The induction of inflammation in macrophages by LPS has been extensively studied for the past three decades. Although there are still questions that remain to be resolved, it is clear that LPS/TLR4 signaling involves the NF-κB, MAPK and PI3-Akt pathways (reviewed in 
). In contrast, very little is known about the effects of MA on macrophages and the signaling pathways that mediate these effects. Our results demonstrate that LPS-treatment of macrophages exposed to MA for 48–72 h result in the induction of IL-8, TNF-α and IL-1β to levels that were greater than were seen by treatment of macrophages with LPS alone. Although, as is to be expected, the actual fold-differences were different for RNA and protein, the expression profiles obtained at the level of RNA were virtually identical to the expression patterns observed at the level of protein. At the level of protein, MA alone induced significantly higher levels of TNF-α and IL-1β. For all three cytokines/chemokines examined, the level of protein induced by treatment with both MA and LPS was significantly greater than the additive effects of each agent alone. This suggests that, at least in some cases, MA and LPS may interact synergistically to increase cytokine or chemokine production. This has significant clinical implications for the effects of drugs of abuse on inflammation, particularly with respect to that caused by infectious agents. Definitive proof of synergy requires treatment with additional doses of these agents and these studies are currently in progress. The induction of increased levels of IL-1β by concomitant treatment with MA and LPS confirms a previous report that demonstrated a similar phenomenon in IL-1β production as measured by ELISA of cell culture supernatants 
The results described above led us to inquire as to the mechanisms responsible for the cytokine induction. LPS/TLR4 signaling pathways have been well described, but the signaling pathways that mediate the effects of MA in macrophages remain largely unexplored. However, it is known that macrophages express D1 and D2 dopamine receptors. Through the use of selective D1 or D2 inhibitors, it has been demonstrated that the effects of MA in macrophages are mediated through either D1 or D2 dopamine receptors 
. Signaling through the D1 and D2 receptors has been demonstrated to be mediated through a number of pathways, including the NF-κB pathway 
, the MAPK pathways 
, and the PI3-Akt pathway 
The first pathway we investigated was the NF-κB pathway. Through the use of cytoplasmic and nuclear extracts, we determined that treatment of U937 macrophages with MA for 48 h alters the nuclear
cytoplasmic ratio of the p50 subunit of NF-κB. Cells treated with either LPS, or a combination of LPS and MA showed an even greater effect in terms of the increase in p50 nuclear
cytoplasmic ratio. Although there was little difference between the nuclear
cytoplasmic ratios observed between the cells treated with LPS and those treated with both LPS and MA, we postulate that this may be due to the ratio having reached its maximum. The role of the NF-κB pathway in mediating the induction of inflammatory cytokines in response to LPS and MA was confirmed using SC-514, an inhibitor of IKK2. Although the effects of SC-514 treatment on the induction of IL-8, TNF-α and IL-1β by MA at the level of RNA were slight, the inhibitory effect of SC-514 as shown by decreased cytokine production was more apparent at the protein level. It is worthwhile to note that while SC-514 inhibits IKK-2 (i.e. IKK-β), this kinase is thought to be dispensable for LPS-mediated activation of NF-κB in macrophages and monocyte-derived dendritic cells 
. In our system the activity of IKK-α (i.e. the non-canonical NF-κB pathway, reviewed in 
), or activation of cytokine production by an alternative pathway (e.g. MAPK) may be the cause of the lack of efficacy of SC-514 in inhibiting cytokine production in LPS-stimulated macrophages. Furthermore, the effects of SC-514 were statistically significant for all cytokines in terms of induction by concomitant treatment with both LPS and MA, at both protein and RNA levels. Taken together with the changes produced by the different treatments on the nuclear-cytoplasmic distribution of p50, this confirms that the NF-κB pathway is a key mediator of the effects of MA on LPS-stimulated macrophages.
