Sandra J. Campbell

Reversible Demyelination, Blood-Brain Barrier Breakdown, and Pronounced Neutrophil Recruitment Induced by Chronic IL-1 Expression in the Brain

Interleukin-1beta (IL-1) expression is associated with a spectrum of neuroinflammatory processes related to chronic neurodegenerative diseases. The single-bolus microinjection of IL-1 into the central nervous system (CNS) parenchyma gives rise to delayed and localized neutrophil recruitment, transient blood-brain barrier (BBB) breakdown, but no overt damage to CNS integrity. However, acute microinjections of IL-1 do not mimic the chronic IL-1 expression, which is a feature of many CNS diseases. To investigate the response of the CNS to chronic IL-1 expression, we injected a recombinant adenovirus expressing IL-1 into the striatum. At the peak of IL-1 expression (days 8 and 14 post-injection), there was a marked recruitment of neutrophils, vasodilatation, and breakdown of the BBB. Microglia and astrocyte activation was evident during the first 14 days post-injection. At days 8 and 14, extensive demyelination was observed but the number of neurons was not affected by any treatment. Finally, at 30 days, signs of inflammation were no longer present, there was evidence of tissue reorganization, the BBB was intact, and the process of remyelination was noticeable. In summary, our data show that chronic expression of IL-1, in contrast to its acute delivery, can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases.

Central Nervous System Injury Triggers Hepatic CC and CXC Chemokine Expression that Is Associated with Leukocyte Mobilization and Recruitment to Both the Central Nervous System and the Liver

The administration of interleukin-1beta to the brain induces hepatic CXC chemokine synthesis, which increases neutrophil levels in the blood, liver, and brain. We now show that such hepatic response is not restricted to the CXC chemokines. CCL-2, a CC chemokine, was released by the liver in response to a tumor necrosis factor (TNF)-alpha challenge to the brain and boosted monocyte levels. Furthermore, a clinically relevant compression injury to the spinal cord triggered hepatic chemokine expression of both types. After a spinal cord injury, elevated CCL-2 and CXCL-1 mRNA and protein were observed in the liver by TaqMan reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay as early as 2 to 4 hours. Simultaneously, we observed elevated levels of these chemokines and circulating leukocyte populations in the blood. Leukocytes were recruited to the liver at this early stage, whereas at the site of challenge in the central nervous system, few were observed until 24 hours. Artificial elevation of blood CCL-2 triggered dose-dependent monocyte mobilization in the blood and enhanced monocyte recruitment to the brain after TNF-alpha challenge. Attenuation of hepatic CCL-2 production with corticosteroids resulted in reduced monocyte levels after the TNF-alpha challenge. Thus, combined production of CC and CXC hepatic chemokines appears to amplify the central nervous system response to injury.

CINC-1 is an acute-phase protein induced by focal brain injury causing leukocyte mobilization and liver injury.

Following injury or infection, the liver releases acute‐phase proteins (APP). After a severe focal injury, this systemic response can be excessive and may lead to multiorgan dysfunction (MODS). CINC‐1 is a neutrophil chemoattractant, and we have now established that it also functions as an early APP after injury to the brain or to peripheral tissues. After induction of a focal inflammatory lesion in the brain, there is rapid hepatic and serum CINC‐1 induction, which is associated with increases in neutrophil numbers within the liver and within the circulation. CINC‐1‐mediated recruitment of neutrophils to organs distant from the primary injury site may contribute to MODS. Indeed, we found that enzyme markers of liver tissue injury are increased in the serum following generation of a focal inflammatory lesion in the brain. Neutralization of CINC‐1 in the periphery reversed brain‐injury‐induced neutrophil mobilization and inhibited recruitment of neutrophils to the brain and to the liver. Thus, a significant component of the hepatic acute‐phase response is the release of chemokines by the liver, which act to amplify the inflammatory response and modulate the subsequent leukocytosis and secondary tissue damage. Hepatic CINC‐1 synthesis following injury presents a novel focus for treatment of inflammation.

