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Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Johnson HL, Willenbring RC, Jin F, Manhart WA, LaFrance SJ, Pirko I, Johnson AJ - PLoS ONE (2014)

Bottom Line: However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood.To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques.Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, Rochester, Minnesota, United States of America; Department of Immunology, Mayo Clinic, Rochester, Minnesota, United States of America; Neurobiology of Disease Graduate Program, Mayo Graduate School, Rochester, Minnesota, United States of America.

ABSTRACT
Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

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Disorganization of tight junction proteins and increased CNS vascular permeability can be induced in perforin−/− mice reconstituted with perforin competent CD8 T cells.Confocal microscopic images from a representative (A) mock E7 peptide-treated C57BL/6 perforin−/− mouse reconstituted with perforin competent CD8 T cells depict a preservation of vascular integrity and intact tight junction proteins claudin-5 and occludin. (B) VP2121–130 peptide-treated C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells display degradation of tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability as seen through FITC-albumin leakage. (C) FITC intensity in brain tissue from two representative confocal microscopic images of each animal was quantified using ImageJ software. VP2121–130 peptide-treated mice (n = 6) displayed a significant higher intensity of FITC-albumin in brain parenchyma when compared to E7 peptide-treated mice (n = 4) (p<0.05).
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pone-0111401-g004: Disorganization of tight junction proteins and increased CNS vascular permeability can be induced in perforin−/− mice reconstituted with perforin competent CD8 T cells.Confocal microscopic images from a representative (A) mock E7 peptide-treated C57BL/6 perforin−/− mouse reconstituted with perforin competent CD8 T cells depict a preservation of vascular integrity and intact tight junction proteins claudin-5 and occludin. (B) VP2121–130 peptide-treated C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells display degradation of tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability as seen through FITC-albumin leakage. (C) FITC intensity in brain tissue from two representative confocal microscopic images of each animal was quantified using ImageJ software. VP2121–130 peptide-treated mice (n = 6) displayed a significant higher intensity of FITC-albumin in brain parenchyma when compared to E7 peptide-treated mice (n = 4) (p<0.05).

Mentions: The increased vascular permeability in perforin−/− mice reconstituted with perforin competent CD8 T cells prompted additional analysis of focal areas of CNS vascular permeability and the state of the BBB tight junction proteins. We determined that mock E7 peptide-treated C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells display preservation of vascular integrity, as shown by lack of FITC-albumin leakage into the brain parenchyma from the brain vasculature, and linear organization of BBB tight junction proteins claudin-5 and occludin (Figure 4A). However, C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells and administered VP2121–130 peptide to induce PIFS presented with a loss of linear organization of tight junction proteins claudin-5 and occluding in the microvessels. Disorganization of BBB tight junctions colocalizes with FITC-albumin leakage from brain vasculature (Figure 4B). FITC intensity from confocal microscopic images was then quantified using ImageJ software in brain tissue slices. Two random representative images from each animal were analyzed. FITC intensity values were normalized through subtraction of background values. We determined using this method that VP2121–130 peptide-treated mice (n = 6) displayed a significant higher intensity of FITC-albumin when compared to E7 peptide-treated mice (n = 4) (p<0.05) (Figure 4C). The colocalization of disorganized tight junction proteins with CNS vascular permeability in this experiment is consistent with previous studies performed in perforin competent wild-type C57BL/6 mice [18], [25]. Vascular leak and disorganization of BBB tight junction proteins is evident with transfer of perforin competent CD8 T cells into a perforin-/- mouse and induced to undergo PIFS.


Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Johnson HL, Willenbring RC, Jin F, Manhart WA, LaFrance SJ, Pirko I, Johnson AJ - PLoS ONE (2014)

Disorganization of tight junction proteins and increased CNS vascular permeability can be induced in perforin−/− mice reconstituted with perforin competent CD8 T cells.Confocal microscopic images from a representative (A) mock E7 peptide-treated C57BL/6 perforin−/− mouse reconstituted with perforin competent CD8 T cells depict a preservation of vascular integrity and intact tight junction proteins claudin-5 and occludin. (B) VP2121–130 peptide-treated C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells display degradation of tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability as seen through FITC-albumin leakage. (C) FITC intensity in brain tissue from two representative confocal microscopic images of each animal was quantified using ImageJ software. VP2121–130 peptide-treated mice (n = 6) displayed a significant higher intensity of FITC-albumin in brain parenchyma when compared to E7 peptide-treated mice (n = 4) (p<0.05).
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Related In: Results  -  Collection

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pone-0111401-g004: Disorganization of tight junction proteins and increased CNS vascular permeability can be induced in perforin−/− mice reconstituted with perforin competent CD8 T cells.Confocal microscopic images from a representative (A) mock E7 peptide-treated C57BL/6 perforin−/− mouse reconstituted with perforin competent CD8 T cells depict a preservation of vascular integrity and intact tight junction proteins claudin-5 and occludin. (B) VP2121–130 peptide-treated C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells display degradation of tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability as seen through FITC-albumin leakage. (C) FITC intensity in brain tissue from two representative confocal microscopic images of each animal was quantified using ImageJ software. VP2121–130 peptide-treated mice (n = 6) displayed a significant higher intensity of FITC-albumin in brain parenchyma when compared to E7 peptide-treated mice (n = 4) (p<0.05).
Mentions: The increased vascular permeability in perforin−/− mice reconstituted with perforin competent CD8 T cells prompted additional analysis of focal areas of CNS vascular permeability and the state of the BBB tight junction proteins. We determined that mock E7 peptide-treated C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells display preservation of vascular integrity, as shown by lack of FITC-albumin leakage into the brain parenchyma from the brain vasculature, and linear organization of BBB tight junction proteins claudin-5 and occludin (Figure 4A). However, C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells and administered VP2121–130 peptide to induce PIFS presented with a loss of linear organization of tight junction proteins claudin-5 and occluding in the microvessels. Disorganization of BBB tight junctions colocalizes with FITC-albumin leakage from brain vasculature (Figure 4B). FITC intensity from confocal microscopic images was then quantified using ImageJ software in brain tissue slices. Two random representative images from each animal were analyzed. FITC intensity values were normalized through subtraction of background values. We determined using this method that VP2121–130 peptide-treated mice (n = 6) displayed a significant higher intensity of FITC-albumin when compared to E7 peptide-treated mice (n = 4) (p<0.05) (Figure 4C). The colocalization of disorganized tight junction proteins with CNS vascular permeability in this experiment is consistent with previous studies performed in perforin competent wild-type C57BL/6 mice [18], [25]. Vascular leak and disorganization of BBB tight junction proteins is evident with transfer of perforin competent CD8 T cells into a perforin-/- mouse and induced to undergo PIFS.

Bottom Line: However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood.To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques.Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, Rochester, Minnesota, United States of America; Department of Immunology, Mayo Clinic, Rochester, Minnesota, United States of America; Neurobiology of Disease Graduate Program, Mayo Graduate School, Rochester, Minnesota, United States of America.

ABSTRACT
Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

Show MeSH
Related in: MedlinePlus