Perforin Competent CD8 T Cells Are Sufficient to Cause Immune-Mediated Blood-Brain Barrier Disruption

Holly L. Johnson; Robin C. Willenbring; Fang Jin; Whitney A. Manhart; Stephanie J. LaFrance; Istvan Pirko; Aaron J. Johnson

doi:10.1371/journal.pone.0111401

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.

Citation: Johnson HL, Willenbring RC, Jin F, Manhart WA, LaFrance SJ, Pirko I, et al. (2014) Perforin Competent CD8 T Cells Are Sufficient to Cause Immune-Mediated Blood-Brain Barrier Disruption. PLoS ONE 9(10): e111401. https://doi.org/10.1371/journal.pone.0111401

Editor: Roberto Furlan, San Raffaele Scientific Institute, Italy

Received: January 3, 2014; Accepted: October 2, 2014; Published: October 22, 2014

Copyright: © 2014 Johnson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work is supported by the National Institutes of Health grant R01 NS060881 and Mayo Graduate School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Numerous devastating neurological disorders, including multiple sclerosis, acute hemorrhagic leukoencephalitis (AHLE), dengue hemorrhagic fever, stroke, glioblastoma multiforme, epilepsy, HIV dementia, and cerebral malaria, are characterized by blood-brain barrier (BBB) disruption [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. Although immune cells have the capacity to initiate CNS vascular permeability, there is relatively little known about how inflammation promotes BBB disruption due to a lack of suitable model systems. This currently undermines the development of therapeutic strategies to ameliorate pathology associated with these disorders. In order to define the mechanisms of BBB disruption, our lab has developed an inducible model of CNS vascular permeability using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model commonly used to study multiple sclerosis [12], [13], [14], [15]. C57BL/6 mice respond to TMEV infection by mounting an antiviral CD8 T cell response that is highly focused on the immunodominant TMEV peptide, VP2_121–130, presented in the context of the D^b class I molecule [16], [17]. However, injection of this immunodominant peptide 7 days post-TMEV infection results in increased astrocyte activation, alteration of BBB tight junctions, severe CNS vascular permeability, and morbidity within 48 hours. This peptide induced fatal syndrome (PIFS) is dependent on virus-specific CD8 T cells and perforin expression [12], [18].

Perforin is a pore forming protein that plays an important role in controlling viral infections and tumors [19]. Perforin has also been shown to play a critical role in an inducible mouse model of seizures, as mice deficient in perforin displayed reduced BBB disruption [6]. When analyzing the effector functions of CD8 T cells in our PIFS model system, we found that perforin, but not Fas ligand, was required for pathology associated with PIFS to develop. In these experiments, we determined C57BL/6 perforin^−/− mice are resistant to PIFS and are devoid of CNS vascular permeability as measured by magnetic resonance imaging (MRI) analysis and leakage of FITC-albumin into the CNS parenchyma. Astrocyte activation, as measured by glial fibrillary acidic protein (GFAP) expression, was also found to be dependent on perforin expression in the PIFS model. Events indicative of BBB disruption are dependent on perforin expression [18]. However, the cellular source of perforin required for promoting BBB disruption is unknown. In addition to CD8 T cells, natural killer (NK) cells and γδ T cells express perforin and have been shown to use perforin-mediated cytotoxicity during viral infections [20], [21], [22]. Neutrophils have also recently been shown to express perforin to regulate immune responses in allergic contact dermatitis [23]. Therefore, while we have previously demonstrated that both CD8 T cells and perforin are critical factors causing BBB disruption, it remained unknown the extent other perforin-expressing immune cell types assisted in the development of PIFS.

Since PIFS is initiated by class I-restricted virus antigen, we hypothesized that CD8 T cells directly use perforin to cause BBB disruption independent of other immune cell types. We tested this hypothesis using adoptive transfer techniques to isolate the CD8 T cell as the sole perforin-expressing cell type in the PIFS model. After reconstituting perforin^−/− mice with perforin competent CD8 T cells, mice were intracranially infected with TMEV and administered either PIFS-inducing VP2_121–130 peptide or mock E7 peptide 7 days post-infection. Mice were then evaluated for activation of astrocytes, disruption of the tight junction organization, and CNS vascular permeability in order to determine whether perforin competent CD8 T cells alone are sufficient to cause BBB disruption.

Materials and Methods

Animals and ethics statement

C57BL/6 perforin deficient male mice (strain #002407) were obtained from Jackson laboratories (Bar Harbor, ME) at 5 weeks of age. C57BL/6-Tg(UBC-GFP) (strain #004353) male mice were bred in-house at Mayo Clinic. C57BL/6 Ly5.1 male mice were provided by Dr. Adam Schrum, Mayo Clinic. All experiments were approved by the Institutional Animal Care and Use Committee of Mayo Clinic (protocol number A57913).

Adoptive transfer

Spleens of GFP+ or Ly5.1+ mice were removed and strained through a nylon mesh 100 µm filter. CD8+ cells were purified from the resulting lymphocyte population using MACS LS cell purification columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Positive cell sorting was used to obtain GFP+ CD8+ cells and negative cell sorting was used to obtain Ly5.1+ CD8+ cells. Both methods resulted in ∼90% purity as determined by flow cytometric analysis (data not shown). C57BL/6 perforin^−/− mice were irradiated with 400 rads of irradiation, given one day to recover, and then intravenously injected with 10⁷ Ly5.1+ CD8+ or GFP+ CD8+ splenocytes (Figure 1A).

Download:

Figure 1. Adoptive transfer of CD8+ cells successfully migrate to brain post induction of PIFS.