A contrast agent recognizing activated platelets reveals murine cerebral malaria pathology undetectable by conventional MRI

Human and murine cerebral malaria are associated with elevated levels of cytokines in the brain and adherence of platelets to the microvasculature. Here we demonstrated that the accumulation of platelets in the brain microvasculature can be detected with MRI, using what we believe to be a novel contrast agent, at a time when the pathology is undetectable by conventional MRI. Ligand-induced binding sites (LIBS) on activated platelet glycoprotein IIb/IIIa receptors were detected in the brains of malaria-infected mice 6 days after inoculation with Plasmodium berghei using microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody specific for the LIBS (LIBS-MPIO). No binding of the LIBS-MPIO contrast agent was detected in uninfected animals. A combination of LIBS-MPIO MRI, confocal microscopy, and transmission electron microscopy revealed that the proinflammatory cytokine TNF-alpha, but not IL-1beta or lymphotoxin-alpha (LT-alpha), induced adherence of platelets to cerebrovascular endothelium. Peak platelet adhesion was found 12 h after TNF-alpha injection and was readily detected with LIBS-MPIO contrast-enhanced MRI. Temporal studies revealed that the level of MPIO-induced contrast was proportional to the number of platelets bound. Thus, the LIBS-MPIO contrast agent enabled noninvasive detection of otherwise undetectable cerebral pathology by in vivo MRI before the appearance of clinical disease, highlighting the potential of targeted contrast agents for diagnostic, mechanistic, and therapeutic studies.

The systemic and local acute phase response following acute brain injury.

It is not known whether acute brain injury results in a systemic acute phase response (APR) or whether an APR influences outcome after an insult to the CNS. The present study sought to establish whether brain injury elicits a systemic or local APR. The expression of acute phase protein (APP) mRNA in liver and brain tissues was measured by Taqman reverse transcriptase-polymerase chain reaction after an excitotoxic lesion in the striatum or challenge with a proinflammatory cytokine. N-methyl-D-aspartate (NMDA)–induced brain lesion did not elicit a systemic APR. In contrast, proinflammatory challenge with mouse recombinant interleukin-1β (mrIL-1β) resulted in a significant hepatic APP mRNA expression within 6 hours. Thus, an inflammatory challenge that results in a meningitis leads to a hepatic APR, whereas acute brain injury alone, with no evidence of a meningitis, does not produce an APR. This is surprising because NMDA leads to an increase in endogenous IL-1β synthesis. This suggests that the brain has an endogenous antiinflammatory mechanism, which protects against the spread of inflammation after an acute injury. In the brain, both excitotoxic lesions and proinflammatory challenge resulted in a profound parenchymal upregulation of APP mRNA after 6 and 12 hours in the injected hemisphere. These results suggest that the local APR may play a role as an antiinflammatory mechanism. These findings indicate a potentially pivotal role for peripheral and local APP production on outcome after brain injury.

Hdm2 Recruits a Hypoxia-Sensitive Corepressor to Negatively Regulate p53-Dependent Transcription

The transcription factor p53 lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. p53 is inactive in proliferating cells, largely because of negative regulation by the Hdm2/Mdm2 oncoprotein, with which it physically associates. Release from this negative regulation is sufficient to activate p53 and can be triggered in cells by multiple stimuli through diverse pathways. This diversity is achieved in part because Hdm2 uses multiple mechanisms to inactivate p53; it targets p53 for ubiquitination and degradation by the proteosome, shuttles it out of the nucleus and into the cytoplasm, prevents its interaction with transcriptional coactivators, and contains an intrinsic transcriptional repressor activity. Here we show that Hdm2 can also repress p53 activity through the recruitment of a known transcriptional corepressor, hCtBP2. This interaction, and consequent repression of p53-dependent transcription, is relieved under hypoxia or hypoxia-mimicking conditions that are known to increase levels of intracellular NADH. CtBP proteins can undergo an NADH-induced conformational change, which we show here results in a loss of their Hdm2 binding ability. This pathway represents a novel mechanism whereby p53 activity can be induced by cellular stress.

Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment.

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT–PCR and enzyme‐liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin‐1β (IL‐1β) injection resulted in up‐regulated chemokine expression in both brain and spinal cord. CINC‐3 mRNA was elevated above CINC‐1 and CINC‐2α, with expression levels for each higher in spinal cord than in brain. By ELISA, IL‐1β induced greater CINC‐1 and CINC‐2α expression compared to CINC‐3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC‐2α protein were observed in response to IL‐1β injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC‐2β mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.

