Experimental Neurology 258 (2014) 105–111
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Review
The systemic response to CNS injury Daniel C. Anthony ⁎, Yvonne Couch Department of Pharmacology, University of Oxford, Oxford, UK
a r t i c l e
i n f o
Article history: Received 28 November 2013 Revised 11 March 2014 Accepted 21 March 2014
a b s t r a c t Inflammation within the brain or spinal cord has the capacity to damage neurons and is known to contribute to long-term disability in a spectrum of central nervous system (CNS) pathologies. However, there is a more profound increase in the recruitment of potentially damaging populations of leukocytes to the spinal cord than to the brain after equivalent injuries. Increased levels of inflammatory cytokines and chemokines in the spinal cord underpin this dissimilarity after injury, which also appears to be very sensitive to processes that operate within organs distant from the primary injury site such as the liver, lung and spleen. Indeed, CNS injury per se can generate profound changes in gene expression and the cellularity of these organs, which, as a consequence, gives rise to secondary organ damage. Our understanding of the local inflammatory processes that can damage neurons is becoming clearer, but our understanding of how the peripheral immune system coordinates the response to CNS injury and how any concomitant infections or injury might impact on the outcome of CNS injury is not so well developed. It is clear that the orientation of the response to peripheral challenges, be it a pro- or anti-inflammatory effect, appears to be dependent on the nature and timing of events. Here, the importance of the inter-relationship between inflammation in the CNS and the consequent inflammatory response in peripheral tissues is highlighted. © 2014 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . The CNS inflammatory response . . . . . . The hepatic response to CNS inflammation . The spinal cord . . . . . . . . . . The brain . . . . . . . . . . . . . Systemic infection and disease resolution . . Targeting the immune system for CNS injury Summary . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .
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Introduction It is well known that the inflammatory response observed in the CNS is unlike the inflammatory response observed in other tissues (Perry et al., 1995). One prominent feature that illustrates the atypical nature of CNS inflammatory responses is the paucity of leukocyte recruitment following injury. Fewer leukocytes accumulate in the brain and spinal cord than in peripheral tissues when subjected to an equivalent
⁎ Corresponding author at: Department of Pharmacology, University of Oxford, Mansfield Road Oxford, OX1 3QT, UK. Fax: +44 01865 271853. E-mail address: [email protected] (D.C. Anthony).
http://dx.doi.org/10.1016/j.expneurol.2014.03.013 0014-4886/© 2014 Elsevier Inc. All rights reserved.
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inflammatory stimulus. This observation can be generalised to a broad set of different inflammatory stimuli. If excitotoxins, such as kainic acid, are microinjected into the CNS, very little leukocyte recruitment occurs with few neutrophils and very delayed (N 48 h) monocyte infiltration, despite rapid microglial activation and blood–brain-barrier (BBB) disruption (Andersson et al., 1991a, 1991b; Marty et al., 1991). Furthermore, in models of Wallerian degeneration, acute degeneration of CNS neurons completely fails to induce leukocyte recruitment to the brain, but in the periphery monocyte recruitment is a clear feature of the process (Lawson et al., 1994). These responses are not restricted to direct models of neuronal injury, but also occur in models of both acute inflammation, such as by the microinjection of cytokines or chemokines, as well as in contusion-
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type injuries. Injection of a low dose of lipopolysaccharide (LPS) into the skin induces a florid inflammatory response with rapid monocyte and neutrophil infiltration. When an equivalent dose of LPS is injected into the brain, very few leukocytes accumulate in the brain parenchyma, although recruitment is more apparent in the choroid plexus and meninges. Increasing the dose of LPS only results in minimal and delayed parenchymal monocyte recruitment (Andersson et al., 1992). These observations all raise a common question, ‘why is the CNS parenchyma so different from peripheral tissue?’
The CNS inflammatory response Cytokines such as interleukin-1 beta (IL-1β) and tumour necrosis factor (TNFα) are both important mediators of inflammation in peripheral tissues, and macrophages are one of the main cell types involved in their production (Dinarello, 1996). Reduced production of cytokines due to the quiescent nature of microglia, or a more generalised downregulation of CNS cytokine synthesis could contribute to the atypical inflammatory response seen in the brain and spinal cord. However, cytokines are readily produced by microglia in vivo in response to proinflammatory stimuli (Blond et al., 2002; Woodroofe et al., 1991). IL1β and TNFα expression has also been shown in the brain, in models of both ischaemia and traumatic injury, where leukocyte recruitment is a prominent feature (Fan et al., 1995; Feuerstein et al., 1994; Galea and Brough, 2013; Liu et al., 1994; Yabuuchi et al., 1994). In the periphery, cytokines such as IL-1β and TNF induce both self and cross amplification, resulting in the recruitment of both neutrophils and macrophages to the site of injury, indicative of cross-talk in their signalling mechanisms (Yang et al., 2010). In the CNS these cytokines are also produced in response to stimuli such as LPS, but their expression results in minimal leukocyte infiltration (Breder et al., 1994; Higgins and Olschowka, 1991; Quan et al., 1994). Thus, it appears that the origin of the resistance of the brain to leukocyte recruitment lies at least in part, downstream of pro-inflammatory cytokine production. While proinflammatory cytokine expression is a feature of inflammation in the brain and spinal cord, there is growing evidence to support the idea that cross talk and cytokine self-amplification do not occur, at least to the same degree (Blond et al., 2002). The microinjection of inflammatory agents into the CNS has been shown to cause differential expression in cytokine vs chemokine production (Blond et al., 2002; Campbell et al., 2007b). For example, IL-1β does not induce TNFα or downstream CC-chemokines when it is injected into the brain parenchyma but rather induces CXC-chemokines (Blond et al., 2002). In equivalent spinal cord injuries, chemokine induction is greatly increased (Campbell et al., 2002). While these types of injuries to both the brain and spinal cord result in lower leukocyte recruitment than to an equivalent peripheral injury, there is also a significant difference between the brain and spinal cord parenchyma. Contusion-type injuries to the cord result in rapid microglial activation and a greater increase in infiltrating neutrophils, whereas a similar injury to the brain results in a delayed microglial response and muted neutrophil recruitment (Schnell et al., 1999a). Similarly, the injection of cytokines such as IL-1 into the spinal cord also results in an augmented neutrophil recruitment when compared to equivalent challenges in the brain (Schnell et al., 1999b). Some important factors to consider in these cases are the anatomical and cellular differences between the spinal cord and brain (see Fig. 1). The spinal cord has almost twice the numbers of microglia as the brain, and the ratio of microglia in the grey and white matter is equal, where it is lower in brain white (Lawson et al., 1990; Vela et al., 1995). Similarly, the brain appears to exhibit an inflammatory neuraxis, with higher numbers of microglia in some regions, such as the hippocampus and substantia nigra. While the spinal cord appears to have a relatively homogenous distribution of microglia, their activation pattern is different depending on the cord segment (Gwak et al., 2012; Olson, 2010; Schomberg and Olson, 2012).
