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Cited by 244Neuroinflammation and Neurodegeneration MechanismsBarrier Structure and Function StudiesTryptophan and brain disorders

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Chronic Interleukin-1β Expression in Mouse Brain Leads to Leukocyte Infiltration and Neutrophil-Independent Blood–Brain Barrier Permeability without Overt Neurodegeneration

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Shaftel, Solomon S., Carlson, Thaddeus J., Olschowka, John A., Kyrkanides, Stephanos, Matousek, Sarah B., O'Banion, M. Kerry·2007·The Journal of Neuroscience
DOI 10.1523/jneurosci.1418-07.2007PMC PMC6673122
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Abstract

The proinflammatory cytokine interleukin-1beta (IL-1beta) plays a significant role in leukocyte recruitment to the CNS. Although acute effects of IL-1beta signaling in the mouse brain have been well described, studies elucidating the downstream effects of sustained upregulation have been lacking. Using the recently described IL-1beta(XAT) transgenic mouse model, we triggered sustained unilateral hippocampal overexpression of IL-1beta. Transgene induction led to blood-brain barrier leakage, induction of MCP-1 (monocyte chemoattractant protein 1) (CCL2), ICAM-1 (intercellular adhesion molecule 1), and dramatic infiltration of CD45-positive leukocytes comprised of neutrophils, T-cells, macrophages, and dendritic cells. Despite prolonged cellular infiltration of the hippocampus, there was no evidence of neuronal degeneration. Surprisingly, neutrophils were observed in the hippocampal parenchyma as late as 1 year after transgene induction. Their presence was coincident with upregulation of the potent neutrophil chemotactic chemokines KC (keratinocyte-derived chemokine) (CXCL1) and MIP-2 (macrophage inflammatory protein 2) (CXCL2). Knock-out of their sole receptor CXCR2 abrogated neutrophil infiltration but failed to reduce leakage of the blood-brain barrier.

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Figures

Figure1 Activation of a transcriptionally silent hIL-1 𝛃 transgenewithinthehippocampusdrivesleukocyterecruitment.Adult IL-1 𝛃 XAT mice received intrahippocampal injections of either FIV-Cre or FIV-GFP control virus unilaterally. A , Structure of the dormantIL-1 𝛃 XAT transgene and subsequent conversion to an activated state after exposure to FIV-Cre. B , A coronal section from an IL-1 𝛃 XAT line B/b mouse 2 weeks after FIV-Cre injection (black arrow) into the dentate gyrus demonstrates spatially restricted CD45 staining in the ipsilateral (right) hemisphere. C , CD45 staining of leukocytes was performed in parallel groups of animals within the ipsilateral dentate gyrus represented by the white box in B . Representative images were captured from WT, IL-1 𝛃 XAT line A/a (A/a), IL-1 𝛃 XAT line B/b (B/b), and IL-1 𝛃 XAT line B/b animals lacking IL-1R1 (B/b, IL-1R1 -/ -) injected with FIV encoding the protein designated in parentheses. Background CD45 staining reflects low-affinity binding to microglia within the hippocampus (scale bar, 10 𝛍 m). D , Hippocampal BBB leakage, as evidenced by Evans Blue concentration, in IL-1 𝛃 XAT line B/b versus WT animals 2 weeks after transgene activation. Graph represents mean ± SEM. n = 3 animals per group. * p ≤ 0.05.
