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. 2011 Sep 26:8:124.
doi: 10.1186/1742-2094-8-124.

Lipocalin 2 in the central nervous system host response to systemic lipopolysaccharide administration

Jacque P K Ip  1 , Aline L Noçon, Markus J Hofer, Sue Ling Lim, Marcus Müller, Iain L Campbell
Affiliations

Lipocalin 2 in the central nervous system host response to systemic lipopolysaccharide administration

Jacque P K Ip et al. J Neuroinflammation. .

Abstract

Background: Lipocalin 2 (Lcn2) is a bacteriostatic factor that may also modulate cellular function, however, little is known concerning the expression or role of Lcn2 in CNS inflammation. Therefore, here we investigated the regulation and possible function of Lcn2 in the CNS following peripheral lipopolysaccharide (LPS) injection in mice.

Methods: A murine model for systemic endotoxemia was used in this study. Wild type or Lcn2 KO mice (both genotypes C57BL/6 strain) were given either a single or dual, staggered intraperitoneal injections of purified E. coli LPS or vehicle alone. The brain was examined for the expression and location of Lcn2 mRNA and protein and various markers for neuroinflammation were analyzed.

Results: Although undetectable under physiological conditions, both Lcn2 mRNA and protein were induced to high levels in the brain after LPS injection. By contrast, RNA corresponding to the putative Lcn2 (termed 24p3R) receptor was present at high levels in the normal brain and remained unaltered by LPS injection. Differences between Lcn2 and 24p3R mRNA expression were found at the anatomic and cellular level. Endothelial cells, microglia and the choroid plexus but not neurons were identified as the main cellular sources for Lcn2 mRNA in the CNS. By contrast, 24p3R mRNA was detected in neurons and the choroid plexus only. Lcn2 protein was found to have a similar cellular localization as the corresponding RNA transcripts with the exception that subsets of neurons were also strongly positive. Various inflammatory, glial, and iron handling markers were analyzed and found to have similar alterations between WT and Lcn2 KO animals.

Conclusions: 1) Lcn2 production is strongly induced in the CNS by systemic LPS injection, 2) in addition to Lcn2 production at key gateways of bacterial entry to the CNS, neurons may be a target for the actions of Lcn2, which is apparently taken up by these cells, and 3) the cellular functions of Lcn2 in the CNS remain enigmatic.

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Figures

Figure 1
Figure 1
Lcn2 and 24p3R mRNA levels in the brain and liver following intraperitoneal injection of LPS. Mice were injected i.p. with sterile 0.15 M NaCl with or without LPS (50 μg) and euthanized after 4 h or 24 h. An extra group of mice received a second LPS injection (dual) 16 h later, and were euthanized 4 h after the second injection. Total RNA was extracted from the brain (A) and the liver (B) and 5 μg analyzed by RPA as described in the Materials and Methods. Quantification of the Lcn2 and 24p3R RNA levels by densitometry (C). For statistical analysis an unpaired, two-tailed t-test was used. For statistical significance * p < 0.05 for LPS- versus vehicle-treated control mice (p < 0.05).
Figure 2
Figure 2
Lcn2 protein levels in the brain following intraperitoneal injection of LPS. Mice were treated as shown. Total protein was extracted from the brain and 50 μg analyzed by Western blot (A) as described in the Materials and Methods with quantification (B) of the Lcn2 protein relative to GAPDH loading control. Comparison of Lcn2 protein level by Western blot (C) in brain from WT versus Lcn2 KO mice after dual LPS-treated mice. For statistical significance *, p < 0.05 for LPS-treated mice versus control mice (p ≤ 0.05).
Figure 3
Figure 3
Anatomical localization of Lcn2 and 24p3R RNA in the brain following intraperitoneal injection of LPS. Brain was removed from mice at different times following LPS injection, fixed in ice-cold 4% PBS-buffered paraformaldehyde and paraffin-embedded sagittal sections (10 μm) subjected to ISH as described in the Materials and Methods. Hybridization was performed with either 24p3R (A&B) and Lcn2 (D&E) anti-sense or 24pR3 (C) and Lcn2 (F) sense cRNA probes.
Figure 4
Figure 4
Cellular localization of Lcn2 and 24p3R RNA in the brain following intraperitoneal injection of LPS. Paraffin-embedded sagittal sections (10 μm) were subjected to ISH followed by immunohistochemistry as described in the Materials and Methods. A. Brain section from vehicle-injected control mouse hybridized with Lcn2 anti-sense cRNA probe and immunostained for NeuN. B-D. Brain sections from dual LPS-treated mice hybridized with Lcn2 anti-sense cRNA probe and immunostained for NeuN (B) and lectin (C&D). E-F. Brain sections from vehicle-injected control mice hybridised with 24p3R anti-sense cRNA probe and immunostained for NeuN (E) or lectin (F).
Figure 5
Figure 5
Cellular localization of Lcn2 protein in the brain following intraperitoneal injection of LPS. Paraffin embedded sections (5 μm) were stained by immunohistochemistry to detect Lcn2 protein as described in the Materials and Methods. A. Brain section from a PBS-injected control mouse. B-F. Brain sections from mice at 24 h following a single intraperitoneal injection of LPS.
Figure 6
Figure 6
Regulation of Lcn2 mRNA levels in C8 microglial cells. The C8 microglial cell line was grown to 75% confluency and treated for 4 h at 37°C as indicted. Total RNA was isolated and analyzed by RPA as described in the Materials and Methods. For statistical analysis one-way ANOVA with Dunnett's multiple comparison test was used. For statistical significance * p < 0.05 versus untreated control (Con).
Figure 7
Figure 7
The level of various host response, chemokine and cytokine mRNA transcripts in the brain of WT versus Lcn2 KO mice following intraperitoneal injection of LPS. Mice were injected i.p. with sterile PBS with or without LPS and received a second LPS injection (dual) 16 h later and euthanized 4 h after the second injection. Total RNA was extracted from the brain and 10 μg analyzed by RPA as described in the Materials and Methods. For quantification of the mRNA levels, densitometry was performed on film autoradiographs and corrected for loading by reference to the L32 loading control. For statistical analysis, one-way ANOVA with Tukey's multiple comparison test was used. For statistical significance *, p < 0.05 for LPS-treated mice versus vehicle-injected control mice (p ≤ 0.05).
Figure 8
Figure 8
Comparison of the astrocyte and microglial response in the brain of WT versus Lcn2 KO mice following intraperitoneal injection of LPS. Mice were injected i.p. with sterile PBS with or without LPS and received a second LPS injection (dual) 16 h later, and were euthanized 4 h after the second injection. Total protein was extracted from the brain and 30 μg analyzed by Western blot (A) and quantified (B) as described in the Materials and Methods. For immunohistochemistry (C) mice were perfused intracardially with ice-cold 4% PBS-buffered paraformaldehyde the brains removed and immunohistochemistry performed for GFAP or IBA-1 as described in the Materials and Methods.
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