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The Journal of Immunology, 2007, 178, 7334-7343
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Peroxynitrite-Dependent Pathway Is Responsible for Blood-Brain Barrier Permeability Changes during a Central Nervous System Inflammatory Response: TNF-{alpha} Is Neither Necessary nor Sufficient1

Timothy W. Phares, Marzena J. Fabis, Christine M. Brimer, Rhonda B. Kean and D. Craig Hooper2

Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Elevated blood-brain barrier (BBB) permeability is associated with both the protective and pathological invasion of immune and inflammatory cells into CNS tissues. Although a variety of processes have been implicated in the changes at the BBB that result in the loss of integrity, there has been no consensus as to their induction. TNF-{alpha} has often been proposed to be responsible for increased BBB permeability but there is accumulating evidence that peroxynitrite (ONOO–)-dependent radicals may be the direct trigger. We demonstrate here that enhanced BBB permeability in mice, whether associated with rabies virus (RV) clearance or CNS autoimmunity, is unaltered in the absence of TNF-{alpha}. Moreover, the induction of TNF-{alpha} expression in CNS tissues by RV infection has no impact on BBB integrity in the absence of T cells. CD4 T cells are required to enhance BBB permeability in response to the CNS infection whereas CD8 T cells and B cells are not. Like CNS autoimmunity, elevated BBB permeability in response to RV infection is evidently mediated by ONOO–. However, as opposed to the invading cells producing ONOO– that have been implicated in the pathogenesis of CNS inflammation, during virus clearance ONOO– is produced without pathological sequelae by IFN-{gamma}-stimulated neurovascular endothelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The blood-brain barrier (BBB),3 a specialization of the vasculature in the CNS that renders it less permeable than the vasculature of the periphery, regulates the exchange of soluble factors and cells between the blood and CNS tissues (1). Alterations in BBB integrity are seen in various noninflammatory neurological diseases, such as certain types of brain cancer (2), and in association with the invasion of circulating immune/inflammatory cells into CNS tissues, as is the case for multiple sclerosis (3). Although enhanced BBB permeability is central to the development of a pathological CNS inflammatory response (4, 5, 6, 7), changes in BBB permeability can also occur without sequelae during a protective CNS immune response (8). For example, the clearance of the attenuated rabies virus (RV) CVS-F3 from the CNS follows increased BBB permeability that is associated with the infiltration of immune effectors into CNS tissues (8). Understanding the mechanisms that enhance BBB permeability in protective vs pathological CNS immunity is essential for the development of methodologies to deliver therapeutics to CNS tissues as well as to maintain BBB integrity where appropriate.

TNF-{alpha}, a proinflammatory cytokine that is a key regulator of a number of biological processes related to immune function, has been proposed as a trigger of enhanced BBB permeability (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). TNF-{alpha} is known to enhance expression of ICAM-1 on neurovascular endothelial cells (20, 21) which are constitutively positive for TNF-{alpha} receptors (22). Although this undoubtedly facilitates leukocyte extravasation into CNS tissues (23), the possibility that TNF-{alpha} induces a change in neurovascular endothelial cells that negatively impacts their barrier function is contentious (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27). In a number of animal studies, TNF-{alpha} administration to the CNS via different routes was found to result in elevated BBB permeability to a variety of markers (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In a number of contrasting studies, comparable administration of TNF-{alpha} did not increase BBB permeability (10, 14, 24, 25, 26, 27). The results of studies of the effect of TNF-{alpha} on in vitro models of the BBB have also been as polar (28, 29, 30, 31, 32, 33). Although it is possible that these discrepancies may be a consequence of variations in the models studied, it should be noted that none of this work examines conditions under which BBB permeability naturally occurs. Studies of the relationship between TNF-{alpha} and the loss of BBB integrity in multiple sclerosis, its animal correlate experimental allergic encephalomyelitis (EAE), the clearance of attenuated RV from CNS tissues, and bacterial meningitis have provided only correlative data (8, 34, 35). It is conceivable that the effects of TNF-{alpha} on permeability are indirect, mediated through the effects of this cytokine on the inflammatory response. For example, TNF-{alpha} is known to induce the expression of inducible NO synthase (iNOS; NOS-2) and may therefore promote BBB permeability changes through a NO-dependent pathway (5, 6, 7, 36). We and others have implicated peroxynitrite (ONOO–), the product of NO and superoxide, in the loss of BBB integrity in diverse in vivo and in vitro models (5, 6, 7, 37, 38). To provide further insight into the mechanism of enhanced BBB permeability, we have used mice lacking TNF-{alpha} (TNF–/–), B cells (JHD–/–), as well as T and B cells (RAG-2–/–) to establish the minimum requirements for BBB permeability changes in CNS immunity. Because TNF-{alpha} may contribute differently to BBB function in diverse CNS immune responses, we have studied two models of CNS immunity that differ in their etiology and pathogenicity: 1) EAE, an autoimmune CNS inflammatory condition where the loss of BBB integrity is critical to the development of the disease (5, 6) and 2) CVS-F3 RV infection where the development of enhanced BBB permeability is associated with the apathogenic clearance of virus from CNS tissues (8, 39).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

