In this study, we found that LPS stimulated the intracellular accumulation of ROS and the expression of the MOR in TPA-HL-60 cells, a macrophage cell model, and that CM from those cultures significantly increased MOR expression in SH-SY5Y cells, a neuronal cell model. These findings suggest that an indirect ROS signaling mechanism could be responsible, at least in part, for the modulation of LPS-stimulated MOR expression in SH-SY5Y cells.
Cytokines have been shown to regulate MOR expression (Borner et al., 2004
; Chang et al., 1998
; Kraus, 2009
; Kraus et al., 2003
; Wei and Loh, 2002
). Our lab previously demonstrated that co-treatment of microvascular endothelial cells with IL-1α and IL-1β increases MOR expression (Vidal et al., 1998
), and that IL-1 is the cytokine responsible for LPS-induced up-regulation of MOR expression in the rat mesentery (Chang et al., 2001
). In this study, our data indicated that cytokines present in the CM from LPS-treated TPA-HL-60 cells are involved in the modulation of MOR mRNA expression in the SH-SY5Y cells. However, it remains to be determined whether the increases in MOR mRNA observed in this study correlate to changes at either the translational or functional level.
To test our hypothesis that ROS plays a role in the LPS-stimulated cytokine secretion in TPA-HL-60 cells, we examined the levels of pro-inflammatory cytokines secreted by TPA-HL-60 cells in response to LPS-induced ROS accumulation. Of the cytokines measured, both TNF-α and GM-CSF levels were found to be significantly decreased when ROS accumulation was blocked by the antioxidant, VE.
TNF-α appears to have a positive effect on SH-SY5Y MOR expression. TNF-α stimulates transcription initiation sites on the MOR gene (Borner et al., 2002
), and TNF-α increases MOR expression in human T lymphocytes, Raji B cells, U937 monocytes, primary human polymorphonuclear leukocytes, and mature dendritic cells (Kraus et al., 2003
GM-CSF down-regulates MOR expression in dendritic cells, which may involve inhibitory actions on IL-4 (Kraus et al., 2003
). We observed an increase in MOR expression in response to the neutralization of GM-CSF in vehicle control+anti-GM-CSF and LPS+anti-GM-CSF treatment groups when compared to the vehicle control. These data suggest that only low levels of GM-CSF, present in the CM, are necessary to modulate MOR expression since the concentration of GM-CSF in the vehicle control was significantly less than that of the LPS treatment group ().
We also observed that neutralization of TNF-α in the LPS treatment group only partially attenuated MOR expression. This partial attenuation of MOR expression may be due to the effects of pro-inflammatory cytokines other than TNF-α in the up-regulation of the MOR. Interestingly, when both TNF-α and GM-CSF were neutralized simultaneously (LPS+anti GM-CSF+anti TNF-α), MOR expression was not significantly decreased compared to the LPS treatment group. This indicates that GM-CSF’s inhibitory effects on MOR expression only becomes apparent when cytokines capable of increasing MOR expression are neutralized, e.g., TNF-α. This again underscores the possibility that cytokines capable of increasing MOR expression can compensate for one another since the simultaneous neutralization of both GM-CSF and TNF-α (LPS+anti GM-CSF+anti TNF-α) did not significantly reduce MOR expression when compared to the LPS treatment group.
We hypothesized that, since morphine potentiates LPS cytotoxicity, activation of the opioid pathway by morphine in a clinical setting could cause an adverse physiological response, i.e., acceleration of sepsis to septic shock (Chang et al., 2001
; Ocasio et al., 2004
). The damaging consequences of endotoxic shock resulting from exposure to LPS, and the subsequent signaling actions mediated by ROS may, in fact, be exacerbated as a result of the immunosuppressive effects associated with MOR activation in neuronal and non-neuronal cells (Gaveriaux-Ruff, Matthes et al., 1998
; Wang, Charboneau et al., 2002
Our findings suggest that the LPS-induced ROS signaling that occurs in immune cells may indirectly regulate the opiodergic pathway by modulating MOR expression in neurons. Our data also indicate that ROS, produced in LPS challenged TPA-HL-60 cells, is involved in modulating the secretion of TNF-α and GM-CSF, two cytokines that have previously been shown to modulate MOR expression. This mechanism, i.e., LPS-induced ROS production coupled to cytokine secretion in immune cells, can impact molecular events in neurons, and highlights one possible way endotoxin exposure resulting from bacterial infection promotes an interaction between the nervous and immune systems.