(A) Schematic illustrating the adoptive transfer of sorted perforin competent CD8+ cells into perforin^−/− mice followed by induction of PIFS. C57BL/6 perforin^−/− mice were irradiated with 400 rads of irradiation and then intravenously injected with 10⁷ GFP+ CD8+ splenocytes (n = 10) or 10⁷ Ly5.1+ CD8+ splenocytes (n = 10). Mice were intracranially infected with TMEV on the following day. PIFS-inducing VP2_121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion. MRI analysis was performed on the following day to visualize the extent of CNS vascular permeability and then the CNS was harvested for additional assays. Both negative (Experiment I) and positive (Experiment II) sort experiments yielded high purity of CD8+ T cell transfer. (B) Representative confocal microscopic images illustrating co-localization of Ly5.1 and CD8 protein (Experiment I). CNS-infiltrating Ly5.1+ cells colocalized with CD8+ cells 85.7% of the time as measured by confocal analysis. (C) Confocal microscopy showing of representative brain tissue slice showing successful transfer of GFP+ CD8+ T cells (Experiment II). Purity of transfer was analyzed via flow cytometry, 98.0%+− 0.5% of cells that were CD8 positive and GFP positive.

https://doi.org/10.1371/journal.pone.0111401.g001

Induction of CNS vascular permeability using the PIFS model

CNS vascular permeability was induced using the PIFS model as previously described [12], [18], [24]. Briefly, all mice were intracranially infected with 2 x 10⁶ PFU Daniel's strain of TMEV. Seven days post-TMEV infection, during the peak of CD8 T cell expansion in the brain, mice were intravenously administered 0.1 mg VP2_121–130 (FHAGSLLVFM) peptide or mock control E7 (RAHYNIVTF) peptide (Genescript) (Figure 1A).

Injection of rhodamine-dextran or FITC-albumin to assess permeability

Mice were given an intravenous injection of 10 mg rhodamine B isothiocyanate-dextran (Sigma #R9379) or 10 mg FITC-albumin (Sigma #A9771) 23 hours after VP2_121–130 or E7 peptide administration. Brains were harvested at 24 hours.

Flow Cytometry

Brain-infiltrating lymphocytes were isolated from the left hemisphere through collagenase digestion and a percoll gradient as previously described [25]. Isolated brain lymphocytes were then incubated with anti-CD45 PerCP (BD Biosciences, 557235) and anti-CD8 PE-Cy7 (BD Biosciences, 552877) for 40 minutes. Cells were washed twice with FACS buffer and then fixed in 4% paraformaldehyde and 1X PBS. Samples were analyzed on a BD LSRII flow cytometer (BD Biosciences) using FACS Diva software (BD Biosciences).

Rhodamine-dextran permeability assay

Mice were intravenously injected with 10 mg rhodamine B isothiocyanate-dextran (Sigma #R9379) 23 hours after VP2_121–130 or E7 peptide administration. Brains were harvested one hour later and frozen on aluminum foil on dry ice. The right hemisphere was homogenized with radioimmunoprecipitation assay (RIPA) buffer [10 mmol/L Tris, 140 mmol/L NaCl, 1% Triton X-100, 1% Na dexycholate, 0.1% SDS and protease inhibitor cocktail (Pierce #78410) pH 7.5] and centrifuged for 10 minutes at 10,000 rpm. Protein concentration was assessed using the BCA protein assay (Pierce #23223) and then samples were normalized for protein content. Homogenates were read on a fluorescent plate reader at 540 nm excitation and 625 nm emission to detect rhodamine-dextran leakage into the brain. Data were collected using SpectraMax software (Molecular Devices).

Confocal Microscopy

Fresh frozen coronal slices from mice injected with FITC-albumin, were cut on a cryostat and placed onto positively charged slides. Slides were washed twice with PBS and then fixed in 3% paraformaldehyde for 15 minutes. After the fixation step, slides were rinsed three times in PBS and then incubated for 1 hour in 5% normal goat serum + 0.5% Igepal CA-630 (Sigma I3021) in PBS. To determine the percentage of CNS-infiltrating Ly5.1+ CD8 T cells, slides were incubated with the following primary antibodies at a concentration of 1:100 overnight at room temperature: anti-mouse CD45.1 PE (eBioscience, 12-0453-82) and rat anti-mouse CD8 (AbD Serotec, MCA2694). The following secondary antibodies were used at a concentration of 1:250 for 1 hour at room temperature: AlexaFluor 532 goat anti-mouse IgG (Invitrogen, A-11002) and AlexaFluor 647 goat anti-rat IgG (Invitrogen, A21247). To visualize the tight junction organization, slides were incubated with the following primary antibodies at a concentration of 1:100 overnight at room temperature: mouse anti-claudin-5 (Invitrogen, 35–2500) and rabbit anti-occludin (Invitrogen, 71–1500). The following secondary antibodies were then added at a concentration of 1:250 for 1 hour at room temperature: AlexaFluor 532 goat anti-mouse IgG (Invitrogen, A-11002) and AlexaFluor647 goat anti-rabbit IgG (Invitrogen, A-21244). To assess astrocyte activation, slides were incubated with mouse anti-GFAP-Cy3 (Sigma, 106K4775) at a concentration of 1:500 overnight at room temperature. All slides were rinsed 5 times in PBS before and after incubation with secondary antibodies. This was followed by Hoechst staining at a concentration of 1:500 in PBS for 5 minutes. Slides were then rinsed 5 times in PBS, dried, and covered with Vectashield medium (Vector lab, H-1000). Images were acquired using a Leica (Germany) DM 2500 confocal microscope equipped with a 63x oil immersion objective (numerical aperture 1.30). All images were collected at room temperature using Type F immersion liquid (Leica Microsystems) and analyzed using LAS AF stimulator AF 6000 acquisition software. All images shown in manuscript are at 63x magnification.

MRI Acquisition

A Bruker Avance II 7 Tesla vertical bore small animal MRI system (Bruker Biospin) was used for image acquisition to evaluate CNS vascular permeability as previously described [9], [26]. Briefly, inhalation anesthesia was induced and maintained using 3–4% isoflurane. An MRI compatible vital sign monitoring system (Model 1030, SA Instruments, Inc., Stony Brook NY) was used to monitor respiratory rate during acquisition. Gadolinium was intraperitoneally administered to mice using weight based dosing of 100 mg/kg. After a standard delay of 15 minutes, a volume acquisition T1-weighted spin echo sequence was used (TR-150 ms, TE = 8 ms, FOV: 32 mm x 19.2 mm x 19.2 mm, Matrix: 160 x 96 x 96, number of averages = 1) to obtain T1-weighted images.

Image Analysis

3D volumetric analysis was performed as previously described [25], [27], [28], [29]. Briefly, Analyze 11.0 software (Biomedical Imaging Resource, Mayo Clinic) was used to quantify the 3D volume of CNS vascular permeability. The 3D volume extractor tool was used to extract brains from gadolinium-enhanced T1-weighted images. The 3D ROI tool was then used to define areas of gadolinium leakage using semi-automated methods. The volume of these areas, which corresponds to the volume of gadolinium leakage, was calculated using the 3D sampling tool. 3D object rendering using the volume rendering tool was performed to visualize the identified volumes of contrast enhancement. All 3D volumes were standardized to the mean of control (mice receiving E7 peptide on day 7) within an experiment and plotted as individual mice, shown as Ratio of Gadolinium Volume.