TNF–/– and wild-type control B6129F2/J mice were purchased from The Jackson Laboratory. RAG-2–/– and wild-type control 129/SvEv mice were obtained from Taconic Farms, whereas JHD–/– mice on a C57BL/6 background were provided by R. Hardy (Fox Chase Cancer Center, Philadelphia, PA). Mice were maintained under pathogen-free conditions and 8- to 12-wk-old mice of both sexes were used in the experiments. All procedures were conducted in accordance with federal guidelines under animal protocols approved by the Thomas Jefferson University Institutional Animal Care and Use Committee.

Virus infection

Mice were either infected intranasally (i.n.) or i.m. in the gastrocnemius with 105 focus-forming units of the attenuated CVS-F3 strain of RV in PBS. CVS-F3 is a laboratory RV strain that is not pathogenic for immunocompetent adult mice regardless of the route of infection (8, 40, 41). Despite spreading throughout the CNS, the virus is cleared by RV-specific immune mechanisms in the absence of any signs of neurological disease (8, 39).

Induction of EAE

Mice were immunized s.c. with 150 µg of rat myelin oligodendrocyte glycoprotein 33–55 (MOG35–55) peptide in CFA supplemented with 4 mg/ml Mycobacterium tuberculosis H37RA (Difco Laboratories). Mice also received 400 ng of pertussis toxin (List Biological Laboratories) i.p. on days 0 and 2. Mice were boosted 1 wk after the initial immunization with MOG35–55 peptide in CFA. The animals were scored for clinical signs of EAE daily. Scores were assigned on the basis of the presence of the following symptoms: 0, normal; 1, piloerection, tail weakness; 2, tail paralysis; 3, tail paralysis and hindlimb weakness; 4, tail paralysis and partial hindlimb paralysis; 5, complete hindlimb paralysis; 6, hind and forelimb paralysis. Where noted in the figure legends, MOG-immunized mice were infected i.m. with CVS-F3 5 days postimmunization.

Detection of virus-specific Abs

Sera from mice infected with CVS-F3 10 days previously were assessed for rabies-specific Abs by direct ELISA. Briefly, plates (Polysorb; Nalge Nunc International) were coated at room temperature with 100 µl of a 5 µg/ml stock solution of UV-inactivated Evelyn-Rockitnicki-Ableseth strain of RV and incubated overnight in a humidified chamber. The plates were washed with PBS containing 0.05% Tween 20 and blocked with 1% BSA in PBS for 2 h before the addition of the serum samples. Samples were diluted 1/4 in PBS and titrated 4-fold. Following a 2-h incubation at room temperature, plates were washed with PBS containing 0.05% Tween 20. Bound Ab was detected using peroxidase-conjugated anti-mouse IgG whole molecule (Sigma-Aldrich) with 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) as a substrate. Absorbance was read at 450 nm in a microplate spectrophotometer (Biotek) and the titer was calculated as the last dilution of serum in ELISA which gave an OD greater than one-half maximal absorbance.