Differential induction of interleukin-1beta and tumour necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain.

Abstract In peripheral tissue, IL-1β has been shown to induce TNFα expression and vice versa, resulting in mixed neutrophil and mononuclear cell recruitment to the site of injury. This has led to the concept of crosstalk in peripheral cytokine signalling pathways. In the brain parenchyma, however, restricted patterns of leukocyte recruitment following the focal injection of pro-inflammatory agents into the brain are observed. This study investigates the expression of the principal pro_inflammatory cytokines—IL-1β and TNFα—in the brain after IL-1β, TNFα, NMDA or endotoxin injection into the brain parenchyma of rats. Each of these agents gives rise to a distinct pattern of acute leukocyte recruitment at 24 h. We found that IL-1β induces de novo synthesis of additional IL-1β but not TNFα, as determined by RT-PCR and ELISA, and TNFα does not induce either itself or IL-1β. Injection of NMDA results in IL-1β, but not TNFα up-regulation. Injection of IL-1β or NMDA is associated with neutrophil recruitment whereas injection of TNFα is associated with mononuclear cell recruitment. Following injection of endotoxin, both TNFα and IL-1β levels are elevated and neutrophils and mononuclear cells are recruited to the brain. These data suggest that the signalling pathways that are present in the periphery are modified in the brain and that differential induction of TNFα and IL-1β may have a role in the atypical pattern of leukocyte recruitment observed in the brain.

VCAM-1-targeted magnetic resonance imaging reveals subclinical disease in a mouse model of multiple sclerosis

Diagnosis of multiple sclerosis (MS) currently requires lesion identification by gadolinium (Gd)‐enhanced or T2‐weighted magnetic resonance imaging (MRI). However, these methods only identify late‐stage pathology associated with blood‐brain barrier breakdown. There is a growing belief that more widespread, but currently undetectable, pathology is present in the MS brain. We have previously demonstrated that an anti‐VCAM‐1 antibody conjugated to microparticles of iron oxide (VCAM‐MPIO) enables in vivo detection of VCAM‐1 by MRI. Here, in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we have shown that presymptomatic lesions can be quantified using VCAM‐MPIO when they are undetecTABLE by Gd‐enhancing MRI. Moreover, in symptomatic animals VCAM‐MPIO binding was present in all regions showing Gd‐DTPA enhancement and also in areas of no Gd‐DTPA enhancement, which were confirmed histologically to be regions of leukocyte infiltration. VCAM‐MPIO binding correlated significantly with increasing disability. Negligible MPIO‐induced contrast was found in either EAE animals injected with an equivalent nontargeted contrast agent (IgG‐MPIO) or in control animals injected with the VCAM‐MPIO. These findings describe a highly sensitive molecular imaging tool that may enable detection of currently invisible pathology in MS, thus accelerating diagnosis, guiding treatment, and enabling quantitative disease assessment.—Serres, S., Mardiguian, S., Campbell, S. J., McAteer, M. A., Akhtar, A., Krapitchev, A., Choudhury, R. P., Anthony, D. C., Sibson, N. R. VCAM‐1‐targeted magnetic resonance imaging reveals subclinical disease in a mouse model of multiple sclerosis. FASEB J. 25, 4415–4422 (2011). www.fasebj.org

Overexpression of IL-1beta by adenoviral-mediated gene transfer in the rat brain causes a prolonged hepatic chemokine response, axonal injury and the suppression of spontaneous behaviour.

Acute brain injury induces early and transient hepatic expression of chemokines, which amplify the injury response and give rise to movement of leukocytes into the blood and subsequently the brain and liver. Here, we sought to determine whether an ongoing injury stimulus within the brain would continue to drive the hepatic chemokine response and how it impacts on behaviour and CNS integrity. We generated chronic IL-1beta expression in rat brain by adenoviral-mediated gene transfer, which resulted in chronic leukocyte recruitment, axonal injury and prolonged depression of spontaneous behaviour. IL-1beta could not be detected in circulating blood, but a chronic systemic response was established, including extended production of hepatic and circulating chemokines, leukocytosis, liver damage, weight loss, decreased serum albumin and marked liver leukocyte recruitment. Thus, hepatic chemokine synthesis is a feature of active chronic CNS disease and provides an accessible target for the suppression of CNS inflammation.