Statistical Analysis

Mean and standard error values were calculated using SigmaStat software (SYSTAT Software Inc). GraphPad Prism Software was used to construct graphs with standard error bars. A Student's t-test was performed using SigmaStat to evaluate CNS vascular permeability, as detected by leakage of rhodamine-dextran into the CNS, and FITC intensity from confocal microscopic images. A Welch test was used to evaluate 3D volumetric analysis of vascular leak visible on T1-weighted MRI scans.

Results

Adoptively transferred perforin competent CD8 T cells induce CNS vascular permeability

Model and adoptive transfer purity.

CD8 T cells that recognize the immunodominant D^b:VP2_121–130 epitope initiate BBB disruption [12]. However, the extent CD8 T cell expressed perforin contributes to CNS vascular permeability remains unknown. Therefore, we developed an adoptive transfer approach to isolate the CD8 T cell as the sole perforin-expressing cell type (Figure 1A). CD8+ cells were purified from the spleens of Ly5.1+ perforin+/+ mice using negative cell sort (Experiment I), or GFP+ perforin^+/+ mice using positive cell sorting (Experiment II) resulting in ∼90% purity as determined by flow cytometric analysis (data not shown). Recipient C57BL/6 perforin^−/− mice were irradiated with 400 rads of irradiation, given one day to recover, and then intravenously injected with 10⁷ sorted CD8+ splenocytes. The following day mice were intracranially infected with TMEV. PIFS-inducing VP2_121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion (Figure 1). Gadolinium-enhanced T1-weighted MRI was performed on the following day to visualize CNS vascular permeability. Immediately following MRI, the CNS was harvested to be further analyzed by confocal microscopy, flow cytometry and CNS vascular permeability was measured by fluorescent molecule diffusion across the BBB.

To determine the percentage of CNS-infiltrating Ly5.1+ CD8 T cells post-induction of PIFS, tissue slices were stained with antibody to Ly5.1 and CD8 and observed under a confocal microscope. Comparable numbers of CNS-infiltrating Ly5.1+ CD8 T cells were observed in both VP2_121–130 peptide (n = 6) and E7 peptide-treated (n = 4) groups, with the average being 85.7% (Figure 1B, data not shown). Flow cytometric analysis was used to determine the percentage of CNS-infiltrating GFP+ CD8 T cells post-induction of PIFS. Isolated CNS-infiltrating lymphocytes were stained with antibody to CD45 and CD8. The CD45^hi population was then gated and analyzed for the percentage of GFP+ CD8 T cells. Comparable numbers of CNS-infiltrating CD8+ cells as a percentage of GFP+ cells were observed in both VP2_121–130 peptide (n = 6) and E7 peptide-treated (n = 4) groups, with the average being 98.0%±0.5% (data not shown). Furthermore, tissue slices were stained with antibody to CD8 and GFP and observed using confocal microscopy and defined to be high purity (Figure 1C).

CNS permeability measured by small animal MRI and fluorescent molecule diffusion.

In order to quantitatively evaluate the full volume of CNS vascular leak in animals undergoing PIFS, where CD8 T cells were the sole perforin-expressing cell type, we performed volumetric analysis of 3D gadolinium-enhanced T1-weighted MRI scans using Analyze 11.0 software developed by Mayo Clinic's Biomedical Imaging Resource [30], [31]. Quantification of the 3D volume of gadolinium leakage from vasculature revealed that VP2_121–130 peptide-treated mice reconstituted with perforin competent CD8 T cells had increased CNS vascular permeability (Figure 2 B,D) when compared to mock E7 peptide-treated controls (Figure 2 A,C) (p<0.05, Figure 2E). CD8+ cells were selected using a negative (Figure 2 A,B) or positive (Figure 2 C,D) sort method. Importantly, an additional VP2_121–130 peptide-treated mouse, not included in this data set, was moribund and unable to be scanned. Post-mortem analysis revealed that this mouse had the highest transfer of CD8 T cells (data not shown).

Download:

Figure 2. CD8 T cells as sole perforin wielding source are sufficient to induce CNS vascular permeability.

The extent of CNS vascular permeability is illustrated by raw image and 3D transparency rendering of gadolinium-enhancing areas from T1-weighted MRI scans in mice administered (A,C) mock E7 control peptide or (B,D) VP2_121–130 peptide. MRI images are separated to show both methods of CD8+ T cell selection (A,B) negative sort or (C,D) positive sort to isolate and transfer CD8+ cells. (E) Quantification of the 3D volume of vascular leakage from all mice (both negative and positive sort) revealed that VP2_121–130 peptide-treated mice having CD8 T cells as the sole perforin-expressing cell type have a significantly more amount of gadolinium leakage (p<0.05) (n = 5,9 per group). (F) A significant increase in CNS vascular permeability detectable by intravenously injected rhodamine dextran accumulation in brain homogenates was observed in VP2_121–130 peptide-treated animals (n = 6) compared to mock E7 peptide-treated animals (n = 4) (p<0.05). Results are depicted as mean ± SEM.

https://doi.org/10.1371/journal.pone.0111401.g002

Another group of animals was injected with rhodamine-dextran 23 hours post-administration of mock E7 peptide or VP2_121–130 peptide to induce PIFS. Rhodamine-dextran was allowed to circulate for one hour. Brains were then harvested and the right hemisphere was used to assess CNS vascular permeability. In concordance with the T1-weighted MRI analysis, a significant increase in vascular permeability was observed in the VP2_121–130 peptide-treated group (n = 6) compared to the E7 peptide-treated group (n = 4), as detected by leakage of rhodamine-dextran into the CNS using a fluorescent plate reader (A₅₄₀) (p<0.05) (Figure 2F).

Increased astrocyte GFAP expression in perforin^−/− mice reconstituted with perforin competent CD8 T cells

Activation of astrocytes, as measured by GFAP expression, co-localizes with CNS vascular permeability in C57BL/6 mice induced to undergo PIFS [18]. To determine the extent astrocytes were activated in perforin^−/− mice reconstituted with perforin competent CD8 T cells, we stained for astrocyte expression of GFAP and performed confocal microscopy. We found that mice treated with VP2_121–130 peptide displayed an increase in astrocyte size consistent with astrogliosis (Figure 3B), when compared to E7 peptide-treated mice (Figure 3A). We also observed colocalization of increased astrocyte activation with CNS vascular permeability. This is consistent with previous studies in which PIFS was evaluated in perforin competent wild-type C57BL/6 mice [18]. In this study, in accordance with previous studies, there is a clear increase in astrocyte activation in areas with vascular leak as compared to unaffected tissue (Figure 3B).