Preparation of CNS tissues and assessment of BBB permeability

CNS tissues were prepared for quantitative RT-PCR analysis and BBB permeability was assessed as previously described using sodium-fluorescein (Na-F), a low molecular mass molecule (376 Da), to detect fluid-phase shifts between the circulation and CNS tissue (8). Briefly, mice received 100 µl of 10% Na-F in PBS i.p., cardiac blood was collected 10 min later, transcardially perfused, and spinal cords or brains were removed, with the latter separated into cerebral cortex and cerebellum. Tissues were homogenized in PBS and centrifuged. The clarified supernatant was used to assess Na-F content while the pellet was retained for RNA isolation. The amount of fluorescein in each sample was determined on a Cytofluor II fluorometer (PerSeptive Biosystems) using standards ranging from 125 to 4000 µg. Na-F accumulation in CNS tissue was normalized to the Na-F content in sera by (microgram fluorescence brain tissue/milligram of protein)/(microgram fluorescence sera/microliter of blood) and is expressed as a fold increase in fluorescence by comparison with the results obtained with normal control mice.

Real-time quantitative RT-PCR

Tissue pellets obtained during the assessment of BBB permeability were used to isolate RNA and quantify the expression levels of specific mRNAs in CNS tissues as previously described (8). Briefly, RNA was isolated from each tissue pellet and cDNA was synthesized from total RNA using dT15 primers and Moloney murine leukemia reverse transcriptase (Promega). Real-time quantitative RT-PCR was conducted on equal volumes of cDNA using specific primer and probe sets (8) and the TaqMan PCR Core Reagent kit (Applied Biosystems). Real-time quantitative PCR was performed using a Bio-Rad iCycler iQ Real-Time Detection System. Data were calculated based on a threshold cycle determined as the PCR cycle at which the fluorescent signal becomes higher than that of the background (cycles 2–10) plus 10 times the SD of the background. The number of copies of specific mRNAs in each sample was determined as previously described (8) and normalized to the mRNA copy number of the housekeeping gene L13 in that sample. Data are expressed as a fold increase ± SEM in copies of specific mRNA in each test group with the copy number obtained from unmanipulated mice taken as 1. To estimate virus replication, virus-specific nucleoprotein mRNA expression was measured by quantitative RT-PCR and expressed as a number of mRNA copies per copy of L13.

Immunohistochemistry

For immunohistochemical analysis, brains and spinal cords were snap-frozen in Tissue-Tek OCT Compound (Sakura Finetex) and sectioned at 10 µm using a Thermo Shandon cryostat. Sections to be assessed for ICAM-1 and CD4 expression were fixed in cold 80% acetone and then stained with either FITC-anti-ICAM (cat. no. 553252; BD Pharmingen) or PE-anti-CD4 (cat. no. 01064; BD Pharmingen) for 1 h. Nitrotyrosine (NT) was detected as previously described (5). Briefly, sections were fixed in 4% paraformaldehyde, incubated for 1.5 h with polyclonal rabbit anti-NT (cat. no. 06-284; Upstate Biotechnology), and then developed using the peroxidase antiperoxidase method (Sternberger Monoclonals) and 3'3-diaminobenzidine (brown) as substrate. Sections assessed for lesion formation were fixed in 4% paraformaldehyde and then stained with Harris’ hematoxylin (0.1%) and eosin Y (1%). Photographs were taken with a Nikon digital camera on a Leitz Microlab microscope and are reproduced at the final magnification noted in the figure legends.

