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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 22, 2003 as doi:10.1096/fj.02-0757fje. |
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* Molecular Neuropathology Laboratory, School of Biological Sciences, University of Southampton, Southampton, S016 7PX, UK;
# CNS Inflammation Group, School of Biological Sciences, University of Southampton, Southampton, S016 7PX, UK; and
Tissue Remodelling and Repair, Hepatology, School of Medicine, University of Southampton, Southampton SO16 6YD, UK
2Correspondence: Molecular Neuropathology Laboratory, School of Biological Sciences, University of Southampton, Biomedical Sciences Building, Southampton S016 7PX, UK. E-mail: s.j.campbell{at}soton.ac.uk
SPECIFIC AIMS
We sought to identify mediators that could account for the rapid systemic mobilization of leukocytes observed after acute brain injury, which cannot be accounted for by induction of the classical acute-phase proteins (APP). We show that acute hepatic chemokine production is responsible for amplifying the brain injury response and results in mobilization of neutrophils to the blood and recruitment of neutrophils to the brain, liver, and to secondary liver damage, which, if paralleled in humans, may contribute to the multiorgan dysfunction syndrome seen after acute brain injury.
PRINCIPAL FINDINGS
1. CINC-1 is a neutrophil chemoattractant that we have established has all the characteristics of an early acute-phase protein
Working with a well-established rat model, we injected the classical inducers of the acute-phase response-endotoxin (LPS), interleukin-1ß (IL-1ß), or interleukin-6 (IL-6) intraperitoneally (i.p.)—and analyzed hepatic and serum levels of CINC-1 and CINC-3 by Taqman RT-PCR and ELISA. There were profound elevations of hepatic CINC-1 and CINC-3 mRNA 2 h after i.p. injection of LPS, IL-1ß, or IL-6 compared with vehicle controls. The i.p. injection of LPS, IL-1ß, or IL-6 also resulted in the rapid appearance of the CINC-1 protein in serum at levels well above controls. CINC-3 protein was present in the serum at 2 h after injection of LPS or IL-1ß, but not IL-6. The acute and transient increases in expression levels of hepatic and serum CINC-1 and CINC-3 in response to proinflammatory challenges are consistent with characteristics of an APP.
2. Levels of hepatic and serum CINC-1 are proportional to the severity of tissue injury
We examined hepatic CINC expression and serum CINC levels after injuries of increasing severity. We found that hepatic CINC-1 mRNA and serum CINC-1 protein were up-regulated in proportion to the degree of tissue damage. CINC-3 protein was not detected in the serum of any of the experimental groups when investigating the injury response. Thus CINC-1, but not CINC-3, satisfies the criteria of an APP.
3. CINC-1 is rapidly elevated in the liver after focal injury to the brain
Previous studies from our laboratory have demonstrated delayed induction of APP in the striatum and liver after a focal proinflammatory challenge to the mouse brain. When we microinjected IL-1ß into the rat striatum using a minimally invasive atraumatic technique, we discovered that hepatic CINC-1 and CINC-3 mRNA and serum CINC-1 were significantly increased in a dose-dependent manner 2 h after injection of IL-1ß into the striatum (Fig. 1
a–c). Despite elevations in hepatic CINC-3 mRNA after intrastriatal injections of IL-1ß, we observed only low levels of CINC-3 protein in the serum (with high-dose IL-1ß). Injection of IL-1ß into the striatum induces significant local production of CINC-3 in the brain, but this protein does not enter the circulation.
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4. The liver produces CINC-1 protein after focal brain injury
To determine whether the time course of elevated serum CINC levels was coincident with elevated hepatic levels and thus identify the liver as a source of the serum CINC, we assayed CINC protein levels in the liver by ELISA (Fig. 1d
). We could not detect CINC-1 protein in the liver of naive rats or in the liver 2 or 6 h after injection of saline into the striatum, but we observed elevated hepatic CINC-1 protein levels after 2 and 6 h in a dose-dependent manner in rats injected with either 1 ng IL-1ß or 1 µg IL-1ß into the striatum. CINC-3 was not detected in the liver after focal injection of IL-1ß at either dose or at any time-point.
5. IL-1ß injection in the brain mobilizes neutrophils into the circulation
IL-1ß injected into the striatum gave rise to a significant increase in the number of neutrophils into the circulation. The increase was observed as early as 2 h after the intracerebral challenge.
6. Neutrophil recruitment to the liver is observed after focal brain injury and is associated with increased levels of serum transaminases
We used immunohistochemistry to determine whether the presence of hepatic CINC-1 protein in the liver was associated with neutrophil recruitment. Neutrophils were recruited to the liver in a dose-dependent manner from 2 h after microinjection of IL-1ß into the brain, before the appearance of neutrophils in the brain. Recruitment of neutrophils to the liver in models of endotoxic shock is associated with increased levels of the serum transaminases. We therefore examined whether recruitment of neutrophils to the liver after intrastriatal injection of IL-1ß could increase the levels of serum transaminases. Levels of aspartate transaminase (AST) and alanine transaminase (ALT) in the serum were significantly elevated after the intrastriatal injection of IL-1ß, but the level of alkaline phosphatase was unchanged.
