The present series of studies extend existing knowledge by demonstrating that two stressors, CORT and laparotomy, similarly affected the responses to subsequent low dose systemic LPS: both potentiated sickness-induced fever and pain but not lethargy. Interestingly, prior laparotomy, but nor prior CORT, produced a potentiated production of nitric oxide in peritoneal macrophages when stimulated with LPS ex vivo. Neither pretreatment affected LPS-induced IL-1β release ex vivo.
Potentiation of the fever response has been shown previously with a “two-hit” paradigm, inescapable tail shock, 24 h before low dose LPS (O’Connor et al., 2003
). An acute series of inescapable tail shocks causes the release of glucocorticoids equivalent to the dose of exogenous CORT administered in our studies (O’Connor et al., 2003
). Both inescapable shock and exogenous CORT potentiate the LPS-induced proinflammatory cytokine production peripherally (Frank et al., 2010
; Johnson et al., 2002
; O’Connor et al., 2003
). Therefore, it is not surprising that prior CORT produced a potentiated fever comparable to that seen after inescapable shock, but rather clarifies that CORT is a key mediator in the potentiated fever response following an acute stressor.
Interestingly, laparotomy was done 2 wk before LPS administration, a much longer duration before LPS than prior CORT or an acute stressor, and yet the potentiated responses were still observed with the same amplitude of fever and of a similar duration. This effect of potentiated inflammatory response was not seen with a 4 wk delay after laparotomy (Hains et al., 2010
). Therefore, it will be intriguing to define whether a similarly prolonged critical window exists for tail shock, glucocorticoids or other challenges.
We have previously shown that laparotomy 2 wk before low dose systemic LPS administration potentiated sickness-induced pain responses (allodynia). Therefore, we explored not only the classic fever response but also other behaviors such as sickness-induced pain by prior CORT. Prior stress, prior CORT and prior laparotomy each potentiate sickness-induced or injury-induced allodynia (Alexander et al., 2009
; Hains et al., 2010
; Takasaki et al., 2005
). Glia within the spinal cord produce proinflammatory cytokines that potentiate the allodynia (Watkins, 2005). Spinal microglia are activated after laparotomy and enhancement of allodynia due to prior laparotomy is suppressed by intrathecal administration of the microglial inhibitor, minocycline (Hains et al., 2010
). With regards to sensitization by CORT, enhancement of allodynia to LPS administered into the spinal cord is abolished by intrathecal interleukin 1 receptor antagonist (Loram et al., 2011
). Therefore, spinal microglia likely have a role to play in this pain potentiation.
While there are common mechanisms inducing the sickness responses such as lethargy and fever, there is some suggestion that certain sickness responses are more responsive to some released pro-inflammatory cytokines than others (Harden et al.). Lethargy is a clinical symptom of infection and is identified in animal models by measuring either voluntary cage activity or voluntary running wheel activity. In both the CORT and laparotomy studies, there was a significant lethargy induced within the first 6 h of the dark phase by LPS when it was administered within the first 4 h after lights on. However, neither challenge enhanced the effect of LPS on lethargy. It is possible that if LPS was administered within the dark phase of the cycle that a greater suppressive effect would have been identified. However, activity following LPS, with a prior inescapable shock showed a main effect of LPS and a main effect of shock, irrespective of whether the LPS was administered at the beginning of the light phase or at the beginning of the dark phase (Johnson et al., 2003
). While there was a trend of further suppression of activity by prior CORT in our study, it was not significant.
Previous work also has shown that the lethargy following systemic LPS outlasted the effects of the fever (Skinner et al., 2009
). We tested on the second day and the subsequent night and found no delayed effects of either laparotomy or CORT on the activity (data not shown). It is possible that voluntary wheel running may have been a more sensitive measure of activity compared to voluntary cage activity. While both measures were suppressed following two different doses of LPS, the wheel running was a more sensitive and showed a more robust suppression (Hopwood et al., 2009
). Therefore, it is possible that sickness-induced lethargy is not as sensitive to the effects of prior surgery and prior CORT as compared to fever and allodynia.
Most studies investigate the effect of infection on fever or tissue response with only a few studies examining the effect on both fever and other sickness-induced behaviors including lethargy. Blocking peripheral IL-6 partially attenuated the lethargy induced by LPS but completely blocked the fever (Harden et al., 2006
). In addition, diclofenac, a cyclooxygenase inhibitor blocking the synthesis of PGE2, only partially blocked LPS induced lethargy but completely blocked the fever (Harden et al., 2010
). Therefore, it is possible that laparotomy and CORT sensitize pathways contributing predominantly to fever and sickness-induced allodynia and less to the pathways inducing lethargy.