Download:

Figure 3. Increased astrocyte activation colocalizes with CNS vascular permeability post-induction of PIFS in perforin^−/− mice reconstituted with perforin competent CD8 T cells.

Representative confocal microscopic images illustrating astrocyte expression of GFAP in (A) E7 peptide-treated (n = 4) and (B) VP2_121–130 peptide-treated (n = 6) perforin^−/− mice reconstituted with perforin competent CD8 T cells. Mice treated with (B) VP2_121–130 peptide displayed increased astrocyte activation, as measured by heightened GFAP expression, in areas of CNS vascular permeability when compared to (A) mock E7 peptide-treated negative control mice. CNS permeability is determined by leakage of intravenously administered FITC-albumin.

https://doi.org/10.1371/journal.pone.0111401.g003

Disorganized tight junction proteins colocalize with increased CNS vascular permeability in perforin^−/− mice reconstituted with perforin competent CD8 T cells

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 VP2_121–130 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 VP2_121–130 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.

Download:

Figure 4. 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) VP2_121–130 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. VP2_121–130 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).

https://doi.org/10.1371/journal.pone.0111401.g004

Discussion

Investigating the underlying molecular mechanisms leading to pathology associated with BBB disruption is of critical importance for the development of therapeutic approaches to treat diseases characterized by CNS vascular permeability. Using the PIFS model system, our lab has previously demonstrated critical roles for CD8 T cells and perforin in promoting activation of astrocytes, loss of linear organization of the tight junction, and extensive CNS vascular permeability. We have also shown that inhibiting the functions of these critical players improves pathology and survival [12], [18]. Furthermore, we have demonstrated that other molecular players implicated in causing BBB disruption, such as GR-1+ neutrophils, CD4 T cells, TNF-α, IFN-γ, LTβR, and IL-1, do not contribute to lethality in CD8 T cell-initiated BBB disruption [16], [25]. However, whether CD8 T cells are the cellular source of perforin required for promoting BBB disruption was not known.

In addition to CD8 T cells, NK cells, γδ T cells, and neutrophils also use perforin as an effector mechanism. [20], [21], [22], [23], [32], [33], [34]. Mice deficient in NK cells displayed a higher degree of inflammation and demyelination after induction of experimental autoimmune encephalomyelitis (EAE) [32], [33], [34]. Depletion of NK cells has also been shown to result in more severe encephalitis in mice post-TMEV infection [21]. Additionally, mice deficient in γδ T cells exhibited increased viral load and were more susceptible to West Nile Virus infection. These mice also displayed decreased levels of intracellular perforin in splenocytes post-infection with West Nile Virus and had a lower cytotoxicity compared to wild-type mice [22]. Furthermore, neutrophils have been shown to use perforin to regulate CD8 T cell infiltration into the skin to mediate allergic contact dermatitis [23]. This raises the question as to whether other molecular players such as NK cells, γδ T cells, and neutrophils contribute to perforin-dependent CD8 T cell-mediated BBB disruption during neuroinflammation.

In this study, we tested our hypothesis that CD8 T cells employ perforin to cause BBB disruption independent of other immune cell types. We developed an adoptive transfer approach to isolate perforin competent CD8 T cells and determine if they are sufficient to cause activation of astrocytes, disorganization of BBB tight junction proteins, and CNS vascular permeability. Using this adoptive transfer approach, we found that perforin^−/− mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited a significant increase in CNS vascular permeability, measured by fluorescent molecule diffusion across the BBB, compared to mock E7 peptide-treated controls. This prompted a more comprehensive analysis using small animal MRI and confocal microscopy. Quantification of the 3D volume of gadolinium leakage from vasculature visible on T1-weighted MRI scans illustrated that PIFS-inducing VP2_121–130 peptide-treated mice reconstituted with CD8 T cells as the sole perforin-expressing cell type displayed significant increase in CNS vascular permeability when compared to mock E7 peptide-treated controls. This analysis showed that perforin competent CD8 T cells are sufficient to induce CNS vascular permeability.

We then used confocal microscopy to analyze BBB disruption in more detail by examining astrocyte activation, the organization of BBB tight junction proteins claudin-5 and occludin, and focal areas of vascular permeability. This analysis demonstrated that perforin competent CD8 T cells are sufficient to cause increased astrocyte activation and disorganization of the tight junctions in areas of CNS vascular permeability.

Studies using the EAE model have also put forward a role for astrocytes in causing disorganization of BBB tight junction proteins and ensuing CNS vascular permeability. These studies have proposed a mechanism in which CD4 T cells induce astrocytes to release vascular endothelial growth factor (VEGF), resulting in BBB disruption [35]. However, in contrast to EAE, CD4 T cells do not contribute to lethality in the PIFS model [36]. Furthermore, while we have also demonstrated a critical role for VEGF in promoting BBB disruption, we found that neurons were the major source of VEGF expression during CNS vascular permeability [24]. However, since we have observed increased astrocyte activation colocalizing with CNS vascular permeability, there is still a potential role for astrocytes in the PIFS model. It is possible that increased astrocyte activation causes a permeable state or is a consequence of BBB disruption. This will be a topic of further investigation.

This study has expanded our knowledge on the mechanism of BBB disruption by illustrating that perforin competent CD8 T cells are sufficient to cause traits associated with immune-mediated BBB disruption. It remains necessary to investigate the functional mechanism used by perforin to cause BBB disruption. In experimental cerebral malaria, granzyme B has been defined as a critical factor in promoting fatal cerebral pathology [37]. The extent granzyme B contributes to BBB disruption in PIFS remains to be defined. Furthermore, the complete mechanism of how perforin enables delivery of granzymes to cause apoptosis of target cells also remains to be defined. It was originally thought that perforin created pores on target cells to allow entry of granzymes, resulting in cell death [38]. Recently it has been suggested that the perforin pore actively, and selectively, passes cationic molecules, such as granzyme B, more efficiently that anionic and neutral molecules [39]. Alternative models propose that granzymes are endocytosed independent of perforin. This is followed by perforin acting on the endosomal membrane to form pores, causing the release of granzymes, which in turn promotes apoptosis of target cells [40], [41]. A recent proposed mechanism involves a combination of these two pathways. In this third model, perforin forms pores in the plasma membrane of the target cell. This results in an influx of Ca²⁺ that triggers repair of the plasma membrane. Granzymes and perforin are then endocytosed together into large endosomes, and perforin acts on the endosomal membrane to release granzymes into the cytosol. However, the mechanism of how perforin causes release of granzymes from the endosome remains unclear [42].