Adoptive transfers

Donor 129/SvEv mice were infected i.n. with CVS-F3 and spleens and lymph nodes harvested 7 days later. Single-cell suspensions were obtained by teasing the tissues through a stainless steel mesh and erythrocytes were removed by isotonic shock. Spleen and lymph node cells were pooled. Cell subsets were isolated by negative selection using MACS technology with either the CD4+ T Cell Isolation kit (cat. no. 130-090-860; Miltenyi Biotec) or the CD8+ T Cell Isolation kit (cat. no. 130-090-859; Miltenyi Biotec). Recipient RAG-2–/– mice, infected 7 days previously, were either left untreated or injected i.v. in the tail vein with 8 x 106 CD4+8– T lymphocytes, CD4–8+ lymphocytes, or unselected mononuclear cells.

Isolation of primary murine CNS microvessel cells

Primary murine CNS microvessel cells were isolated and cultured as previously described (42). Briefly, brains were removed from either 129/SvEv or TNF–/– mice and rinsed with MCDB 131 medium (Invitrogen Life Technologies) supplemented with 2% FBS (Atlanta Biologicals), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). Tissues were homogenized and centrifuged at 10,000 x g for 15 min at 4°C in 15% dextran. The pellet was then digested in 0.1% collagenase/dispase (Roche) for 1.5 h at 37°C, centrifuged, resuspended in 45% Percoll (Sigma-Aldrich), and centrifuged at 20,000 x g for 10 min at 4°C. Microvessels were collected from the top of the 45% Percoll, washed three times with PBS, and cultured in MCDB 131 medium supplemented with 30 µg/ml ECGS (Sigma-Aldrich), 10% FBS, 15 U/ml heparin, 325 µg/ml glutathione (Sigma-Aldrich), 50 µM 2-ME (Sigma-Aldrich), 4 mM L-glutamine (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin. After 10–22 days of culture, dispersed cells were plated on either rat tail collagen, type 1 (BD Biosciences) coated 0.8-µm pore size transwells (Corning) or in 48-well flat-bottom plates at 37°C with 5% CO2.

Assessment of BBB permeability and ONOO– production in vitro

To examine the effects of cytokine treatment on BBB permeability, monolayers of primary murine CNS microvessel cells cultured on transwells were treated with either 20 ng/ml mouse recombinant IFN-{gamma} (BD Pharmingen) or TNF-{alpha} (eBioscience) for 8 min. Permeability was assessed by measuring the amount of Na-F (50 µg/ml) diffusing from the inner transwell chamber across cell monolayers into the outer chamber. Uric acid (UA) (200 µM), a known scavenger of ONOO–-dependent radicals (6), was added to the inner and outer chambers of transwell cultures to determine whether the IFN-{gamma}-induced elevation in permeability is mediated through the production of ONOO–. The production of ONOO– by IFN-{gamma}-treated CNS microvessel cells grown in flat-bottom plates was assessed by measuring the conversion of added dihydrorhodamine123 (DHR123) (Molecular Probes) to fluorescent rhodamine 123 as described previously (6). Briefly, DHR123 (5 µM) was added to cultures 6 min subsequent to the addition of IFN-{gamma} (40 ng/ml) and incubated for 2 min at 37°C. Supernatants were then collected and fluorescence was measured in a microplate fluorometer with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Statistical analyses

Results are expressed as the mean ± SEM for groups of 5–39 mice. Evaluation of the significance of differences between groups was performed using the unpaired t test or, for nonparametric results, the Mann-Whitney U test. Graphs were plotted and statistics were assessed using GraphPad Prism 3.0 (GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The inflammatory responses and BBB permeability changes associated with the clearance of virus from CNS tissues are intact in mice lacking TNF-{alpha}