While anatomical differences between the brain and the spinal cord (white matter vs grey matter, ease of access for surgery, etc.) must be taken into consideration when studying the inflammatory response in these tissues, the response of the vasculature to injury remains largely similar. It appears unlikely that restricted leukocyte entry to the brain or spinal cord is the result of deficient adhesion molecule expression. Endothelial cells of the cerebral vasculature possess the full complement of adhesion molecules found in the periphery (Sobel et al., 1990). The spinal cord presents a slightly more interesting target. While there are similar adhesion molecules expressed in the cord, there is a decrease in the expression of specific tight junction proteins such as ZO-1 (Ge and Pachter, 2006), suggesting a ‘looser’ barrier. If LPS or IL-1β is injected into the brain, or if neuronal degeneration is induced by kainic acid, adhesion molecule expression occurs in a manner comparable to the periphery, but without the downstream leukocyte recruitment (Bell and Perry, 1995; Bernardes-Silva et al., 2001). Similar adhesion molecule upregulation occurs in the spinal cord (Schnell et al., 1999a). These observations suggest that the expression patterns of adhesion molecules in the CNS are not able to account for the refractory nature of the brain to leukocyte recruitment. Despite the reported differences in the barrier structure it should be remembered that the blood–brain or blood–spinal cord barriers are barriers to solutes, but all immune cells are still able to traffic across an intact barrier for routine immune surveillance (Williams and Hickey, 1995) and in disease (such as developing multiple sclerosis lesions). It is also not the case that leukocytes will automatically cross a damaged CNS barrier. In the kainic acid induced excitotoxic lesion described above, neutrophils do not enter the brain in significant numbers or in the circumventricular organs that lack a BBB (Bolton and Perry, 1998). This discussion of the resistance of the CNS to the infiltration of immune cells, simply serves to highlight our failure to identify the molecular basis for the atypical inflammatory response in the brain and spinal cord. We also considered the possibility that signals, which are normally released from peripheral injury sites to activate leukocyte mobilisation and priming, may not be released from the CNS after injury. However, experiments designed to test this hypothesis revealed that not only was the acute phase response (APR) activated after the generation of focal inflammatory lesions in the brain, but it is just as rapid as the response to comparable peripheral challenges (if not greater). These observations led to the identification of chemokines as a new class of acute phase protein (Campbell et al., 2003, 2005). The hepatic response to CNS inflammation Inflammation, resulting from injury or infection, is predominantly a local phenomenon, but if the stimulus is sufficient, local inflammation is also accompanied by more far-reaching responses, which may include leukocytosis (elevated circulating leukocyte numbers), fever, and changes in the serum levels of many factors including electrolytes, metal ions, clotting agents, complement, and glucocorticoids. Collectively these changes are referred to as the acute phase response (APR) and the liver is the principal organ involved in coordinating the response (Baumann and Gauldie, 1994). The primary purpose of the acute phase protein production by the liver is considered to be to effect a return to homeostasis, by removing inflammatory stimuli, attenuating local inflammation, and promoting tissue repair and regeneration. Hepatic acute phase protein production is coordinated by a range of inflammatory mediators, including cytokines, glucocorticoids, and anaphylotoxins (Jensen and Whitehead, 1998; Koj, 1998; Szalai et al., 2000). It is widely accepted that the pro-inflammatory cytokines IL1β, IL-6, and TNF are critical mediators of APR initiation (Koj, 1996; Kushner, 1993). Despite this, an array searching for the key mediators that accompany the hepatic APR to CNS injury revealed that the changes in gene expression in the liver are more extensive than previously thought, and include more genes than just the “traditional” acute phase proteins (Campbell et al., 2007). In particular, the list of induced
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Fig. 1. The systemic response to CNS injury. Injury to the spinal cord triggers the induction of an acute phase response that includes marked hepatic CXC and CC chemokine induction. The release of hepatic chemokines results in leukocyte mobilization from the marrow. Chemokines are also expressed in other organs as a consequence of SCI. However, some of this production is likely to reflect an increase in leukocyte recruitment within the liver, lung and kidney in particular. As a consequence of this inappropriate recruitment markers of heptocellular damage can be detected and this is likely to contribute to multiorgan dysfunction syndrome (MODS) and systemic inflammatory response syndrome (SIRS), which are often features of CNS injury.
genes encoded chemokines, adhesion molecules, and genes that are not normally associated with the inflammatory response including lipocalin-2 and jun-B oncogene (Campbell et al., 2007c). It has also become clear that the production of acute phase proteins after CNS injury can have unexpected consequences resulting in systemic inflammatory response syndrome (SIRS), in which inflammatory cells from the circulation accumulate in organs such as the liver, lung and kidney, leading to organ damage (Bao et al., 2011). The spinal cord Many studies have sought to explore the contribution of the inflammatory response to the outcome of spinal cord injury and there is no doubt that contribution will be different from brain injury (Table 1). However, remarkably little attention has been paid to the manner in
which the injury signals are communicated to the rest of the body and the impact this has on the function of organs distant form the injury site. Injury to the spinal cord has been shown to generate a significant hepatic chemokine response and downstream hepatocellular injury (Campbell et al., 2005; Fleming et al., 2012). Interestingly, spinal cord injury (SCI)-induced damage to the liver appears to be dependent on the site of the injury; upper thoracic SCI is more effective than a lower thoracic SCI at inducing hepatic injury (Fleming et al., 2012). The recruitment of leukocytes to the liver after SCI is also very rapid (30 min) compared to the recruitment to the cord (Hundt et al., 2011). Thus, early intervention is likely to be important. Liver CCL-2 and CXCL-1 mRNA and protein are found to be elevated in the liver in association with leukocyte mobilization before any significant leucocyte recruitment to the cord occurs (Fig. 1). In humans, SCI is also associated
Table 1 A comparison of the inflammatory response to brain and spinal cord injury.