Perforin is commonly defined as providing cytolytic activity against target cells [38]. Recently new, non-classical, mechanisms have been put forward that perforin can function in non-cytolytic pathways. Perforin has been identified, along with granzyme, as having a noncytolytic role in controlling the reactivation of HSV-1 neuronal infections without inducing cytotoxicity [43]. Additionally, in mouse model of obesity related insulin resistance and visceral adipose tissue (VAT), a lack of perforin showed reduced insulin sensitivity and changed the inflammation status within the VAT lesions, suggesting an immunoregulatory role for perforin in this disease state. Importantly, in this study, the formation of crown like structures, indicative of adipocyte death, was not changed in mice lacking perforin, further emphasizing a noncytolytic role of perforin [44]. Other noncytolytic roles for perforin extend to contributing to CD8 T cell activation during arenavirus infection and regulating antigen presentation of dendritic cells (DCs) [45], [46]. Previous work, in our lab, shows caspase-3 cleavage is not present until after BBB disruption has already occurred [18]. Given these new roles for perforin and lack of evidence for apoptosis, it is possible that during BBB disruption, perforin is acting in a non-classical mechanism. However, the exact mechanism by which perforin wielding CD8 T cells can induce BBB disruption is yet to be defined.

The contribution of molecules delivered by perforin beyond granzyme B also needs to be evaluated. For example, orphan granzymes have recently been proposed to play a role in pathogen clearance and cell-mediated death [47]. Nevertheless, based on the findings put forward in this manuscript, investigating mechanisms of perforin-dependent cytotoxicity are important to the development of novel therapeutic strategies to ameliorate pathology associated with BBB disruption in several devastating neurological disorders. The demonstration that CD8 T cells can serve as a sole source of perforin to induce BBB disruption will greatly aid in this process.

Acknowledgments

We would like to thank Dr. Adam Schrum (Mayo Clinic, Rochester, MN) for providing us with Ly5.1 congenic mice and Michael Hansen (Mayo Clinic, Rochester, MN) for technical assistance.

Author Contributions

Conceived and designed the experiments: HLJ RCW IP AJJ. Performed the experiments: HLJ RCW WAM FJ SJL. Analyzed the data: HLJ RCW WAM IP AJJ. Wrote the paper: HLJ RCW AJJ.