In a prior investigation, we established that TNF-{alpha} expression is elevated in the CNS of RV-infected mice before the onset of the increased BBB permeability (8). To examine more comprehensively the putative roles of TNF-{alpha} in the development of enhanced BBB permeability and CNS inflammation, we first determined whether the clearance of CVS-F3 RV from the CNS is compromised in TNF–/– mice. As shown in Fig. 1A, the absence of TNF-{alpha} has no impact on the replication and spread of RV in the CNS. Nucleoprotein-specific mRNA, an index of RV replication, peaks at comparable levels in the CNS tissues of wild-type and TNF–/– mice at 8 days postinfection. Nucleoprotein mRNA levels then drop >100-fold in both strains of mice by day 18 postinfection (Fig. 1A). This evidently coincides with the development of antiviral immunity in wild-type and TNF–/– mice as various immune parameters including BBB permeability are elevated by 10 days postinfection (Figs. 1B and 2). BBB permeability becomes significantly enhanced in both wild-type and TNF–/– mice by 8 days postinfection, becoming maximal 2 days later (Fig. 1B). However, extensive changes in BBB integrity appear to be prolonged through day 12 postinfection in the TNF–/– mice (Fig. 1B) and RV nucleoprotein message levels remain slightly higher in the TNF–/– mice between days 18 and 24 postinfection (Fig. 1A). Nevertheless, no difference in either the proinflammatory response or cell accumulation in the CNS tissues is seen between wild-type and TNF–/– mice at day 10 postinfection (Fig. 2). Equivalent levels of mRNAs specific for ICAM-1, IFN-{gamma}-inducible protein-10 (IP-10), MCP-1, RANTES, MIP-1{alpha}, and MIP-1beta are expressed in the CNS tissues of CVS-F3 RV-infected wild-type and TNF–/– mice (Fig. 2A). Elevations in CD4, CD8, CD11b, CD19 mRNA (Fig. 2B), and virus-specific Ab (Fig. 2C) are also comparable in both strains of mice.


Figure 1
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  		FIGURE 1. The extent of BBB permeability changes and kinetics of RV CVS-F3 replication in CNS tissues are comparable in wild-type and TNF–/– mice. Groups of wild-type and TNF–/– mice (n = 5 or 6) were infected i.n. with CVS-F3 RV and euthanized at the indicated time points to assess virus replication and the extent of BBB permeability changes in the cerebellum as described in Materials and Methods. Virus replication was assessed by quantifying RV nucleoprotein mRNA levels and is expressed as the mean ± SEM copies of rabies nucleoprotein mRNA per copy of the housekeeping gene L13 mRNA in the CNS tissues (A). In B, BBB permeability changes are presented as the mean ± SEM fold increase in Na-F uptake in the tissues with the levels from uninfected mice taken as 1. Statistically significant differences between the measures in infected, by comparison with uninfected, mice determined by the Mann-Whitney U test are denoted by * (p < 0.01) and ** (p < 0.005). Statistically significant differences in the mean expression levels between the two strains of mice at a given time point established by the unpaired t test are denoted by # (p < 0.05) and ## (p < 0.005).

 

Figure 2
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  		FIGURE 2. TNF–/– mice show no deficit in the CNS inflammatory and humoral responses to CVS-F3 infection. The CVS-F3 RV-infected wild-type and TNF–/– mice studied at the day 10 postinfection time point in Fig. 1 were assessed for RV-specific serum Ab levels and the expression of select mRNAs in the cerebellum as detailed in Materials and Methods. mRNA expression levels were determined by quantitative RT-PCR and are expressed as the fold increase ± SEM in copies of specific mRNA with the copies detected in uninfected mouse cerebellum taken as 1, all values corrected for total mRNA content using the housekeeping gene L13. A, The results for ICAM-1, IP-10, MCP-1, RANTES, MIP-1{alpha}, and MIP-1beta while B shows those for CD4, CD8, CD11b, and CD19. Levels of virus-specific Abs are presented in C as the geometric mean ± SEM of the half maximal titer. Statistically significant differences in the mean expression levels between the two strains of mice established by the unpaired t test are denoted by # (p < 0.05) and ## (p < 0.005).