Figure1 Activation of a transcriptionally silent hIL-1 𝛃 transgenewithinthehippocampusdrivesleukocyterecruitment.Adult IL-1 𝛃 XAT mice received intrahippocampal injections of either FIV-Cre or FIV-GFP control virus unilaterally. A , Structure of the dormantIL-1 𝛃 XAT transgene and subsequent conversion to an activated state after exposure to FIV-Cre. B , A coronal section from an IL-1 𝛃 XAT line B/b mouse 2 weeks after FIV-Cre injection (black arrow) into the dentate gyrus demonstrates spatially restricted CD45 staining in the ipsilateral (right) hemisphere. C , CD45 staining of leukocytes was performed in parallel groups of animals within the ipsilateral dentate gyrus represented by the white box in B . Representative images were captured from WT, IL-1 𝛃 XAT line A/a (A/a), IL-1 𝛃 XAT line B/b (B/b), and IL-1 𝛃 XAT line B/b animals lacking IL-1R1 (B/b, IL-1R1 -/ -) injected with FIV encoding the protein designated in parentheses. Background CD45 staining reflects low-affinity binding to microglia within the hippocampus (scale bar, 10 𝛍 m). D , Hippocampal BBB leakage, as evidenced by Evans Blue concentration, in IL-1 𝛃 XAT line B/b versus WT animals 2 weeks after transgene activation. Graph represents mean ± SEM. n = 3 animals per group. * p ≤ 0.05.CC-BY-NC-SA · From the PDF
Figure 2 Induction of IL-1 𝛃 expression drives chronic leukocyte recruitment to the mouse hippocampus. IL-1 𝛃 XAT line B/b micereceived unilateral intrahippocampal injections of FIV-Cre and were analyzed over a prolonged time course. A , CD45 staining in the ipsilateral (injected) hemispheres at the times indicated after FIV-Cre injection identifies infiltrating leukocytes. A white dashed line approximates the location of the granule cell layer of the dentate gyrus. qRT-PCR ( B , C ) generated a ratio of gene expression in the ipsilateral hippocampi of FIV-Cre injected IL-1 𝛃 XAT mice compared with wild-type control animals at the same time point for ICAM-1 ( B ) and MCP-1 (CCL2) ( C ). D , Immunohistochemical detection of MCP-1 protein expression in the ipsilateral and contralateral hemispheres 2 weeks after FIV-Cre. n = 3-5 animals per time point. Scale bars: A , 25 𝛍 m; D , 50 𝛍 m. Graphs represent mean ± SEM. ns, Not significant. * p ≤ 0.05.
Figure 2 Induction of IL-1 𝛃 expression drives chronic leukocyte recruitment to the mouse hippocampus. IL-1 𝛃 XAT line B/b micereceived unilateral intrahippocampal injections of FIV-Cre and were analyzed over a prolonged time course. A , CD45 staining in the ipsilateral (injected) hemispheres at the times indicated after FIV-Cre injection identifies infiltrating leukocytes. A white dashed line approximates the location of the granule cell layer of the dentate gyrus. qRT-PCR ( B , C ) generated a ratio of gene expression in the ipsilateral hippocampi of FIV-Cre injected IL-1 𝛃 XAT mice compared with wild-type control animals at the same time point for ICAM-1 ( B ) and MCP-1 (CCL2) ( C ). D , Immunohistochemical detection of MCP-1 protein expression in the ipsilateral and contralateral hemispheres 2 weeks after FIV-Cre. n = 3-5 animals per time point. Scale bars: A , 25 𝛍 m; D , 50 𝛍 m. Graphs represent mean ± SEM. ns, Not significant. * p ≤ 0.05.CC-BY-NC-SA · From the PDF
Figure 3 IL-1 𝛃 expression recruits diverse leukocyte populations to the mouse hippocampus. Leukocyte populations were defined in the ipsilateral hippocampi of IL-1 𝛃 XAT line B/b mice 2 weeks after gene activation by immunohistochemistry ( A-C , E ), flow cytometry ( D ), and tissue staining ( F ). A , B , T-cell populations were identified and classified by their expression of CD3(red)andeitherCD4( A ; green) or CD8 ( B ; green), where coexpressing cells appear yellow in merged images. C , The presence of dendritic cells was established by expression of both CD11c (green) and MHC-II (red), appearing yellow in merged images. D , An increase in the number of infiltrating macrophages is demonstrated 2 weeks after FIV-Cre versus FIV-LacZ injection by expansion of the CD45 hi , CD11b + cell population. E , F , Neutrophils were identified by cellular labeling with the 7/4 antibody ( E ) and after hematoxylin and eosin (H&E) tissue staining ( F , arrows). N, Neuronal nuclei. Scale bars, 5 𝛍 m.