References

1. Brown H, Hien TT, Day N, Mai NT, Chuong LV, et al. (1999) Evidence of blood-brain barrier dysfunction in human cerebral malaria. Neuropathology and applied neurobiology 25: 331–340.
- View Article
- Google Scholar
2. Eugenin EA, Osiecki K, Lopez L, Goldstein H, Calderon TM, et al. (2006) CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS. The Journal of neuroscience : the official journal of the Society for Neuroscience 26: 1098–1106.
- View Article
- Google Scholar
3. Huber JD, Egleton RD, Davis TP (2001) Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends in neurosciences 24: 719–725.
- View Article
- Google Scholar
4. Lacerda-Queiroz N, Rodrigues DH, Vilela MC, Rachid MA, Soriani FM, et al. (2012) Platelet-activating factor receptor is essential for the development of experimental cerebral malaria. The American journal of pathology 180: 246–255.
- View Article
- Google Scholar
5. Marchi N, Granata T, Ghosh C, Janigro D (2012) Blood-brain barrier dysfunction and epilepsy: pathophysiologic role and therapeutic approaches. Epilepsia 53: 1877–1886.
- View Article
- Google Scholar
6. Marchi N, Johnson AJ, Puvenna V, Johnson HL, Tierney W, et al. (2011) Modulation of peripheral cytotoxic cells and ictogenesis in a model of seizures. Epilepsia 52: 1627–1634.
- View Article
- Google Scholar
7. Medana IM, Turner GD (2006) Human cerebral malaria and the blood-brain barrier. International journal for parasitology 36: 555–568.
- View Article
- Google Scholar
8. Minagar A, Alexander JS (2003) Blood-brain barrier disruption in multiple sclerosis. Multiple sclerosis 9: 540–549.
- View Article
- Google Scholar
9. Pirko I, Suidan GL, Rodriguez M, Johnson AJ (2008) Acute hemorrhagic demyelination in a murine model of multiple sclerosis. Journal of neuroinflammation 5: 31.
- View Article
- Google Scholar
10. Schneider SW, Ludwig T, Tatenhorst L, Braune S, Oberleithner H, et al. (2004) Glioblastoma cells release factors that disrupt blood-brain barrier features. Acta Neuropathologica 107: 272–276.
- View Article
- Google Scholar
11. Talavera D, Castillo AM, Dominguez MC, Gutierrez AE, Meza I (2004) IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers. The Journal of general virology 85: 1801–1813.
- View Article
- Google Scholar
12. Johnson AJ, Mendez-Fernandez Y, Moyer AM, Sloma CR, Pirko I, et al. (2005) Antigen-specific CD8+ T cells mediate a peptide-induced fatal syndrome. Journal of immunology 174: 6854–6862.
- View Article
- Google Scholar
13. McDole J, Johnson AJ, Pirko I (2006) The role of CD8+ T-cells in lesion formation and axonal dysfunction in multiple sclerosis. Neurological research 28: 256–261.
- View Article
- Google Scholar
14. Rodriguez M, Oleszak E, Leibowitz J (1987) Theiler's murine encephalomyelitis: a model of demyelination and persistence of virus. Critical reviews in immunology 7: 325–365.
- View Article
- Google Scholar
15. Johnson HL, Jin F, Pirko I, Johnson AJ (2013) Theiler's murine encephalomyelitis virus as an experimental model system to study the mechanism of blood-brain barrier disruption. Journal of neurovirology.
16. Johnson AJ, Njenga MK, Hansen MJ, Kuhns ST, Chen LP, et al. (1999) Prevalent class I-restricted T-cell response to the Thelier's virus epitope D-b : VP2(121-130) in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28. Journal of Virology 73: 3702–3708.
- View Article
- Google Scholar
17. Johnson AJ, Upshaw J, Pavelko KD, Rodriguez M, Pease LR (2001) Preservation of motor function by inhibition of CD8+ virus peptide-specific T cells in Theiler's virus infection. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 15: 2760–2762.
- View Article
- Google Scholar
18. Suidan GL, McDole JR, Chen Y, Pirko I, Johnson AJ (2008) Induction of blood brain barrier tight junction protein alterations by CD8 T cells. PloS one 3: e3037.
- View Article
- Google Scholar
19. Janigro D (2012) Are you in or out? Leukocyte, ion, and neurotransmitter permeability across the epileptic blood-brain barrier. Epilepsia 53: 26–34.
- View Article
- Google Scholar
20. Bukowski JF, Woda BA, Habu S, Okumura K, Welsh RM (1983) Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo. Journal of immunology 131: 1531–1538.
- View Article
- Google Scholar
21. Paya CV, Patick AK, Leibson PJ, Rodriguez M (1989) Role of natural killer cells as immune effectors in encephalitis and demyelination induced by Theiler's virus. Journal of immunology 143: 95–102.
- View Article
- Google Scholar
22. Wang T, Scully E, Yin Z, Kim JH, Wang S, et al. (2003) IFN-gamma-producing gamma delta T cells help control murine West Nile virus infection. Journal of immunology 171: 2524–2531.
- View Article
- Google Scholar
23. Kish DD, Gorbachev AV, Parameswaran N, Gupta N, Fairchild RL (2012) Neutrophil expression of Fas ligand and perforin directs effector CD8 T cell infiltration into antigen-challenged skin. Journal of immunology 189: 2191–2202.
- View Article
- Google Scholar
24. Suidan GL, Dickerson JW, Chen Y, McDole JR, Tripathi P, et al. (2010) CD8 T cell-initiated vascular endothelial growth factor expression promotes central nervous system vascular permeability under neuroinflammatory conditions. Journal of immunology 184: 1031–1040.
- View Article
- Google Scholar
25. Johnson HL, Chen Y, Jin F, Hanson LM, Gamez JD, et al. (2012) CD8 T Cell-Initiated Blood-Brain Barrier Disruption Is Independent of Neutrophil Support. Journal of immunology 189: 1937–1945.
- View Article
- Google Scholar
26. Denic A, Macura SI, Mishra P, Gamez JD, Rodriguez M, et al. (2011) MRI in rodent models of brain disorders. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 8: 3–18.
- View Article
- Google Scholar
27. Pirko I, Gamez J, Johnson AJ, Macura SI, Rodriguez M (2004) Dynamics of MRI lesion development in an animal model of viral-induced acute progressive CNS demyelination. NeuroImage 21: 576–582.
- View Article
- Google Scholar
28. Pirko I, Johnson AJ, Chen Y, Lindquist DM, Lohrey AK, et al. (2011) Brain atrophy correlates with functional outcome in a murine model of multiple sclerosis. NeuroImage 54: 802–806.
- View Article
- Google Scholar
29. Pirko I, Nolan TK, Holland SK, Johnson AJ (2008) Multiple sclerosis: pathogenesis and MR imaging features of T1 hypointensities in a [corrected] murine model. Radiology 246: 790–795.
- View Article
- Google Scholar
30. Robb RA (1999) 3-D visualization in biomedical applications. Annual review of biomedical engineering 1: 377–399.
- View Article
- Google Scholar
31. Robb RA (2001) The Biomedical Imaging Resource at Mayo Clinic. Ieee Transactions on Medical Imaging 20: 854–867.
- View Article
- Google Scholar
32. Hao J, Liu R, Piao W, Zhou Q, Vollmer TL, et al. (2010) Central nervous system (CNS)-resident natural killer cells suppress Th17 responses and CNS autoimmune pathology. The Journal of experimental medicine 207: 1907–1921.
- View Article
- Google Scholar
33. Xu W, Fazekas G, Hara H, Tabira T (2005) Mechanism of natural killer (NK) cell regulatory role in experimental autoimmune encephalomyelitis. Journal of neuroimmunology 163: 24–30.
- View Article
- Google Scholar
34. Zhang B, Yamamura T, Kondo T, Fujiwara M, Tabira T (1997) Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells. The Journal of experimental medicine 186: 1677–1687.
- View Article
- Google Scholar
35. Argaw AT, Gurfein BT, Zhang Y, Zameer A, John GR (2009) VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown. Proceedings of the National Academy of Sciences of the United States of America 106: 1977–1982.
- View Article
- Google Scholar
36. Johnson AJ, Njenga MK, Hansen MJ, Kuhns ST, Chen L, et al. (1999) Prevalent class I-restricted T-cell response to the Theiler's virus epitope Db:VP2121-130 in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28. Journal of virology 73: 3702–3708.
- View Article
- Google Scholar
37. Haque A, Best SE, Unosson K, Amante FH, de Labastida F, et al. (2011) Granzyme B expression by CD8+ T cells is required for the development of experimental cerebral malaria. Journal of immunology 186: 6148–6156.
- View Article
- Google Scholar
38. Tschopp J, Masson D, Stanley KK (1986) Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis. Nature 322: 831–834.
- View Article
- Google Scholar
39. Stewart SE, Kondos SC, Matthews AY, D'Angelo ME, Dunstone MA, et al. (2014) The perforin pore facilitates the delivery of cationic cargos. The Journal of biological chemistry 289: 9172–9181.
- View Article
- Google Scholar
40. Froelich CJ, Orth K, Turbov J, Seth P, Gottlieb R, et al. (1996) New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. The Journal of biological chemistry 271: 29073–29079.
- View Article
- Google Scholar
41. Metkar SS, Wang B, Aguilar-Santelises M, Raja SM, Uhlin-Hansen L, et al. (2002) Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity 16: 417–428.
- View Article
- Google Scholar
42. Pipkin ME, Lieberman J (2007) Delivering the kiss of death: progress on understanding how perforin works. Current opinion in immunology 19: 301–308.
- View Article
- Google Scholar
43. Knickelbein JE, Khanna KM, Yee MB, Baty CJ, Kinchington PR, et al. (2008) Noncytotoxic lytic granule-mediated CD8+ T cell inhibition of HSV-1 reactivation from neuronal latency. Science 322: 268–271.
- View Article
- Google Scholar
44. Revelo XS, Tsai S, Lei H, Luck H, Ghazarian M, et al. (2014) Perforin is a Novel Immune Regulator of Obesity Related Insulin Resistance. Diabetes.
45. Lykens JE, Terrell CE, Zoller EE, Risma K, Jordan MB (2011) Perforin is a critical physiologic regulator of T-cell activation. Blood 118: 618–626.
- View Article
- Google Scholar
46. Terrell CE, Jordan MB (2013) Perforin deficiency impairs a critical immunoregulatory loop involving murine CD8(+) T cells and dendritic cells. Blood 121: 5184–5191.
- View Article
- Google Scholar
47. Grossman WJ, Revell PA, Lu ZH, Johnson H, Bredemeyer AJ, et al. (2003) The orphan granzymes of humans and mice. Current opinion in immunology 15: 544–552.
- View Article
- Google Scholar