 
BBB permeability changes induced by MOG immunization are comparable in wild-type and mice lacking TNF-{alpha}

Although TNF-{alpha} expression is evidently not required for the development of enhanced BBB permeability in the clearance of RV, it is conceivable that this cytokine may make a necessary contribution to alterations in BBB integrity where the neuroinflammatory response has a different etiology and outcome. Unlike CVS-F3 infection which is apathogenic in normal adult mice (8, 39), immunization with myelin Ags such as myelin basic protein and MOG in EAE elicits clinical signs of neurological disease in association with the appearance of TNF-{alpha} in the CNS tissues and the loss of BBB integrity (43). The onset of clinical signs of EAE is significantly delayed and disease severity substantially reduced in MOG-immunized TNF–/– mice by comparison with MOG-immunized wild-type mice (Fig. 3A). We were concerned that differences in the course of EAE between wild-type and TNF–/– mice may negatively impact the interpretation of BBB permeability data. Therefore, we investigated whether CVS-F3 infection, with its strong CNS proinflammatory components, could be used to promote the more rapid onset of severe EAE in MOG-immunized TNF–/– mice. As shown in Fig. 3A, infection of TNF–/– mice with CVS-F3 5 days after immunization with MOG results in the development of clinical signs of EAE with kinetics and severity indistinguishable from wild-type mice. Although CVS-F3 RV infection at 5 days post-MOG immunization accelerates the development of EAE in wild-type mice, there is no effect on the peak severity of the disease (Fig. 3A). Nevertheless, all four groups of mice showed comparable extents of BBB permeability to Na-F when signs of EAE appeared, regardless of onset or severity (Fig. 3B). To establish whether cell accumulation in the CNS tissues, as opposed to the loss of BBB integrity, may be responsible for the clinical severity of EAE, we assessed spinal cord tissues from TNF–/– mice infected with CVS-F3, immunized with MOG, or infected with CVS-F3 and immunized with MOG, as well as MOG-immunized wild-type mice (Fig. 4). Lesion activity, which is not seen in CVS-F3 infected mice (Fig. 4A), is limited in MOG-immunized TNF–/– mice (Fig. 4B) by comparison with similarly immunized wild-type controls (Fig. 4D). CVS-F3 infection considerably exacerbates lesion activity in MOG-immunized TNF–/– mice (Fig. 4C).


Figure 3
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  		FIGURE 3. Despite no difference in the loss of BBB integrity MOG-immunized TNF–/– mice fail to develop EAE with severity equivalent to wild-type mice unless infected with CVS-F3. Wild-type (n = 13–39) and TNF–/– (n = 8–22) mice were either immunized with MOG (EAE) or immunized and then 5 days later infected with CVS-F3 RV (EAE + RV) as detailed in Materials and Methods. The mice were scored daily and euthanized at peak disease severity (wild-type EAE, 4.6 ± 0.28 SEM; wild-type EAE + RV, 4.4 ± 0.63 SEM; TNF–/– EAE, 1.8 ± 0.47 S.E.M; TNF–/– EAE + RV, 4.5 ± 0.30 S.E.M.) to assess BBB permeability in spinal cord tissue as described in the Materials and Methods. In A, clinical signs of EAE were scored using a severity scale from 0 to 6 and are presented as the mean clinical score ± SEM. In B, BBB permeability changes are presented as the mean ± SEM fold increase in Na-F uptake in the tissues with the levels from uninfected mice taken as 1. The BBB permeability of each of the test groups is significantly higher statistically than that of unmanipulated mice as determined by the Mann-Whitney U test (p < 0.001) while there is no significant difference between the test groups.

 

Figure 4
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  		FIGURE 4. EAE severity is associated with the extent of lesion activity in spinal cord tissues. Sections from the spinal cord tissues of CVS-F3 RV-infected TNF–/– mice (A), MOG-immunized TNF–/– mice (B), MOG-immunized, CVS-F3 RV-infected TNF–/– mice (C), or MOG-immunized wild-type mice (D) were prepared and stained with Harris’ hematoxylin and eosin Y as described in Materials and Methods. The general extent of lesion formation found throughout the tissues is shown in the primary photomicrographs. Single lesions, identified by arrows, are shown at higher magnification in the insets. Photomicrographs are presented at a magnification of ~x30 (primary) or ~x80 (inset).