Anatomy Blood–brain barrier response Microglial numbers Microglial phenotype Neutrophil infiltration
Effects on mood Systemic APR Effect of systemic infection
Brain
Spinal cord
Grey matter surrounds white matter Very little breakdown after trauma or proinflammatory cytokine injection 20–150/mm2grey matter N white matter. Region specific differences in cell numbers, e.g. high numbers in hippocampus and substantia nigra Lower levels of CD45 and CDllb than microglia in the cord. Decreased local CXC chemokine production compared to cord. Very few neutrophils recruited to the parenchyma, but many to the meninges after trauma or a cytokine challenge. Following trauma N30% develop depression—usually but not exclusively dependent upon injury location Hepatic chemokines upregulated as rapidly as 1 h post-injury and NFkB activity at 30 min. Rapid neutrophil recruitment to the liver. Significantly impairs functional recovery, but sterile TLR4 agonists can be protective
White matter surrounds grey matter Significant breakdown after trauma or proinflammatory cytokine injection 250/mm2grey = white. No regional differences in numbers, but activation state differs with lesion location. Higher levels of CD45 and CDllb than microglia in the brain. Increased local CXC chemokine production compared to brain. Significant recruitment of neutrophils to the parenchyma Following injury 20–25% develop depression, but no link to injury location Neutrophil recruitment to the liver, and CXCL1 upregulated 2 h after a compression injury Significantly impairs functional recovery, but sterile TLR4 agonists can be protective
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with an increase in the oxidative activity of blood leukocytes from 6 h to 2 weeks after injury (Bao et al., 2009, 2011), but it is still unclear whether the number of liver leukocytes also increases in human SCI. Interestingly, we have discovered that neutrophil recruitment to the liver is a feature of multiple sclerosis and we suspect that the recruitment of leukocytes to distant organs is likely to be a feature of many CNS pathologies (Campbell et al., 2010). The brain Systemic cytokines also play important roles beyond coordinating the cellular response to a local injury. When an individual suffers from a bacterial or viral infection, the situation is often characterised by subjective feelings of ‘sickness’. These include malaise, lassitude, fatigue, numbness, coldness, muscle and joint aches, and reduced appetite. These symptoms can also be generated by chronic inflammatory disease or injury and are a consequence of systemic cytokine production. Originally considered trivial effects, co-ordinated behavioural response to injury or disease has become recognised as a highly organised strategy to alter behaviours to promote the fight against infection and facilitate repair (Dantzer, 2001). These typical behaviours may also play an altruistic role in signaling to others that an individual is sick and should be avoided. Given the nature of these behaviours, it is perhaps not surprising that the similarities between the ‘malaise’ associated with sickness has prompted comparison with the behaviours associated with major depression (De La Garza, 2005), a factor that has the capacity to confound recovery in a number of different CNS diseases. For example, post-stroke depression has been known for some time to be associated with a poorer outcome after injury (Sinyor et al., 1986), and recent studies have shown that early treatment with antidepressants such as fluoxetine has the capacity to significantly improve recovery (Chollet et al., 2011), but the cause or effect arguments require further investigation. Precisely how peripheral cytokine production can trigger these altered behaviours is still unclear. Both neural (vagal) and humoral routes have been proposed (Anthony et al., 2012) with work from our laboratory showing that systemic inflammation can profoundly affect the central serotonergic system by altering 5HT receptor gene expression (Couch et al., 2013a, 2013b). The humoral pathway argues that circulating cytokines such as IL-1β are detected by endothelial cells within the circumventricular organs and choroid plexus for example, and this triggers prostaglandin production and downstream central cytokine production (Dantzer, 2001; Jiang et al., 2008; Konsman et al., 2002). A neural route has also been demonstrated by cutting vagal afferents, which can eliminate the sickness behaviours, but only in certain circumstances (Konsman et al., 2000; Luheshi et al., 2000). The liver contains vagal sensory nerves, and inflammatory cytokines in hepatic cells may activate the vagus to carry signals back to the CNS (Borovikova et al., 2000); however, the literature is very contradictory in this area and studies are often confounded by experimental complications resulting from surgery. Systemic infection and disease resolution Despite the paucity of information regarding the way in which the brain and spinal cord communicate with the liver, there is a plethora of data showing that this communication system is vital for survival and any deviation can have severe consequences for the organism. Systemic infection has long been recognised as a risk factor in a range of conditions that affect the CNS (Anthony et al., 2012). It seems likely, that this is a consequence of the summation of injury signals in the liver, and other organs, from all the focal sites of injury throughout the body. Our experiments suggest that the relationship between the contributing factors may not be as stoichiometric as we previously envisaged. In some cases, summation of injury signals clearly leads to excessive amplification of the APR and increased recruitment of leukocytes to sites of CNS injury and, in particular, to the liver, where they cause hepatocellular damage (Campbell et al., 2005). However, at
other times peripheral inflammation seems to suppress leukocyte recruitment to the brain and spinal cord, which may be either as a consequence of the redirection of leukocytes or perhaps the generation of local ‘anti-inflammatory’ signals in an attempt to suppress the potentially damaging consequences of an excessive APR (Davis et al., 2005). Support for the negative impact of infection on acute CNS injury is highlighted in studies on stroke. In the human population, the prevalence of infection in the month preceding ischaemic stroke has been estimated to be at least 20% (Grau et al., 1995a). Acute periods of infection during the preceding month before a stroke (most often bacterial and affecting the respiratory system), are known to be risk factors for cerebral infarction (Syrjanen et al., 1988). Furthermore, patients with recent infection tend to present with a more severe neurological deficit than patients without infection (Grau et al., 1995b), factors of significance when considering acute inflammatory responses in the brain. The production and release of TNF as part of the hepatic APR in response to infection, in addition to its effects on leukocyte populations, also cause profound changes in the haemodynamic response of many vascular beds. We have shown that TNF, in contrast to its effects in peripheral vascular beds, causes an endothelin-1-dependent reduction in cerebral blood volume, which is likely to contribute to poor reperfusion after an ischaemic or traumatic injury to the CNS (Sibson et al., 2002). There are plenty of examples that demonstrate a deleterious effect of infection on the outcome of CNS injuries such as those incurred during stroke (Chamorro et al., 2007) and multiple sclerosis (Buljevac et al., 2002). However, while peripheral injury or infection is known to have an impact on the pathogenesis of chronic CNS disease, until recently it was much less clear whether the systemic response to brain or spinal cord injury per se would play a significant role in governing the outcome. It was also unclear whether the unusual pattern of inflammation in the CNS might result in an altered systemic inflammatory response to infection. For example, in an individual that is considered brain dead, this state is known to adversely affect both the quantity and quality of organs available for transplant, where the activation of proinflammatory pathways contributes to graft dysfunction in the recipient (de Vries et al., 2011). Targeting the immune system for CNS injury The discovery that there is a peripheral, hepatic component in addition to an inflammatory response in the CNS presented a new concept with which to approach the treatment of such conditions. History, reasonably, has dictated that anti-inflammatory therapy needs to be delivered to the site of the local inflammatory response and many therapeutic strategies have been abandoned due to poor CNS bioavailability. The inhibition of peripheral TNF after a central injection of IL-1β provides a very clear example of how local inflammation can be inhibited by targeting the systemic response. The TNF neutralising therapy etanercept does not cross the intact blood–brain barrier in any appreciable amount, but administration of etanercept following the microinjection of IL-1β into the brain inhibits the recruitment of leukocytes to the CNS (Campbell et al., 2007b). Conversely, the number of leukocytes entering the injured CNS can be increased by priming with systemic injections of chemokines, such as CINC-1, which are responsible for the mobilisation and recruitment of leukocytes (Campbell et al., 2005b). TNF is well known to induce the transcription factor NF-κB (Ali and Mann, 2004). NF-κB is a dimeric transcription factor that regulates the expression of a wide array of immune and inflammatory genes (Hayden and Ghosh, 2004). Gene expression array analysis has proven that inflammatory challenge to the brain induces genes regulated by NF-kB in the periphery (Campbell et al., 2007b). A study using transgenic mice that express a luciferase reporter under transcriptional regulation of NF-κB has shown conclusively that NF-κB activation occurs rapidly in the liver following inflammatory challenge to the brain (Campbell et al., 2008). Selective inhibition of NF-κB activity by intravenous adenoviral-mediated delivery of an IκBalpha super-repressor