[ref1] 1. Brown H, Hien TT, Day N, Mai NT, Chuong LV, et al. (1999) Evidence of blood-brain barrier dysfunction in human cerebral malaria. Neuropathology and applied neurobiology 25: 331–340.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Eugenin EA, Osiecki K, Lopez L, Goldstein H, Calderon TM, et al. (2006) CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS. The Journal of neuroscience : the official journal of the Society for Neuroscience 26: 1098–1106.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Huber JD, Egleton RD, Davis TP (2001) Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends in neurosciences 24: 719–725.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Lacerda-Queiroz N, Rodrigues DH, Vilela MC, Rachid MA, Soriani FM, et al. (2012) Platelet-activating factor receptor is essential for the development of experimental cerebral malaria. The American journal of pathology 180: 246–255.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Marchi N, Granata T, Ghosh C, Janigro D (2012) Blood-brain barrier dysfunction and epilepsy: pathophysiologic role and therapeutic approaches. Epilepsia 53: 1877–1886.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Marchi N, Johnson AJ, Puvenna V, Johnson HL, Tierney W, et al. (2011) Modulation of peripheral cytotoxic cells and ictogenesis in a model of seizures. Epilepsia 52: 1627–1634.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref7] 7. Medana IM, Turner GD (2006) Human cerebral malaria and the blood-brain barrier. International journal for parasitology 36: 555–568.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref8] 8. Minagar A, Alexander JS (2003) Blood-brain barrier disruption in multiple sclerosis. Multiple sclerosis 9: 540–549.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref9] 9. Pirko I, Suidan GL, Rodriguez M, Johnson AJ (2008) Acute hemorrhagic demyelination in a murine model of multiple sclerosis. Journal of neuroinflammation 5: 31.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref10] 10. Schneider SW, Ludwig T, Tatenhorst L, Braune S, Oberleithner H, et al. (2004) Glioblastoma cells release factors that disrupt blood-brain barrier features. Acta Neuropathologica 107: 272–276.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref11] 11. Talavera D, Castillo AM, Dominguez MC, Gutierrez AE, Meza I (2004) IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers. The Journal of general virology 85: 1801–1813.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref12] 12. Johnson AJ, Mendez-Fernandez Y, Moyer AM, Sloma CR, Pirko I, et al. (2005) Antigen-specific CD8+ T cells mediate a peptide-induced fatal syndrome. Journal of immunology 174: 6854–6862.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref13] 13. McDole J, Johnson AJ, Pirko I (2006) The role of CD8+ T-cells in lesion formation and axonal dysfunction in multiple sclerosis. Neurological research 28: 256–261.
View Article
Google Scholar

[38] View Article

[39] Google Scholar

[ref14] 14. Rodriguez M, Oleszak E, Leibowitz J (1987) Theiler's murine encephalomyelitis: a model of demyelination and persistence of virus. Critical reviews in immunology 7: 325–365.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref15] 15. Johnson HL, Jin F, Pirko I, Johnson AJ (2013) Theiler's murine encephalomyelitis virus as an experimental model system to study the mechanism of blood-brain barrier disruption. Journal of neurovirology.

[ref16] 16. Johnson AJ, Njenga MK, Hansen MJ, Kuhns ST, Chen LP, et al. (1999) Prevalent class I-restricted T-cell response to the Thelier's virus epitope D-b : VP2(121-130) in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28. Journal of Virology 73: 3702–3708.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref17] 17. Johnson AJ, Upshaw J, Pavelko KD, Rodriguez M, Pease LR (2001) Preservation of motor function by inhibition of CD8+ virus peptide-specific T cells in Theiler's virus infection. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 15: 2760–2762.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref18] 18. Suidan GL, McDole JR, Chen Y, Pirko I, Johnson AJ (2008) Induction of blood brain barrier tight junction protein alterations by CD8 T cells. PloS one 3: e3037.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref19] 19. Janigro D (2012) Are you in or out? Leukocyte, ion, and neurotransmitter permeability across the epileptic blood-brain barrier. Epilepsia 53: 26–34.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref20] 20. Bukowski JF, Woda BA, Habu S, Okumura K, Welsh RM (1983) Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo. Journal of immunology 131: 1531–1538.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref21] 21. Paya CV, Patick AK, Leibson PJ, Rodriguez M (1989) Role of natural killer cells as immune effectors in encephalitis and demyelination induced by Theiler's virus. Journal of immunology 143: 95–102.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref22] 22. Wang T, Scully E, Yin Z, Kim JH, Wang S, et al. (2003) IFN-gamma-producing gamma delta T cells help control murine West Nile virus infection. Journal of immunology 171: 2524–2531.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref23] 23. Kish DD, Gorbachev AV, Parameswaran N, Gupta N, Fairchild RL (2012) Neutrophil expression of Fas ligand and perforin directs effector CD8 T cell infiltration into antigen-challenged skin. Journal of immunology 189: 2191–2202.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref24] 24. Suidan GL, Dickerson JW, Chen Y, McDole JR, Tripathi P, et al. (2010) CD8 T cell-initiated vascular endothelial growth factor expression promotes central nervous system vascular permeability under neuroinflammatory conditions. Journal of immunology 184: 1031–1040.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref25] 25. Johnson HL, Chen Y, Jin F, Hanson LM, Gamez JD, et al. (2012) CD8 T Cell-Initiated Blood-Brain Barrier Disruption Is Independent of Neutrophil Support. Journal of immunology 189: 1937–1945.
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref26] 26. Denic A, Macura SI, Mishra P, Gamez JD, Rodriguez M, et al. (2011) MRI in rodent models of brain disorders. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 8: 3–18.
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref27] 27. Pirko I, Gamez J, Johnson AJ, Macura SI, Rodriguez M (2004) Dynamics of MRI lesion development in an animal model of viral-induced acute progressive CNS demyelination. NeuroImage 21: 576–582.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref28] 28. Pirko I, Johnson AJ, Chen Y, Lindquist DM, Lohrey AK, et al. (2011) Brain atrophy correlates with functional outcome in a murine model of multiple sclerosis. NeuroImage 54: 802–806.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref29] 29. Pirko I, Nolan TK, Holland SK, Johnson AJ (2008) Multiple sclerosis: pathogenesis and MR imaging features of T1 hypointensities in a [corrected] murine model. Radiology 246: 790–795.
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref30] 30. Robb RA (1999) 3-D visualization in biomedical applications. Annual review of biomedical engineering 1: 377–399.
View Article
Google Scholar