 
TNF-{alpha} production in the CNS fails to trigger BBB permeability changes in the absence of T cells

The fact that TNF-{alpha} is dispensable for the induction of BBB permeability changes in CVS-F3 clearance and EAE does not prove that this proinflammatory cytokine cannot trigger the loss of BBB integrity. Unlike EAE where the appearance of TNF-{alpha} in CNS tissues is evidently dependent on the loss of BBB integrity (43), CVS-F3 infection induces TNF-{alpha} in CNS tissues before the onset of enhanced BBB permeability (8) and is therefore more appropriate to the study of the relationship between TNF-{alpha} and BBB function. Mice deficient in B cells (JHD–/–) or B and T cells (RAG-2–/–), as expected from their inability to produce virus-neutralizing Ab, fail to clear RV from the CNS (39). To determine whether the immunological deficits in these strains of mice also impact the putative relationship between TNF-{alpha} and BBB integrity, we assessed CNS TNF-{alpha} mRNA levels and BBB permeability to Na-F following infection of these mice with CVS-F3. As shown in Fig. 5A, TNF-{alpha} mRNA levels are significantly elevated in the CNS tissues of both RAG-2–/– and JHD–/– mice by 8 days postinfection. Although the elevations in TNF-{alpha} mRNA levels remain constant over the course of infection in RAG-2–/– mice, a continued increase is seen in JHD–/– animals (Fig. 5A). Despite similarly increased TNF-{alpha} mRNA levels in the CNS tissues between days 8 and 10 postinfection, only the JHD–/– mice develop enhanced BBB permeability (Fig. 5B).


Figure 5
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  		FIGURE 5. BBB permeability changes do not occur in the absence of T cells. Groups of RAG-2–/– and JHD–/– mice (n = 5 or 6) were infected i.n. with CVS-F3 RV and euthanized at the indicated time points to assess levels of mRNA specific for TNF-{alpha} and BBB permeability in the cerebellum as described in Materials and Methods. In A, levels of TNF-{alpha} expression are presented as the fold increase ± SEM in copies of specific mRNA in the tissues with the levels from uninfected mice taken as 1. In B, BBB permeability changes are presented as the fold increase in Na-F uptake in the tissues with levels from uninfected mice taken as 1. Statistically significant differences between the measures in infected by comparison with uninfected mice, determined by the Mann-Whitney U test, are denoted by * (p < 0.01) and ** (p < 0.005). Significant differences in the mean TNF-{alpha} mRNA expression levels between the two strains of mice at a given time point established by the unpaired t test are denoted by # (p < 0.05).

 
Conceivably, a defect in the capacity of the neurovascular endothelial cells of RAG-2–/– mice to respond to TNF-{alpha} could be responsible for the dissociation between its production and the induction of elevated BBB permeability. The finding that the expression of ICAM-1 is similar on the neurovascular endothelial cells of both CVS-F3 RV-infected RAG-2–/– and wild-type mice argues against this possibility (Fig. 6). Moreover, the expression levels of mRNAs specific for a variety of proinflammatory chemokines, including IP-10, MCP-1, RANTES, MIP-1{alpha}, and MIP-1beta, as well as ICAM-1, are strongly elevated in CVS-F3 RV-infected RAG-2–/– mice to levels approaching those of CVS-F3 RV-infected wild type (Fig. 7).


Figure 6
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  		FIGURE 6. ICAM-1 is expressed by the neurovasculature of CVS-F3 RV-infected RAG-2–/– mice. Sections from the cerebellum of RAG-2–/– (A and B) and wild-type (C) mice either uninfected (A) or infected with CVS-F3 10 days previously (B and C) were stained with FITC-conjugated ICAM-1 specific Ab and photographed as detailed in Materials and Methods. Photomicrographs are presented at a magnification of ~x220.