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to the liver also markedly reduced the numbers of neutrophils recruited to the brain (Campbell et al., 2008). Taken together, these data illustrate that one of the functions of hepatic NF-κB is, unexpectedly, as a pivotal regulator of CNS inflammation. Downstream of NF-κB activity it has been shown that it is possible to manipulate the number of leukocytes recruited to the CNS after injury by manipulating the processes that lead to the systemic leukocytosis that follows (Campbell et al., 2003b, 2005b). Our laboratory has demonstrated that when chemokines are administered intravenously in conjunction with a CNS lesion, more leukocytes are mobilised and leukocyte recruitment to the brain is exacerbated (Campbell et al., 2005b), and, if the hepatic chemokine response is inhibited, leukocyte recruitment to the CNS after injury is suppressed. Thus, the manipulation of events distant from the site of injury can very effectively modify the recruitment of leukocytes to the CNS, and is an important consideration when peripheral infection is commonplace after acute injury to the CNS (McCormick et al., 2013). For example, in the spinal cord, targeting the systemic response has been shown to have some potential. Liposomes filled with clodronate have been used to deplete Kupffer cells, which appear to be the principal source of the APRs prior to spinal cord injury (Campbell et al., 2008). The clodronate treatment reduced Kupffer cells by 90% and subsequent production of the APR, as well as reducing the numbers of neutrophils and ED-1-positive cells in the spinal cord after contusion injury. While the discovery of the hepatic chemokine response to brain and spinal cord injury strongly supports the principal that any factors that exacerbate the APR are likely to have an adverse effect on CNS injury and disease, it is clear that the situation is rather more complicated. There is also growing evidence to suggest that systemic inflammation can confer a degree of tolerance to brain injury. A range of preconditioning strategies have shown that brief periods of ischaemia, periods of seizure, and anaesthetic exposure confer neuroprotection against a subsequent larger insult (Kapinya et al., 2002; Simon et al., 1993; Towfighi et al., 1999). In addition, the systemic administration of endotoxin has been reported to confer neuroprotection from ischaemia (Bordet et al., 2000; Rosenzweig et al., 2004). Whilst an interesting phenomenon, the clinical application of pre-conditioning may be challenging given the differences between the immune systems of preclinical model rodents and humans. For example, the amount of lipopolysaccharide required to induce ‘flu-like symptoms in rodents (around 2 μg total injection) will result in death in humans (Sauter and Wolfensberger, 1980) suggesting fundamental differences in basic immunology. Furthermore, the timing of conditioning challenges seems to be an important issue and one might predict that a post-conditioning challenge might exacerbate a CNS lesion. As discussed above there is evidence to suggest that lipopolysaccharide (LPS) will accentuate neuronal degeneration, such as in a model of murine prion disease (Combrinck et al., 2002), and also in a middle cerebral artery occlusion model of ischaemia (McColl et al., 2007). We were surprised to find that it is also possible to inhibit the recruitment of leukocytes after SCI using an intravenous injection of LPS before, and, importantly, after, the contusion injury. However, the response appears to be LPS-specific, as we could not achieve the same results with bolus injections of IL-1 (Davis et al., 2005), suggesting a regulatory role for TLR-type signalling. It has been shown that neutrophils in liver sinusoids can modulate the proinflammatory response of Kupffer cells to bacteria cleared from the bloodstream by suppressing cytokine and chemokine mRNA expression (Holub et al., 2009). Thus, the excessive accumulation of neutrophils in the liver following the LPS challenge may actually suppress downstream mobilization signals. However, these results are perhaps less surprising than we first thought. In the 1950s, Windle et al. discovered that systemic injection of Piromen, a crude pyrogen used to induce fever, augmented recovery of sensory function in spinalised cats (Windle and Chambers, 1950). Piromen is predominantly LPS. In the 1990s Guth extended these observations and studied the effects of
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injecting LPS intraperitoneally either alone or in combination with indomethacin and pregnenolone over an extended time period following a compression lesion (Guth et al., 1994b). While the best outcome was generated with a combination therapy. LPS alone reduced lesion cavitation and promoted axon/neurite growth into the lesion site. LPS and indomethacin also marginally improved recovery of motor function in a weight-drop model of spinal contusion injury (Guth et al., 1994a). It is not clear how these therapies affected the immediate histological outcomes and the role of the systemic response was not considered. More recently, another group sought to replicate the experiments of Guth and reported a more modest, though significant outcome (Popovich et al., 2012). It is our view that the use of this combination therapy is likely to generate complex local and systemic effects and that each component is likely to have its own optimal therapeutic window. Clearly, as with all attempts at combination therapy, the number of possible permutations can be overwhelming. There is no doubt that the capacity of pyrogens to modulate the hepatic response will mean that the ability of the liver to respond to systemic infection will have been significantly affected as part of these regimes. Whatever the mechanism, these results all highlight the importance of the nature of the systemic challenge on the outcome of a CNS injury. Summary While it is clear that the systemic immune system plays a significant and diverse role in the response to CNS injury, it is important to determine the precise mediators involved in this reaction before applying potentially harmful immune suppression therapy. By further studying the interactions between the hepatic APR and the CNS APR it may be possible to pinpoint molecular targets to prevent CNS inflammation, and subsequent neurological damage, being exacerbated by the peripheral immune system. References Ali, S., Mann, D.A., 2004. Signal transduction via the NF-kappaB pathway: a targeted treatment modality for infection, inflammation and repair. Cell Biochem. Funct. 22, 67–79. Andersson, P.B., Perry, V.H., Gordon, S., 1991a. The CNS acute inflammatory response to excitotoxic neuronal cell death. Immunol. Lett. 30, 177–182. Andersson, P.B., Perry, V.H., Gordon, S., 1991b. The kinetics and morphological characteristics of the macrophage-microglial response to kainic acid-induced neuronal degeneration. Neuroscience 42, 201–214. Andersson, P.B., Perry, V.H., Gordon, S., 1992. The acute inflammatory response to lipopolysaccharide in CNS parenchyma differs from that in other body tissues. Neuroscience 48, 169–186. Anthony, D.C., Couch, Y., Losey, P., Evans, M.C., 2012. The systemic response to brain injury and disease. Brain Behav. Immun. 26, 534–540. Bao, F., Bailey, C.S., Gurr, K.R., Bailey, S.I., Rosas-Arellano, M.P., Dekaban, G.A., Weaver, L.C., 2009. Increased oxidative activity in human blood neutrophils and monocytes after spinal cord injury. Exp. Neurol. 215, 308–316. Bao, F., Bailey, C.S., Gurr, K.R., Bailey, S.I., Rosas-Arellano, M.P., Brown, A., Dekaban, G.A., Weaver, L.C., 2011. Human spinal cord injury causes specific increases in surface expression of beta integrins on leukocytes. J. Neurotrauma 28, 269–280. Baumann, H., Gauldie, J., 1994. The acute-phase response. Immunol. Today 15, 74–80. Bell, M.D., Perry, V.H., 1995. Adhesion molecule expression on murine cerebral endothelium following the injection of a proinflammagen or during acute neuronal degeneration. J. Neurocytol. 24, 695–710. Bernardes-Silva, M., Anthony, D.C., Issekutz, A.C., Perry, V.H., 2001. Recruitment of neutrophils across the blood–brain barrier: the role of E- and P-selectins. J. Cereb. Blood Flow Metab. 21, 1115–1124. Blond, D., Campbell, S.J., Butchart, A.G., Perry, V.H., Anthony, D.C., 2002. Differential induction of interleukin-1beta and tumour necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain. Brain Res. 958, 89–99. Bolton, S.J., Perry, V.H., 1998. Differential blood–brain barrier breakdown and leucocyte recruitment following excitotoxic lesions in juvenile and adult rats. Exp. Neurol. 154, 231–240. Bordet, R., Deplanque, D., Maboudou, P., Puisieux, F., Pu, Q., Robin, E., Martin, A., Bastide, M., Leys, D., Lhermitte, M., Dupuis, B., 2000. Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance. J. Cereb. Blood Flow Metab. 20, 1190–1196. Borovikova, L.V., Ivanova, S., Zhang, M., Yang, H., Botchkina, G.I., Watkins, L.R., Wang, H., Abumrad, N., Eaton, J.W., Tracey, K.J., 2000. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405, 458–462.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Review
The systemic response to CNS injury Daniel C. Anthony ⁎, Yvonne Couch Department of Pharmacology, University of Oxford, Oxford, UK
a r t i c l e
i n f o
Article history: Received 28 November 2013 Revised 11 March 2014 Accepted 21 March 2014
a b s t r a c t Inflammation within the brain or spinal cord has the capacity to damage neurons and is known to contribute to long-term disability in a spectrum of central nervous system (CNS) pathologies. However, there is a more profound increase in the recruitment of potentially damaging populations of leukocytes to the spinal cord than to the brain after equivalent injuries. Increased levels of inflammatory cytokines and chemokines in the spinal cord underpin this dissimilarity after injury, which also appears to be very sensitive to processes that operate within organs distant from the primary injury site such as the liver, lung and spleen. Indeed, CNS injury per se can generate profound changes in gene expression and the cellularity of these organs, which, as a consequence, gives rise to secondary organ damage. Our understanding of the local inflammatory processes that can damage neurons is becoming clearer, but our understanding of how the peripheral immune system coordinates the response to CNS injury and how any concomitant infections or injury might impact on the outcome of CNS injury is not so well developed. It is clear that the orientation of the response to peripheral challenges, be it a pro- or anti-inflammatory effect, appears to be dependent on the nature and timing of events. Here, the importance of the inter-relationship between inflammation in the CNS and the consequent inflammatory response in peripheral tissues is highlighted. © 2014 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . The CNS inflammatory response . . . . . . The hepatic response to CNS inflammation . The spinal cord . . . . . . . . . . The brain . . . . . . . . . . . . . Systemic infection and disease resolution . . Targeting the immune system for CNS injury Summary . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .
