified amylase, and D shows the relative splenic amylase activity of control and 0.1 mM Pb-exposed mouse pups. Significance of the difference between the groups, indicated by the asterisk was determined by the Student’s t-test at p ≤ 0.05.

Figure 4

Pb promotes Th2 skewing in the spleen as indicated by relative Th1 and Th2 cytokine mRNA levels. Cytokine transcripts for IL-4 and IFN-gamma were measured by an absolute quantification RT-PCR method and were normalized to gapdh transcript level. Results are presented as average (cytokine CN/GAPDH CN) X 100 ± SEM. The results represent data from 3 control and 3 0.1 mM Pb treated mouse pup spleens, representative of 3 litters per condition. Significance of the difference between the groups, indicated by the asterisk was determined by the Student’s t-test at p ≤ 0.05.

Figure 5

Modification of OVA-tg T-cell differentiation with spleen cells from stat4^−/− DO11.10^+/− (a), stat6^−/− DO11.10^+/− (b), or wild type DO11.10^+/− (c) mice. Cells were differentiated with OVA in the absence of various additives (black circle) or presence of TGF-β (open circle), anti-IL-4 (black triangle), anti-IL-12 (open triangle), Pb (black square), TGF-β+anti-IL-4 (open square), TGF-β+anti-IL-12 (black diamond), or Pb+anti-IL-4 (open diamond). Data were obtained from 6 mice assayed individually in six separate experiments, and the results are expressed as the mean amounts (± SEM) of IL-4 and IFN-γ produced.