Methamphetamine increases LPS-mediated expression of IL-8, TNF-α and IL-1β in human macrophages through common signaling pathways

Abstract

The use of methamphetamine (MA) has increased in recent years, and is a major health concern throughout the world. The use of MA has been associated with an increased risk of acquiring HIV-1, along with an increased probability of the acquisition of various sexually transmitted infections. In order to determine the potential effects of MA exposure in the context of an infectious agent, U937 macrophages were exposed to various combinations of MA and bacterial lipopolysaccharide (LPS). Treatment with MA alone caused significant increases in the levels of TNF-α, while treatment with both MA and LPS resulted in significant increases in TNF-α, IL-1β and the chemokine IL-8. The increases in cytokine or chemokine levels seen when cells were treated with both LPS and MA were generally greater than those increases observed when cells were treated with only LPS. Treatment with chemical inhibitors demonstrated that the signal transduction pathways including NF-kB, MAPK, and PI3-Akt were involved in mediating the increased inflammatory response. As discussed in the paper, these pathways appear to be utilized by both MA and LPS, in the induction of these inflammatory mediators. Since these pathways are involved in the induction of inflammation in response to other pathogens, this suggests that MA-exacerbated inflammation may be a common feature of infectious disease in MA abusers.

Figure 1. Treatment of U937 macrophages with MA or/and LPS induced an increase in IL-8, TNF-α, and IL-1β.

Cells were differentiated into macrophages by treatment with 100 nM PMA for 48 h. Cells were treated with MA at 0, 24 and 48 h and then stimulated with 100 ng/ml LPS for 3 h, after which they were harvested. RNA was isolated and analyzed with real-time RT-PCR (A–C). In a separate experiment, cells were treated with MA at 0, 24 and 48 h then stimulated with 100 ng/ml LPS for 24 h and the cytokines in supernatants were measured using Bio-Plex (D–F). The error bars show standard error of three independent experiments. *: p<0.05; **: p<0.01.

Figure 2. Treatment of U937 macrophages with MA ± LPS resulted in increased translocation of the p50 subunit of NF-κB.

1×10⁶ U937 monocytes were differentiated into macrophages with 100 nM PMA for 48 h. Cells were treated with MA at 0, 24 and 48 h then stimulated with 100 ng/ml LPS for 3 h. Cytosolic and nuclear protein fractions were purified by electrophoresis on 10% SDS gel and transfer to a PVDF membrane. The cytosolic fractions and nuclear fractions are shown in a representative western blot. The expression of the p50 subunit was normalized to the appropriate compartmental housekeeping genes (Lamin B for nucleus and GAPDH for cytoplasm). The means and SE values are from three independent experiments.

Figure 3. An inhibitor of NF-kB abrogates cytokine/chemokine expression induced by MA ± LPS.

(A–C)1×10⁶ U937 monocytes were differentiated into macrophages with 100 nM PMA for 48 h. IKK2 inhibitor SC-514 was added 1 h before each MA exposure at 0, 24, 48 h. Cells were treated with LPS and the final dose of MA and then harvested after 3 h for RNA isolation and real time RT-PCR analysis. The mRNA expression levels were compared to untreated control. (D–F) For cytokine protein levels, cell supernatants were harvested 24 h after the last MA ± LPS treatment. The protein concentrations were normalized by subtracting values of control or inhibitor only. The means and SE values are from three independent experiments. *:p<0.05; **: p<0.01 comparing to MA/LPS/LPS+MA treatment as positive controls.

Figure 4. Inhibitors of MAPK or PI3-Akt abrogate cytokine/chemokine expression induced by MA ± LPS.

1×10⁶ U937 monocytes were differentiated into macrophages by treatment with 100 nM PMA for 48 h. p38 inhibitor SB203580, PI3K/Akt inhibitor LY294002 and ERK1/2 inhibitor U-0126 were added 1 h before MA exposure at 0, 24, 48 h. Cells were then challenged with LPS and the final dose of MA. After 3 h cells were harvested, RNA isolated and analyzed by real time RT-PCR. The means and SE values are from three independent experiments. *:p<0.05; **: p<0.01. Expression levels were compared to the identical treatment without inhibitor.