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Introduction It is well known that the inflammatory response observed in the CNS is unlike the inflammatory response observed in other tissues (Perry et al., 1995). One prominent feature that illustrates the atypical nature of CNS inflammatory responses is the paucity of leukocyte recruitment following injury. Fewer leukocytes accumulate in the brain and spinal cord than in peripheral tissues when subjected to an equivalent
⁎ Corresponding author at: Department of Pharmacology, University of Oxford, Mansfield Road Oxford, OX1 3QT, UK. Fax: +44 01865 271853. E-mail address: [email protected] (D.C. Anthony).
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inflammatory stimulus. This observation can be generalised to a broad set of different inflammatory stimuli. If excitotoxins, such as kainic acid, are microinjected into the CNS, very little leukocyte recruitment occurs with few neutrophils and very delayed (N 48 h) monocyte infiltration, despite rapid microglial activation and blood–brain-barrier (BBB) disruption (Andersson et al., 1991a, 1991b; Marty et al., 1991). Furthermore, in models of Wallerian degeneration, acute degeneration of CNS neurons completely fails to induce leukocyte recruitment to the brain, but in the periphery monocyte recruitment is a clear feature of the process (Lawson et al., 1994). These responses are not restricted to direct models of neuronal injury, but also occur in models of both acute inflammation, such as by the microinjection of cytokines or chemokines, as well as in contusion-
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type injuries. Injection of a low dose of lipopolysaccharide (LPS) into the skin induces a florid inflammatory response with rapid monocyte and neutrophil infiltration. When an equivalent dose of LPS is injected into the brain, very few leukocytes accumulate in the brain parenchyma, although recruitment is more apparent in the choroid plexus and meninges. Increasing the dose of LPS only results in minimal and delayed parenchymal monocyte recruitment (Andersson et al., 1992). These observations all raise a common question, ‘why is the CNS parenchyma so different from peripheral tissue?’
The CNS inflammatory response Cytokines such as interleukin-1 beta (IL-1β) and tumour necrosis factor (TNFα) are both important mediators of inflammation in peripheral tissues, and macrophages are one of the main cell types involved in their production (Dinarello, 1996). Reduced production of cytokines due to the quiescent nature of microglia, or a more generalised downregulation of CNS cytokine synthesis could contribute to the atypical inflammatory response seen in the brain and spinal cord. However, cytokines are readily produced by microglia in vivo in response to proinflammatory stimuli (Blond et al., 2002; Woodroofe et al., 1991). IL1β and TNFα expression has also been shown in the brain, in models of both ischaemia and traumatic injury, where leukocyte recruitment is a prominent feature (Fan et al., 1995; Feuerstein et al., 1994; Galea and Brough, 2013; Liu et al., 1994; Yabuuchi et al., 1994). In the periphery, cytokines such as IL-1β and TNF induce both self and cross amplification, resulting in the recruitment of both neutrophils and macrophages to the site of injury, indicative of cross-talk in their signalling mechanisms (Yang et al., 2010). In the CNS these cytokines are also produced in response to stimuli such as LPS, but their expression results in minimal leukocyte infiltration (Breder et al., 1994; Higgins and Olschowka, 1991; Quan et al., 1994). Thus, it appears that the origin of the resistance of the brain to leukocyte recruitment lies at least in part, downstream of pro-inflammatory cytokine production. While proinflammatory cytokine expression is a feature of inflammation in the brain and spinal cord, there is growing evidence to support the idea that cross talk and cytokine self-amplification do not occur, at least to the same degree (Blond et al., 2002). The microinjection of inflammatory agents into the CNS has been shown to cause differential expression in cytokine vs chemokine production (Blond et al., 2002; Campbell et al., 2007b). For example, IL-1β does not induce TNFα or downstream CC-chemokines when it is injected into the brain parenchyma but rather induces CXC-chemokines (Blond et al., 2002). In equivalent spinal cord injuries, chemokine induction is greatly increased (Campbell et al., 2002). While these types of injuries to both the brain and spinal cord result in lower leukocyte recruitment than to an equivalent peripheral injury, there is also a significant difference between the brain and spinal cord parenchyma. Contusion-type injuries to the cord result in rapid microglial activation and a greater increase in infiltrating neutrophils, whereas a similar injury to the brain results in a delayed microglial response and muted neutrophil recruitment (Schnell et al., 1999a). Similarly, the injection of cytokines such as IL-1 into the spinal cord also results in an augmented neutrophil recruitment when compared to equivalent challenges in the brain (Schnell et al., 1999b). Some important factors to consider in these cases are the anatomical and cellular differences between the spinal cord and brain (see Fig. 1). The spinal cord has almost twice the numbers of microglia as the brain, and the ratio of microglia in the grey and white matter is equal, where it is lower in brain white (Lawson et al., 1990; Vela et al., 1995). Similarly, the brain appears to exhibit an inflammatory neuraxis, with higher numbers of microglia in some regions, such as the hippocampus and substantia nigra. While the spinal cord appears to have a relatively homogenous distribution of microglia, their activation pattern is different depending on the cord segment (Gwak et al., 2012; Olson, 2010; Schomberg and Olson, 2012).