7. Neutrophil recruitment to the brain is delayed
We had found earlier that after injection of IL-1ß into the striatum, neutrophils are recruited to the brain parenchyma only after a delay. Again, we found that 1 ng of IL-1ß failed to recruit neutrophils to the brain parenchyma by 4 h despite marked, though unilateral, recruitment to the meninges and liver. We increased the dose of IL-1ß to 1 µg to determine whether the intrinsic resistance of the brain could be overcome. The 1 µg dose also failed to elicit neutrophil recruitment to the brain parenchyma after 4 h despite the presence of bilateral meningitis and increased neutrophil recruitment to the liver. Six hours after the injection of 1 µg IL-1ß, neutrophil recruitment was more marked in the meninges; neutrophils were now present in the superficial layers of the cortex and striatum around the injection site.
8. Communication from the brain to the liver
We analyzed serum levels of the proinflammatory cytokines IL-1ß, tumor necrosis factor 
(TNF-), and IL-6 by ELISA after injection of 1 ng or 1 µg IL-1ß into the striatum to investigate whether the communication from the brain to the liver is via cytokines in the blood. However, none of these proinflammatory cytokines were detectable in serum by ELISA.
9. Blocking the functional activity of CINC-1 with an i.v. injection of purified neutralizing anti-CINC-1 IgGs significantly reduced neutrophil mobilization and recruitment to the brain and liver
Six hours after i.v. administration of the anti-CINC-1 and microinjection of 1 µg IL-1ß into the striatum, the number of neutrophils recruited to the liver, meninges, and superficial layers of the cortex and striatum was significantly reduced compared with the number recruited after an equivalent injection of IL-1ß and nonimmune goat anti-human IgG. Anti-CINC-1 was effective in reducing the number of neutrophils present in circulating blood after the injection of 1 µg IL-1ß into the striatum (Fig. 2d
),
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CONCLUSIONS AND SIGNIFICANCE
In addition to its established role as a neutrophil chemoattractant, CINC-1 functions as an early APP. Indeed, CINC-1 is produced by the liver well before most other classical APP. Injury increases hepatic CINC-1 synthesis and secretion that leads to raised circulatory CINC-1 levels and neutrophil mobilization to the blood, the magnitude of which is proportional to the degree of initial injury. Thus, a significant component of the hepatic APR after central nervous system (CNS) trauma is the release of chemokines by the liver, which acts to amplify the inflammatory response and modulate the subsequent leukocytosis and secondary tissue damage (Fig. 2
). Hepatic chemokine synthesis after acute brain injury presents a novel focus for the treatment of inflammation. Injury to the brain elicits a peripheral response that is likely to represent a useful target especially attractive for novel anti-inflammatory agents that do not cross the blood–brain barrier.
We were unable to detect increased levels of IL-1ß, IL-6, or TNF- in the blood after focal microinjection of IL-1ß into the brain. Thus, if there is release of cytokine into the circulation, it is insufficient to induce detectable serum levels of the principal proinflammatory cytokines. Neural mechanisms may be operating via vagal efferents to control peripheral cytokine and, indirectly, chemokine synthesis. Parasympathetic pathways have been described showing that the vagal release of acetylcholine can modulate the release of cytokines in the liver. In the present study, we discovered that the amount of CINC-1 mRNA measured after focal microinjection of IL-1ß into the brain parenchyma is 10-fold that measured after the injection of an equivalent quantity into the periphery, suggesting that central IL-1ß induces and amplifies the hepatic CINC signal.
A role for chemokines in the release of cells from bone marrow into the circulation is not without precedent. Circulating IL-5/eotaxin, MCP-1, and IL-8 have been implicated in the mobilization of leukocytes, but this activity was not linked to hepatic chemokine synthesis or the APR. We show that hepatic CINC-1 appears to be operating as a systemic amplifier of the local inflammatory response, with levels of neutrophil mobilization and subsequent neutrophil recruitment to the brain and liver being proportional to the hepatic and circulating CINC-1 levels. A neutralizing anti-CINC-1 antibody was sufficient to reduce neutrophil mobilization and neutrophil recruitment to tissues distant from the initial injury site. In patients with acute brain injury, levels of cytokines and neutrophils are elevated in the blood and are thought to play a role in the pathology of injury that often occurs in organs distant from the brain, and may be a factor determining clinical outcome. AST and ALT levels were significantly elevated and indicate there is hepatocellular injury associated with appearance of neutrophils in the liver after the intrastriatal injection of IL-1ß.
The current hypothesis of neutrophil recruitment describes local production of chemokine at the site of injury, establishing a gradient that attracts circulating leukocytes. When we consider that circulating neutrophils are exposed to CINC-1 in the blood before local chemokine gradients are established, that hypothesis is seen as a somewhat simplified scenario. CINC-1 expression peaks within the first 2 h after injury before falling dramatically to basal levels. We suggest that the acute hepatic production of chemokine is necessary to allow neutrophil mobilization. However, the levels must fall quickly to allow circulating neutrophils to detect increases in local chemokine concentrations. This concept is supported by transgenic studies showing that prolonged exposure of leukocytes to circulating chemokine results in desensitization of the chemokine receptors. Our data provide pivotal and novel evidence that acute brain injury induces a rapid and robust systemic innate immune response.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0757fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0757fje 
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