Figure 3 IL-1 𝛃 expression recruits diverse leukocyte populations to the mouse hippocampus. Leukocyte populations were defined in the ipsilateral hippocampi of IL-1 𝛃 XAT line B/b mice 2 weeks after gene activation by immunohistochemistry ( A-C , E ), flow cytometry ( D ), and tissue staining ( F ). A , B , T-cell populations were identified and classified by their expression of CD3(red)andeitherCD4( A ; green) or CD8 ( B ; green), where coexpressing cells appear yellow in merged images. C , The presence of dendritic cells was established by expression of both CD11c (green) and MHC-II (red), appearing yellow in merged images. D , An increase in the number of infiltrating macrophages is demonstrated 2 weeks after FIV-Cre versus FIV-LacZ injection by expansion of the CD45 hi , CD11b + cell population. E , F , Neutrophils were identified by cellular labeling with the 7/4 antibody ( E ) and after hematoxylin and eosin (H&E) tissue staining ( F , arrows). N, Neuronal nuclei. Scale bars, 5 𝛍 m.CC-BY-NC-SA · From the PDF
Figure 5 Neuronal toxicity is not evident after sustained hippocampal IL-1 𝛃 expression. IL-1 𝛃 XAT line B/b mice received FIV-Cre injections and were analyzed 2 weeks or 2 months thereafter. Determination of neuronal toxicity was made comparing ipsilateral (injected) to noninjected (contralateral) hippocampi within animals. A , Preservation of both the pyramidal and granule cell layers of the hippocampus is demonstrated using NeuN (red) in the ipsilateral and contralateral hippocampi 2 weeks after FIV-Creinjection. B , NeuN(red)andTUNEL + cells (green, white arrows) 2 weeks after FIV-Cre injection. No overlap between these markerswasdetected. C , Leukocytes undergoing apoptosis (yellow, indicated by white arrows) expressed both TUNEL (green) and CD45 (red) markers 2 weeks after FIV-Cre injection. D , Determination of granule cell density within the dentate gyrus of animals 2 weeks and 2 months after FIV-Cre injection. Statistical analysis compared the ipsilateral versus contralateral cellular density betweengroupsofanimalsatthesametimepoint. n = 3-4animalspertimepoint.Scale bars: A , 100 𝛍 m; B , 25 𝛍 m; C , 10 𝛍 m. n = 3-4 animals per time point. Graphs represent mean ± SEM. ns, Not significant.
Figure 5 Neuronal toxicity is not evident after sustained hippocampal IL-1 𝛃 expression. IL-1 𝛃 XAT line B/b mice received FIV-Cre injections and were analyzed 2 weeks or 2 months thereafter. Determination of neuronal toxicity was made comparing ipsilateral (injected) to noninjected (contralateral) hippocampi within animals. A , Preservation of both the pyramidal and granule cell layers of the hippocampus is demonstrated using NeuN (red) in the ipsilateral and contralateral hippocampi 2 weeks after FIV-Creinjection. B , NeuN(red)andTUNEL + cells (green, white arrows) 2 weeks after FIV-Cre injection. No overlap between these markerswasdetected. C , Leukocytes undergoing apoptosis (yellow, indicated by white arrows) expressed both TUNEL (green) and CD45 (red) markers 2 weeks after FIV-Cre injection. D , Determination of granule cell density within the dentate gyrus of animals 2 weeks and 2 months after FIV-Cre injection. Statistical analysis compared the ipsilateral versus contralateral cellular density betweengroupsofanimalsatthesametimepoint. n = 3-4animalspertimepoint.Scale bars: A , 100 𝛍 m; B , 25 𝛍 m; C , 10 𝛍 m. n = 3-4 animals per time point. Graphs represent mean ± SEM. ns, Not significant.CC-BY-NC-SA · From the PDF
Image 5
CC-BY-NC-SA · From the PDF
Figure 7 CXCR2 is required for neutrophil infiltration after hippocampal IL-1 𝛃 expression. IL-1 𝛃 XAT line B/b animals either lacking (CXCR2 -/ -), heterozygous (CXCR2 + / -), or with two copies (CXCR2 + / + ) of CXCR2 were examined 2 weeks after FIV-Cre hippocampal injections. A , Hippocampal cellular infiltrates from bilaterally injected mice were analyzed by flow cytometry. After gating on M-CSF-negative cells, the percentage of neutrophils are indicated in the top right corner of each plot as the Lys6G hi , Lys6C hi cell population. B , 7/4 staining of neutrophils within the dentate gyrus reflects the results from A . C , BBB leakage was determined using hippocampal Evans Blue concentrations and is graphed relative to that observed in CXCR2 + / + animals. BBB leakage was present in all animal groups and was not significantly altered by CXCR2 gene dosage. n = 3-4 animals per group. Scale bar, 20 𝛍 m. Graphs represent mean ± SEM. ns, Not significant.