While anatomical differences between the brain and the spinal cord (white matter vs grey matter, ease of access for surgery, etc.) must be taken into consideration when studying the inflammatory response in these tissues, the response of the vasculature to injury remains largely similar. It appears unlikely that restricted leukocyte entry to the brain or spinal cord is the result of deficient adhesion molecule expression. Endothelial cells of the cerebral vasculature possess the full complement of adhesion molecules found in the periphery (Sobel et al., 1990). The spinal cord presents a slightly more interesting target. While there are similar adhesion molecules expressed in the cord, there is a decrease in the expression of specific tight junction proteins such as ZO-1 (Ge and Pachter, 2006), suggesting a ‘looser’ barrier. If LPS or IL-1β is injected into the brain, or if neuronal degeneration is induced by kainic acid, adhesion molecule expression occurs in a manner comparable to the periphery, but without the downstream leukocyte recruitment (Bell and Perry, 1995; Bernardes-Silva et al., 2001). Similar adhesion molecule upregulation occurs in the spinal cord (Schnell et al., 1999a). These observations suggest that the expression patterns of adhesion molecules in the CNS are not able to account for the refractory nature of the brain to leukocyte recruitment. Despite the reported differences in the barrier structure it should be remembered that the blood–brain or blood–spinal cord barriers are barriers to solutes, but all immune cells are still able to traffic across an intact barrier for routine immune surveillance (Williams and Hickey, 1995) and in disease (such as developing multiple sclerosis lesions). It is also not the case that leukocytes will automatically cross a damaged CNS barrier. In the kainic acid induced excitotoxic lesion described above, neutrophils do not enter the brain in significant numbers or in the circumventricular organs that lack a BBB (Bolton and Perry, 1998). This discussion of the resistance of the CNS to the infiltration of immune cells, simply serves to highlight our failure to identify the molecular basis for the atypical inflammatory response in the brain and spinal cord. We also considered the possibility that signals, which are normally released from peripheral injury sites to activate leukocyte mobilisation and priming, may not be released from the CNS after injury. However, experiments designed to test this hypothesis revealed that not only was the acute phase response (APR) activated after the generation of focal inflammatory lesions in the brain, but it is just as rapid as the response to comparable peripheral challenges (if not greater). These observations led to the identification of chemokines as a new class of acute phase protein (Campbell et al., 2003, 2005). The hepatic response to CNS inflammation Inflammation, resulting from injury or infection, is predominantly a local phenomenon, but if the stimulus is sufficient, local inflammation is also accompanied by more far-reaching responses, which may include leukocytosis (elevated circulating leukocyte numbers), fever, and changes in the serum levels of many factors including electrolytes, metal ions, clotting agents, complement, and glucocorticoids. Collectively these changes are referred to as the acute phase response (APR) and the liver is the principal organ involved in coordinating the response (Baumann and Gauldie, 1994). The primary purpose of the acute phase protein production by the liver is considered to be to effect a return to homeostasis, by removing inflammatory stimuli, attenuating local inflammation, and promoting tissue repair and regeneration. Hepatic acute phase protein production is coordinated by a range of inflammatory mediators, including cytokines, glucocorticoids, and anaphylotoxins (Jensen and Whitehead, 1998; Koj, 1998; Szalai et al., 2000). It is widely accepted that the pro-inflammatory cytokines IL1β, IL-6, and TNF are critical mediators of APR initiation (Koj, 1996; Kushner, 1993). Despite this, an array searching for the key mediators that accompany the hepatic APR to CNS injury revealed that the changes in gene expression in the liver are more extensive than previously thought, and include more genes than just the “traditional” acute phase proteins (Campbell et al., 2007). In particular, the list of induced
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Fig. 1. The systemic response to CNS injury. Injury to the spinal cord triggers the induction of an acute phase response that includes marked hepatic CXC and CC chemokine induction. The release of hepatic chemokines results in leukocyte mobilization from the marrow. Chemokines are also expressed in other organs as a consequence of SCI. However, some of this production is likely to reflect an increase in leukocyte recruitment within the liver, lung and kidney in particular. As a consequence of this inappropriate recruitment markers of heptocellular damage can be detected and this is likely to contribute to multiorgan dysfunction syndrome (MODS) and systemic inflammatory response syndrome (SIRS), which are often features of CNS injury.
genes encoded chemokines, adhesion molecules, and genes that are not normally associated with the inflammatory response including lipocalin-2 and jun-B oncogene (Campbell et al., 2007c). It has also become clear that the production of acute phase proteins after CNS injury can have unexpected consequences resulting in systemic inflammatory response syndrome (SIRS), in which inflammatory cells from the circulation accumulate in organs such as the liver, lung and kidney, leading to organ damage (Bao et al., 2011). The spinal cord Many studies have sought to explore the contribution of the inflammatory response to the outcome of spinal cord injury and there is no doubt that contribution will be different from brain injury (Table 1). However, remarkably little attention has been paid to the manner in
which the injury signals are communicated to the rest of the body and the impact this has on the function of organs distant form the injury site. Injury to the spinal cord has been shown to generate a significant hepatic chemokine response and downstream hepatocellular injury (Campbell et al., 2005; Fleming et al., 2012). Interestingly, spinal cord injury (SCI)-induced damage to the liver appears to be dependent on the site of the injury; upper thoracic SCI is more effective than a lower thoracic SCI at inducing hepatic injury (Fleming et al., 2012). The recruitment of leukocytes to the liver after SCI is also very rapid (30 min) compared to the recruitment to the cord (Hundt et al., 2011). Thus, early intervention is likely to be important. Liver CCL-2 and CXCL-1 mRNA and protein are found to be elevated in the liver in association with leukocyte mobilization before any significant leucocyte recruitment to the cord occurs (Fig. 1). In humans, SCI is also associated
Table 1 A comparison of the inflammatory response to brain and spinal cord injury.