In addition to the NF-κB pathway, the MAPK signaling pathway has also been demonstrated to mediate D1 and D2 signaling 
. MA has been shown to affect phosphorylation of ERK and p38 MAPK in macrophages 
and monocyte-derived dendritic cells 
. We also determined the role of the PI3-Akt pathway, as this pathway has also been shown to modulate TLR4/LPS signaling 
, as well as signaling through D1 and D2 
. Our results demonstrated that p38MAPK and ERK1/2 are involved in mediating MA-induction of TNF-α. Our studies also show that MA-inductions of IL-8 and IL-1β are mediated by the PI3-Akt pathway and ERK1/2.
Taken together with the results discussed above, it is important to note that the use of any one of the signaling inhibitors, i.e. inhibitors for p38 MAPK, PI3-Akt, ERK1/2, or NF-κB, results in significant abrogation of MA-mediated increases of IL-8, IL-1β and TNF-α. This is most probably because the pathways that mediate MA-induced increases in these inflammatory cytokines are also involved in the induction of cytokines in response to LPS. We propose that it is through the utilization of these overlapping pathways that MA exerts its effects on LPS-stimulated macrophages. Furthermore, as we have noted, while the use of an inhibitor of IKK2 results in the inhibition of the LPS/MA cotreatments, the ERK1/2 inhibitor abrogated cytokine induction in response to the cotreatments with LPS and MA, as well as to the treatments with LPS and MA individually. This may suggest that ERK signaling is downstream of NF-κB signaling. A more definitive analysis of the sequence of induction of these pathways is currently in progress.
Although we have demonstrated that the NF-κB and MAPK signaling pathways play key roles in the induction of inflammatory cytokines by LPS and MA, the roles other signaling components and pathways involved in this phenomenon remain to be elucidated. For example, although dopamine receptors has been shown to be expressed in macrophages (10, 11), direct binding of MA to these receptors has not been demonstrated. In addition, while we have demonstrated that the 2 major signaling pathways in the induction of cytokines by LPS and MA, the effects of MA on the upstream components of the TLR4 signaling pathway (e.g. CD14, MyD88, TRAF6, etc.) have yet to be determined. It is quite possible that MA affects LPS signaling by affecting the expression or function of any of these components.
Our finding of the increased production of inflammatory cytokines in response to MA also has important implications in terms of the clinical effects of MA abuse. Numerous studies have demonstrated a strong linkage between MA abuse and HIV infection. MA abuse tends to increase risky sexual behaviors, and is thus a contributing factor to the acquisition of HIV infection 
. In the early stages of HIV infection the virus enters the CNS, most probably through trafficking of infected monocytes across the blood-brain barrier (BBB). In a trend that has been increasing in the past few years, the effects of HIV infection of the CNS, a syndrome known as HIV-associated neurocognitive disorder (HAND), have become more prevalent. Current thought is that one of the major factors responsible for HAND is increased neuroinflammation due to cytokine production induced by HIV-1 neurotoxins. A recent report has demonstrated that MA increases levels of matrix metalloproteinase 9 (MMP9) in LPS-treated macrophages 
. Increased levels of this enzyme are associated with increased trafficking of infected monocytes across the BBB 
. Taken together with our finding of MA-mediated induction of increased levels of inflammatory cytokines, this suggests that MA could operate on several levels to increase the prevalence of HAND in HIV-infected abusers of the drug. Thus, MA may not only increase the level of monocyte trafficking across the BBB, but it may also act to increase the production of inflammatory cytokines that are produced in microglia.
In summary, our results demonstrate that exposure to MA increases the levels of the inflammatory cytokines IL-8, IL-1β and TNF-α in LPS-treated macrophages. This is the first report that has demonstrated the induction of inflammatory cytokines by MA treatment of macrophages. The pathways that we have identified as being involved in MA-mediated induction of these cytokines are also key players in LPS-mediated inflammation. The exacerbation of cytokine production in response to both agents is likely the result of the utilization of overlapping signaling pathways activated by MA and LPS. This finding has important implications because of the potential of MA to exacerbate other inflammatory conditions that also modulate the signaling pathways identified in this study.