Lead (Pb) as a Factor Initiating and Potentiating Inflammation in Human THP-1 Macrophages

Abstract

The aim of this study was to assess the influence of lead (Pb) at low concentrations (imitating Pb levels in human blood in chronic environmental exposure to this metal) on interleukin 1β (IL-1β) and interleukin 6 (IL-6) concentrations and the activity and expression of COX-1 and COX-2 in THP-1 macrophages. Macrophages were cultured in vitro in the presence of Pb at concentrations of: 1.25 μg/dL; 2.5 μg/dL; 5 μg/dL; 10 μg/dL. The first two concentrations of Pb were selected on the basis of our earlier study, which showed that Pb concentration in whole blood (PbB) of young women living in the northern regions of Poland and in the cord blood of their newborn children was within this range (a dose imitating environmental exposure). Concentrations of 5 μg/dL and 10 μg/dL correspond to the previously permissible PbB concentrations in children or pregnant women, and adults. Our results indicate that even low concentrations of Pb cause an increase in production of inflammatory interleukins (IL-1β and IL-6), increases expression of COX-1 and COX-2, and increases thromboxane B2 and prostaglandin E2 concentration in macrophages. This clearly suggests that the development of inflammation is associated not only with COX-2 but also with COX-1, which, until recently, had only been attributed constitutive expression. It can be concluded that environmental Pb concentrations are able to activate the monocytes/macrophages similarly to the manner observed during inflammation.

Keywords: IL-1β; IL-6; THP-1 macrophages; cyclooxygenases; inflammation; lead (Pb).

Figure 1

The effect of lead (Pb) on COX-1 messenger RNA (mRNA) and protein expression in macrophages after 24 and 48 h of incubation. (A) COX-1 mRNA expression following Pb exposure; (B) COX-1 protein expression (densitometric analysis) of protein normalized to Beta-actin (β-actin); (C) representative Western blot following Pb exposure. Macrophages were cultured with Pb solutions for 24 or 48 h. After incubation, cells were harvested by scraping and mRNA was measured using the real-time PCR method (n = 6) and protein expression by using the Western blotting method (n = 3). *Statistically significant differences vs. control (p ≤ 0.05). Control—cells incubated in Roswell Park Memorial Institute (RPMI) medium with 10% fetal bovine serum (FBS) and without Pb.

Figure 2

The effect of Pb on COX-2 mRNA and protein expression in macrophages after 24 and 48 h of incubation. (A) COX-2 mRNA expression following lead exposure; (B) COX-2 protein expression (densitometric analysis) of protein normalized to β-actin; (C) representative Western blot following lead exposure. Macrophages were cultured with lead solutions for 24 or 48 h. After incubation, cells were harvested by scraping and mRNA was measured by using the real-time PCR method (n = 6) and protein expression by using the Western blotting method (n = 3). * Statistically significant differences vs. control (p ≤ 0.05). # Statistically significant differences vs. 1.25 μg/dL (p ≤ 0.05). ^ Statistically significant differences vs. 2.5 μg/dL (p ≤ 0.05). & Statistically significant differences vs. 5 μg/dL (p ≤ 0.05). Control—cells incubated in RPMI medium with 10% FBS and without Pb.

Figure 3

The effect of Pb on quantity of PGE2 and TXB2 in culture supernatants of macrophages cultured with various lead solutions for 24 h (A, C) or 48 h (B, D). Macrophages were cultured with lead solutions for 24 or 48 h. After incubation, cells were harvested by scraping and PGE2 or TXB2 and concentrations were measured by the ELISA method (n = 6). * Statistically significant differences vs. control (p ≤ 0.05). # Statistically significant differences vs. 1.25 μg/dL (p ≤ 0.05). ^ Statistically significant differences vs. 2.5 μg/dL (p ≤ 0.05). & Statistically significant differences vs. 10 μg/dL (p ≤ 0.05). Control—cells incubated in RPMI medium with 10% FBS and without Pb.

Figure 4

The effect of Pb on the quantity of IL-1β and IL-6 in culture supernatants of macrophages cultured with various Pb solutions for 24 h (A, C) or 48 h (B, D). Macrophages were cultured with lead solutions for 24 or 48 h. After incubation, cells were harvested by scraping and IL-1β or IL-6 concentration was measured by the ELISA method (n = 6). * Statistically significant differences vs. control (p ≤ 0.05). # Statistically significant differences vs. 1.25 μg/dL (p ≤ 0.05). ^ Statistically significant differences vs. 2.5 μg/dL (p ≤ 0.05). Control—cells incubated in RPMI medium with 10% FBS and without Pb.

Figure 5

Culture of THP-1 (A) monocytes cultured in RPMI medium with 10% FBS; (B) macrophages after 24 h of incubation with phorbol myristate acetate (PMA). Normal cells are visible.