Anatomy Blood–brain barrier response Microglial numbers Microglial phenotype Neutrophil infiltration
Effects on mood Systemic APR Effect of systemic infection
Brain
Spinal cord
Grey matter surrounds white matter Very little breakdown after trauma or proinflammatory cytokine injection 20–150/mm2grey matter N white matter. Region specific differences in cell numbers, e.g. high numbers in hippocampus and substantia nigra Lower levels of CD45 and CDllb than microglia in the cord. Decreased local CXC chemokine production compared to cord. Very few neutrophils recruited to the parenchyma, but many to the meninges after trauma or a cytokine challenge. Following trauma N30% develop depression—usually but not exclusively dependent upon injury location Hepatic chemokines upregulated as rapidly as 1 h post-injury and NFkB activity at 30 min. Rapid neutrophil recruitment to the liver. Significantly impairs functional recovery, but sterile TLR4 agonists can be protective
White matter surrounds grey matter Significant breakdown after trauma or proinflammatory cytokine injection 250/mm2grey = white. No regional differences in numbers, but activation state differs with lesion location. Higher levels of CD45 and CDllb than microglia in the brain. Increased local CXC chemokine production compared to brain. Significant recruitment of neutrophils to the parenchyma Following injury 20–25% develop depression, but no link to injury location Neutrophil recruitment to the liver, and CXCL1 upregulated 2 h after a compression injury Significantly impairs functional recovery, but sterile TLR4 agonists can be protective
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with an increase in the oxidative activity of blood leukocytes from 6 h to 2 weeks after injury (Bao et al., 2009, 2011), but it is still unclear whether the number of liver leukocytes also increases in human SCI. Interestingly, we have discovered that neutrophil recruitment to the liver is a feature of multiple sclerosis and we suspect that the recruitment of leukocytes to distant organs is likely to be a feature of many CNS pathologies (Campbell et al., 2010). The brain Systemic cytokines also play important roles beyond coordinating the cellular response to a local injury. When an individual suffers from a bacterial or viral infection, the situation is often characterised by subjective feelings of ‘sickness’. These include malaise, lassitude, fatigue, numbness, coldness, muscle and joint aches, and reduced appetite. These symptoms can also be generated by chronic inflammatory disease or injury and are a consequence of systemic cytokine production. Originally considered trivial effects, co-ordinated behavioural response to injury or disease has become recognised as a highly organised strategy to alter behaviours to promote the fight against infection and facilitate repair (Dantzer, 2001). These typical behaviours may also play an altruistic role in signaling to others that an individual is sick and should be avoided. Given the nature of these behaviours, it is perhaps not surprising that the similarities between the ‘malaise’ associated with sickness has prompted comparison with the behaviours associated with major depression (De La Garza, 2005), a factor that has the capacity to confound recovery in a number of different CNS diseases. For example, post-stroke depression has been known for some time to be associated with a poorer outcome after injury (Sinyor et al., 1986), and recent studies have shown that early treatment with antidepressants such as fluoxetine has the capacity to significantly improve recovery (Chollet et al., 2011), but the cause or effect arguments require further investigation. Precisely how peripheral cytokine production can trigger these altered behaviours is still unclear. Both neural (vagal) and humoral routes have been proposed (Anthony et al., 2012) with work from our laboratory showing that systemic inflammation can profoundly affect the central serotonergic system by altering 5HT receptor gene expression (Couch et al., 2013a, 2013b). The humoral pathway argues that circulating cytokines such as IL-1β are detected by endothelial cells within the circumventricular organs and choroid plexus for example, and this triggers prostaglandin production and downstream central cytokine production (Dantzer, 2001; Jiang et al., 2008; Konsman et al., 2002). A neural route has also been demonstrated by cutting vagal afferents, which can eliminate the sickness behaviours, but only in certain circumstances (Konsman et al., 2000; Luheshi et al., 2000). The liver contains vagal sensory nerves, and inflammatory cytokines in hepatic cells may activate the vagus to carry signals back to the CNS (Borovikova et al., 2000); however, the literature is very contradictory in this area and studies are often confounded by experimental complications resulting from surgery. Systemic infection and disease resolution Despite the paucity of information regarding the way in which the brain and spinal cord communicate with the liver, there is a plethora of data showing that this communication system is vital for survival and any deviation can have severe consequences for the organism. Systemic infection has long been recognised as a risk factor in a range of conditions that affect the CNS (Anthony et al., 2012). It seems likely, that this is a consequence of the summation of injury signals in the liver, and other organs, from all the focal sites of injury throughout the body. Our experiments suggest that the relationship between the contributing factors may not be as stoichiometric as we previously envisaged. In some cases, summation of injury signals clearly leads to excessive amplification of the APR and increased recruitment of leukocytes to sites of CNS injury and, in particular, to the liver, where they cause hepatocellular damage (Campbell et al., 2005). However, at
other times peripheral inflammation seems to suppress leukocyte recruitment to the brain and spinal cord, which may be either as a consequence of the redirection of leukocytes or perhaps the generation of local ‘anti-inflammatory’ signals in an attempt to suppress the potentially damaging consequences of an excessive APR (Davis et al., 2005). Support for the negative impact of infection on acute CNS injury is highlighted in studies on stroke. In the human population, the prevalence of infection in the month preceding ischaemic stroke has been estimated to be at least 20% (Grau et al., 1995a). Acute periods of infection during the preceding month before a stroke (most often bacterial and affecting the respiratory system), are known to be risk factors for cerebral infarction (Syrjanen et al., 1988). Furthermore, patients with recent infection tend to present with a more severe neurological deficit than patients without infection (Grau et al., 1995b), factors of significance when considering acute inflammatory responses in the brain. The production and release of TNF as part of the hepatic APR in response to infection, in addition to its effects on leukocyte populations, also cause profound changes in the haemodynamic response of many vascular beds. We have shown that TNF, in contrast to its effects in peripheral vascular beds, causes an endothelin-1-dependent reduction in cerebral blood volume, which is likely to contribute to poor reperfusion after an ischaemic or traumatic injury to the CNS (Sibson et al., 2002). There are plenty of examples that demonstrate a deleterious effect of infection on the outcome of CNS injuries such as those incurred during stroke (Chamorro et al., 2007) and multiple sclerosis (Buljevac et al., 2002). However, while peripheral injury or infection is known to have an impact on the pathogenesis of chronic CNS disease, until recently it was much less clear whether the systemic response to brain or spinal cord injury per se would play a significant role in governing the outcome. It was also unclear whether the unusual pattern of inflammation in the CNS might result in an altered systemic inflammatory response to infection. For example, in an individual that is considered brain dead, this state is known to adversely affect both the quantity and quality of organs available for transplant, where the activation of proinflammatory pathways contributes to graft dysfunction in the recipient (de Vries et al., 2011). Targeting the immune system for CNS injury The discovery that there is a peripheral, hepatic component in addition to an inflammatory response in the CNS presented a new concept with which to approach the treatment of such conditions. History, reasonably, has dictated that anti-inflammatory therapy needs to be delivered to the site of the local inflammatory response and many therapeutic strategies have been abandoned due to poor CNS bioavailability. The inhibition of peripheral TNF after a central injection of IL-1β provides a very clear example of how local inflammation can be inhibited by targeting the systemic response. The TNF neutralising therapy etanercept does not cross the intact blood–brain barrier in any appreciable amount, but administration of etanercept following the microinjection of IL-1β into the brain inhibits the recruitment of leukocytes to the CNS (Campbell et al., 2007b). Conversely, the number of leukocytes entering the injured CNS can be increased by priming with systemic injections of chemokines, such as CINC-1, which are responsible for the mobilisation and recruitment of leukocytes (Campbell et al., 2005b). TNF is well known to induce the transcription factor NF-κB (Ali and Mann, 2004). NF-κB is a dimeric transcription factor that regulates the expression of a wide array of immune and inflammatory genes (Hayden and Ghosh, 2004). Gene expression array analysis has proven that inflammatory challenge to the brain induces genes regulated by NF-kB in the periphery (Campbell et al., 2007b). A study using transgenic mice that express a luciferase reporter under transcriptional regulation of NF-κB has shown conclusively that NF-κB activation occurs rapidly in the liver following inflammatory challenge to the brain (Campbell et al., 2008). Selective inhibition of NF-κB activity by intravenous adenoviral-mediated delivery of an IκBalpha super-repressor