[87] View Article

[88] Google Scholar

[ref31] 31. Robb RA (2001) The Biomedical Imaging Resource at Mayo Clinic. Ieee Transactions on Medical Imaging 20: 854–867.
View Article
Google Scholar

[90] View Article

[91] Google Scholar

[ref32] 32. Hao J, Liu R, Piao W, Zhou Q, Vollmer TL, et al. (2010) Central nervous system (CNS)-resident natural killer cells suppress Th17 responses and CNS autoimmune pathology. The Journal of experimental medicine 207: 1907–1921.
View Article
Google Scholar

[93] View Article

[94] Google Scholar

[ref33] 33. Xu W, Fazekas G, Hara H, Tabira T (2005) Mechanism of natural killer (NK) cell regulatory role in experimental autoimmune encephalomyelitis. Journal of neuroimmunology 163: 24–30.
View Article
Google Scholar

[96] View Article

[97] Google Scholar

[ref34] 34. Zhang B, Yamamura T, Kondo T, Fujiwara M, Tabira T (1997) Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells. The Journal of experimental medicine 186: 1677–1687.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref35] 35. Argaw AT, Gurfein BT, Zhang Y, Zameer A, John GR (2009) VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown. Proceedings of the National Academy of Sciences of the United States of America 106: 1977–1982.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref36] 36. Johnson AJ, Njenga MK, Hansen MJ, Kuhns ST, Chen L, et al. (1999) Prevalent class I-restricted T-cell response to the Theiler's virus epitope Db:VP2121-130 in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28. Journal of virology 73: 3702–3708.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref37] 37. Haque A, Best SE, Unosson K, Amante FH, de Labastida F, et al. (2011) Granzyme B expression by CD8+ T cells is required for the development of experimental cerebral malaria. Journal of immunology 186: 6148–6156.
View Article
Google Scholar

[108] View Article

[109] Google Scholar

[ref38] 38. Tschopp J, Masson D, Stanley KK (1986) Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis. Nature 322: 831–834.
View Article
Google Scholar

[111] View Article

[112] Google Scholar

[ref39] 39. Stewart SE, Kondos SC, Matthews AY, D'Angelo ME, Dunstone MA, et al. (2014) The perforin pore facilitates the delivery of cationic cargos. The Journal of biological chemistry 289: 9172–9181.
View Article
Google Scholar

[114] View Article

[115] Google Scholar

[ref40] 40. Froelich CJ, Orth K, Turbov J, Seth P, Gottlieb R, et al. (1996) New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. The Journal of biological chemistry 271: 29073–29079.
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref41] 41. Metkar SS, Wang B, Aguilar-Santelises M, Raja SM, Uhlin-Hansen L, et al. (2002) Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity 16: 417–428.
View Article
Google Scholar

[120] View Article

[121] Google Scholar

[ref42] 42. Pipkin ME, Lieberman J (2007) Delivering the kiss of death: progress on understanding how perforin works. Current opinion in immunology 19: 301–308.
View Article
Google Scholar

[123] View Article

[124] Google Scholar

[ref43] 43. Knickelbein JE, Khanna KM, Yee MB, Baty CJ, Kinchington PR, et al. (2008) Noncytotoxic lytic granule-mediated CD8+ T cell inhibition of HSV-1 reactivation from neuronal latency. Science 322: 268–271.
View Article
Google Scholar

[126] View Article

[127] Google Scholar

[ref44] 44. Revelo XS, Tsai S, Lei H, Luck H, Ghazarian M, et al. (2014) Perforin is a Novel Immune Regulator of Obesity Related Insulin Resistance. Diabetes.

[ref45] 45. Lykens JE, Terrell CE, Zoller EE, Risma K, Jordan MB (2011) Perforin is a critical physiologic regulator of T-cell activation. Blood 118: 618–626.
View Article
Google Scholar

[130] View Article

[131] Google Scholar

[ref46] 46. Terrell CE, Jordan MB (2013) Perforin deficiency impairs a critical immunoregulatory loop involving murine CD8(+) T cells and dendritic cells. Blood 121: 5184–5191.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref47] 47. Grossman WJ, Revell PA, Lu ZH, Johnson H, Bredemeyer AJ, et al. (2003) The orphan granzymes of humans and mice. Current opinion in immunology 15: 544–552.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Animals and ethics statement

Adoptive transfer

Induction of CNS vascular permeability using the PIFS model

Injection of rhodamine-dextran or FITC-albumin to assess permeability

Flow Cytometry

Rhodamine-dextran permeability assay

Confocal Microscopy

MRI Acquisition

Image Analysis

Statistical Analysis

Results

Adoptively transferred perforin competent CD8 T cells induce CNS vascular permeability

Model and adoptive transfer purity.

CNS permeability measured by small animal MRI and fluorescent molecule diffusion.

Increased astrocyte GFAP expression in perforin−/− mice reconstituted with perforin competent CD8 T cells

Disorganized tight junction proteins colocalize with increased CNS vascular permeability in perforin−/− mice reconstituted with perforin competent CD8 T cells

Discussion

Acknowledgments

Author Contributions

References

Increased astrocyte GFAP expression in perforin^−/− mice reconstituted with perforin competent CD8 T cells

Disorganized tight junction proteins colocalize with increased CNS vascular permeability in perforin^−/− mice reconstituted with perforin competent CD8 T cells