Figure 7 CXCR2 is required for neutrophil infiltration after hippocampal IL-1 𝛃 expression. IL-1 𝛃 XAT line B/b animals either lacking (CXCR2 -/ -), heterozygous (CXCR2 + / -), or with two copies (CXCR2 + / + ) of CXCR2 were examined 2 weeks after FIV-Cre hippocampal injections. A , Hippocampal cellular infiltrates from bilaterally injected mice were analyzed by flow cytometry. After gating on M-CSF-negative cells, the percentage of neutrophils are indicated in the top right corner of each plot as the Lys6G hi , Lys6C hi cell population. B , 7/4 staining of neutrophils within the dentate gyrus reflects the results from A . C , BBB leakage was determined using hippocampal Evans Blue concentrations and is graphed relative to that observed in CXCR2 + / + animals. BBB leakage was present in all animal groups and was not significantly altered by CXCR2 gene dosage. n = 3-4 animals per group. Scale bar, 20 𝛍 m. Graphs represent mean ± SEM. ns, Not significant.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Shaftel, S. S., Carlson, T. J., Olschowka, J. A., Kyrkanides, S., Matousek, S. B., & O'Banion, M. K.
Affiliations
University of Rochester Medicine (US)
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2007-08-29
Volume
27
Issue
35
Pages
9301-9309
ISSN
  • 0270-6474 (ISSN-L)
  • 1529-2401
DOI
10.1523/jneurosci.1418-07.2007
PMC
PMC6673122
OpenAlex
W2094676206
Cited by
243
References
55
Citations by year
2026: 6 · 2025: 7 · 2024: 5 · 2023: 9 · 2022: 14 · 2021: 13 · 2020: 10 · 2019: 18 · 2018: 22 · 2017: 9 · 2016: 15 · 2015: 13 · 2014: 19 · 2013: 19 · 2012: 10
FWCI
8.68
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • University of Rochester
  • National Institutes of Health
  • National Institute of General Medical Sciences (GM 07356, T32GM007356)
  • National Institute of Neurological Disorders and Stroke (NS048522, R29NS033553, NS 33553, R21NS048522, R01NS033553)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Life Sciences › Neuroscience › Biological Psychiatry › Tryptophan and brain disorders
Keywords
IL-1β, chronic neuroinflammation, blood-brain barrier permeability, leukocyte infiltration, neutrophils, CXCR2, CXCL1, CXCL2, MCP-1, ICAM-1, hippocampus, transgenic mouse model, neurodegeneration, chemokines
MeSH terms
Animals, Blood-Brain Barrier, Hippocampus, Leukocytes, Mice, Inbred C57BL, Mice, Transgenic, Neutrophils, Monokines, Up-Regulation, Leukocyte Common Antigens, Intercellular Adhesion Molecule-1, Chemokine CCL2, Mice, Interleukin-1beta, Chemokine CXCL2

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