A pathogen-host interaction where immune cells release IL-1, NO and other pro-inflammatory substances initiate the sickness-induced pain and fever pathways. Peripheral to central signaling triggers immune cells in the brain and spinal cord to also release pro-inflammatory cytokines. Both the peripheral and central immune cells involved in the sickness response can become sensitized. Cellular sensitization is defined as the effect of an initial insult on a cell’s response to a subsequent insult. Sensitized cells are hypervigiliant, but do not exhibit an activated phenotype. Thus, while they respond to subthreshold stimuli and exhibit an exaggerated response to suprathreshold stimuli, they are not producing proinflammatory cytokines or engaging in respiratory burst activity (Ma et al., 2003
Laparotomy involves both tissue damage and an introduction of enteric bacteria into the peritoneal cavity, and thus there is an activation of local immune cells in the period immediately following surgery with an increase in pro-inflammatory cytokines (Kim and Yoon, 2010
; Lee et al., 2003
). It is possible that peritoneal macrophages can become sensitized as an effect of this initial immune response. Peritoneal cells stimulated within 24 h after surgery display decreased phagocytic activity, decreased TNFα release, and reduced endotoxin clearance (Pitombo, 2006
; Moehrlen, 2005
). However, we observed enhanced NO release to LPS in these cells two weeks post-surgery. Although no effect was found on IL-1 release to ex vivo
LPS, it is possible that IL-1 was produced intracellularly but not released from cells under the experimental cell culture conditions. Increased IL-1 mRNA was in fact noted in unstimulated peritoneal cells collected 2 wk after laparotomy compared to sham surgery (Hains, Unpub. Observ.).
The current finding that CORT did not affect peritoneal macrophage responses is consistent with previous studies. Even when administered in vitro
, pretreatment with CORT caused a significant reduction in TNF secretion following LPS stimulation (Du et al., 2010
). Unlike laparotomy, CORT treatment does not cause inflammation or local immune activation. It is more likely that the effect of prior CORT on fever and pain is from an enhanced CNS immune response, involving sensitized microglia. Laparotomy may also lead to microglial sensitization (Hains et al., 2010
), in addition to the effect on peritoneal macrophages, as demonstrated here.
Along with perivascular and meningeal macrophages, microglia represent a primary source for IL-1 in the CNS (van Dam et al., 1992
). Sensitized microglia are characterized by exaggerated IL-1 production in response to stimulation. Our group has recently shown that both stress and CORT caused increased IL-1 production in microglia following ex vivo
LPS (Frank et al., 2007
; Frank et al., 2010
). In addition, microglial activation within the spinal cord is noted 2 wk after laparotomy (Hains et al., 2010
), suggesting that microglia were not quescient at the time of LPS injection in the present studies. While microglial phenotype was not examined here and no conclusions can be made from the current findings regarding the role of microglia in sensitized sickness responses, it certainly warrants further investigation.
An immune system sensitized by prior surgery or prior CORT results in potentiation of some but not all potentiated sickness-induced behaviors. While the two challenges under investigation are distinct in their site and underlying mechanism, the potentiation of the fever and pain induced by low dose LPS are comparable. This is despite the fact that laparotomy was done 2 wk before the LPS while CORT was administered 24 h before the LPS. Through immune-to-brain-to-spinal cord communication, systemic LPS activates immune cells both in the periphery and within the central nervous system. During infection, there is a release by peripheral immune cells of pro-inflammatory cytokines and other proinflammatory mediators such as nitric oxide and reactive oxygen species. The communication of these peripheral immune signals to the brain and spinal cord result in fever and other sickness behaviors. While laparotomy induces an injury and subsequent inflammation, CORT alone does not elevate proinflammatory cytokines, but rather is classically anti-inflammatory and suppresses inflammatory responses when administered in the presence of inflammation (Sorrells et al., 2009
). Therefore, while CORT must sensitize the immune system, as demonstrated by work from our lab and others (Frank et al., 2010
; Munhoz et al., 2006
) such that a subsequent immune challenge results in exaggerated sickness behaviors including pain, it must do so in a different manner to that of prior surgery.