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to the liver also markedly reduced the numbers of neutrophils recruited to the brain (Campbell et al., 2008). Taken together, these data illustrate that one of the functions of hepatic NF-κB is, unexpectedly, as a pivotal regulator of CNS inflammation. Downstream of NF-κB activity it has been shown that it is possible to manipulate the number of leukocytes recruited to the CNS after injury by manipulating the processes that lead to the systemic leukocytosis that follows (Campbell et al., 2003b, 2005b). Our laboratory has demonstrated that when chemokines are administered intravenously in conjunction with a CNS lesion, more leukocytes are mobilised and leukocyte recruitment to the brain is exacerbated (Campbell et al., 2005b), and, if the hepatic chemokine response is inhibited, leukocyte recruitment to the CNS after injury is suppressed. Thus, the manipulation of events distant from the site of injury can very effectively modify the recruitment of leukocytes to the CNS, and is an important consideration when peripheral infection is commonplace after acute injury to the CNS (McCormick et al., 2013). For example, in the spinal cord, targeting the systemic response has been shown to have some potential. Liposomes filled with clodronate have been used to deplete Kupffer cells, which appear to be the principal source of the APRs prior to spinal cord injury (Campbell et al., 2008). The clodronate treatment reduced Kupffer cells by 90% and subsequent production of the APR, as well as reducing the numbers of neutrophils and ED-1-positive cells in the spinal cord after contusion injury. While the discovery of the hepatic chemokine response to brain and spinal cord injury strongly supports the principal that any factors that exacerbate the APR are likely to have an adverse effect on CNS injury and disease, it is clear that the situation is rather more complicated. There is also growing evidence to suggest that systemic inflammation can confer a degree of tolerance to brain injury. A range of preconditioning strategies have shown that brief periods of ischaemia, periods of seizure, and anaesthetic exposure confer neuroprotection against a subsequent larger insult (Kapinya et al., 2002; Simon et al., 1993; Towfighi et al., 1999). In addition, the systemic administration of endotoxin has been reported to confer neuroprotection from ischaemia (Bordet et al., 2000; Rosenzweig et al., 2004). Whilst an interesting phenomenon, the clinical application of pre-conditioning may be challenging given the differences between the immune systems of preclinical model rodents and humans. For example, the amount of lipopolysaccharide required to induce ‘flu-like symptoms in rodents (around 2 μg total injection) will result in death in humans (Sauter and Wolfensberger, 1980) suggesting fundamental differences in basic immunology. Furthermore, the timing of conditioning challenges seems to be an important issue and one might predict that a post-conditioning challenge might exacerbate a CNS lesion. As discussed above there is evidence to suggest that lipopolysaccharide (LPS) will accentuate neuronal degeneration, such as in a model of murine prion disease (Combrinck et al., 2002), and also in a middle cerebral artery occlusion model of ischaemia (McColl et al., 2007). We were surprised to find that it is also possible to inhibit the recruitment of leukocytes after SCI using an intravenous injection of LPS before, and, importantly, after, the contusion injury. However, the response appears to be LPS-specific, as we could not achieve the same results with bolus injections of IL-1 (Davis et al., 2005), suggesting a regulatory role for TLR-type signalling. It has been shown that neutrophils in liver sinusoids can modulate the proinflammatory response of Kupffer cells to bacteria cleared from the bloodstream by suppressing cytokine and chemokine mRNA expression (Holub et al., 2009). Thus, the excessive accumulation of neutrophils in the liver following the LPS challenge may actually suppress downstream mobilization signals. However, these results are perhaps less surprising than we first thought. In the 1950s, Windle et al. discovered that systemic injection of Piromen, a crude pyrogen used to induce fever, augmented recovery of sensory function in spinalised cats (Windle and Chambers, 1950). Piromen is predominantly LPS. In the 1990s Guth extended these observations and studied the effects of
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injecting LPS intraperitoneally either alone or in combination with indomethacin and pregnenolone over an extended time period following a compression lesion (Guth et al., 1994b). While the best outcome was generated with a combination therapy. LPS alone reduced lesion cavitation and promoted axon/neurite growth into the lesion site. LPS and indomethacin also marginally improved recovery of motor function in a weight-drop model of spinal contusion injury (Guth et al., 1994a). It is not clear how these therapies affected the immediate histological outcomes and the role of the systemic response was not considered. More recently, another group sought to replicate the experiments of Guth and reported a more modest, though significant outcome (Popovich et al., 2012). It is our view that the use of this combination therapy is likely to generate complex local and systemic effects and that each component is likely to have its own optimal therapeutic window. Clearly, as with all attempts at combination therapy, the number of possible permutations can be overwhelming. There is no doubt that the capacity of pyrogens to modulate the hepatic response will mean that the ability of the liver to respond to systemic infection will have been significantly affected as part of these regimes. Whatever the mechanism, these results all highlight the importance of the nature of the systemic challenge on the outcome of a CNS injury. Summary While it is clear that the systemic immune system plays a significant and diverse role in the response to CNS injury, it is important to determine the precise mediators involved in this reaction before applying potentially harmful immune suppression therapy. By further studying the interactions between the hepatic APR and the CNS APR it may be possible to pinpoint molecular targets to prevent CNS inflammation, and subsequent neurological damage, being exacerbated by the peripheral immune system. References Ali, S., Mann, D.A., 2004. Signal transduction via the NF-kappaB pathway: a targeted treatment modality for infection, inflammation and repair. Cell Biochem. Funct. 22, 67–79. Andersson, P.B., Perry, V.H., Gordon, S., 1991a. The CNS acute inflammatory response to excitotoxic neuronal cell death. Immunol. Lett. 30, 177–182. Andersson, P.B., Perry, V.H., Gordon, S., 1991b. The kinetics and morphological characteristics of the macrophage-microglial response to kainic acid-induced neuronal degeneration. Neuroscience 42, 201–214. Andersson, P.B., Perry, V.H., Gordon, S., 1992. The acute inflammatory response to lipopolysaccharide in CNS parenchyma differs from that in other body tissues. Neuroscience 48, 169–186. Anthony, D.C., Couch, Y., Losey, P., Evans, M.C., 2012. The systemic response to brain injury and disease. Brain Behav. Immun. 26, 534–540. Bao, F., Bailey, C.S., Gurr, K.R., Bailey, S.I., Rosas-Arellano, M.P., Dekaban, G.A., Weaver, L.C., 2009. Increased oxidative activity in human blood neutrophils and monocytes after spinal cord injury. Exp. Neurol. 215, 308–316. Bao, F., Bailey, C.S., Gurr, K.R., Bailey, S.I., Rosas-Arellano, M.P., Brown, A., Dekaban, G.A., Weaver, L.C., 2011. Human spinal cord injury causes specific increases in surface expression of beta integrins on leukocytes. J. Neurotrauma 28, 269–280. Baumann, H., Gauldie, J., 1994. The acute-phase response. Immunol. Today 15, 74–80. Bell, M.D., Perry, V.H., 1995. Adhesion molecule expression on murine cerebral endothelium following the injection of a proinflammagen or during acute neuronal degeneration. J. Neurocytol. 24, 695–710. Bernardes-Silva, M., Anthony, D.C., Issekutz, A.C., Perry, V.H., 2001. Recruitment of neutrophils across the blood–brain barrier: the role of E- and P-selectins. J. Cereb. Blood Flow Metab. 21, 1115–1124. Blond, D., Campbell, S.J., Butchart, A.G., Perry, V.H., Anthony, D.C., 2002. Differential induction of interleukin-1beta and tumour necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain. Brain Res. 958, 89–99. Bolton, S.J., Perry, V.H., 1998. Differential blood–brain barrier breakdown and leucocyte recruitment following excitotoxic lesions in juvenile and adult rats. Exp. Neurol. 154, 231–240. Bordet, R., Deplanque, D., Maboudou, P., Puisieux, F., Pu, Q., Robin, E., Martin, A., Bastide, M., Leys, D., Lhermitte, M., Dupuis, B., 2000. Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance. J. Cereb. Blood Flow Metab. 20, 1190–1196. Borovikova, L.V., Ivanova, S., Zhang, M., Yang, H., Botchkina, G.I., Watkins, L.R., Wang, H., Abumrad, N., Eaton, J.W., Tracey, K.J., 2000. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405, 458–462.
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