 

Figure 7
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  		FIGURE 7. CVS-F3 infection induces the expression of a variety of proinflammatory markers in the CNS of RAG-2–/– mice. Levels of mRNAs specific for ICAM-1, IP-10, MCP-1, RANTES, MIP-1{alpha}, and MIP-1beta were assessed in the cerebellum of wild-type and RAG-2–/– mice infected with CVS-F3 RV 10 days previously as detailed in Materials and Methods. mRNA expression is presented as the fold increase ± SEM in copies of specific mRNA in the cerebellum with the copy number detected in similar tissues from uninfected mice taken as 1. Copies of mRNA in each of the test groups are significantly higher statistically than that of uninfected mice as determined by the Mann-Whitney U test (p < 0.005). Statistically significant differences in the mean expression levels between the two strains of mice at the given time point established by the unpaired t test are denoted by # (p < 0.05) and ## (p < 0.005).

 
CD4 T cells are required for the induction of enhanced BBB permeability in response to neurotrophic virus infection

The fact that CVS-F3 RV infection triggers BBB permeability changes in JHD–/– but not RAG-2–/– mice implies that the activity of a T cell subset may be necessary for this process. To determine whether this is the case, we adoptively transferred different lymphocyte populations from CVS-F3 RV-infected wild-type mice into CVS-F3 RV-infected RAG-2–/– recipients. As shown in Fig. 8A, increased BBB permeability to Na-F develops in CVS-F3 RV-infected RAG-2–/– mice reconstituted with either unselected mononuclear cells or isolated CD4+8– T lymphocytes, but not with CD4–8+ T cells. Of note, with respect to the putative relationship between BBB function and TNF-{alpha}, the induction of BBB permeability in RV-infected RAG-2–/– mice subsequent to CD4+8– T cell transfer is not accompanied by an increase in the expression of TNF-{alpha} mRNA in CNS tissues (Fig. 8B).


Figure 8
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  		FIGURE 8. BBB permeability changes in response to CVS-F3 infection are CD4 T cell dependent. Groups of wild-type and RAG-2–/– mice (n = 5–10) were infected i.n. with CVS-F3 RV. At 7 days postinfection, lymphocytes from infected wild-type mice were isolated and either unselected cells, CD4+8– T cells, or CD4–8+ T cells were transferred into the infected RAG-2–/– mice as detailed in Materials and Methods. Six days following adoptive transfer, mice were euthanized and BBB permeability and levels of TNF-{alpha}-specific mRNA were assessed in the cerebellum as described in Materials and Methods. BBB permeability changes in (A) are presented as the fold increase in Na-F uptake in the tissues with the levels from uninfected mice taken as 1. Levels of TNF-{alpha} expression are expressed as the fold increase ± SEM in copies of specific mRNA in the tissues with the levels from uninfected mice taken as 1 (B). Statistically significant differences in Na-F uptake between the different groups of CVS-F3-infected RAG-2–/– mice and uninfected RAG-2–/– mice, determined by the Mann-Whitney U test, are denoted by *** (p < 0.001). Significant differences in the mean expression levels of TNF-{alpha} mRNA between CVS-F3-infected RAG-2–/– mice that received cells and those that did not, established by the unpaired t test, are denoted by ### (p < 0.001).

 
IFN-{gamma} induces enhanced BBB permeability through a ONOO–-dependent pathway

In our previous studies of CVS-F3 clearance from the CNS, we noted regional and temporal correlations between increases in CD4 T cell accumulation, IFN-{gamma} expression, and BBB permeability (8). The finding that the adoptive transfer of CD4 T cells is required to enhance BBB permeability in CVS-F3 RV-infected RAG-2–/– mice led us to investigate the potential relationship between the T cell product IFN-{gamma} and BBB integrity further. At different times after CVS-F3 infection, wild-type mice were assessed for the extent of BBB permeability to Na-F and for the expression of mRNAs specific for IFN-{gamma} and markers of other potential triggers of BBB permeability changes including TNF-{alpha}, iNOS, and IL-6. As shown in Fig. 9A there is a linear correlation between the extent of BBB permeability to Na-F and the appearance of IFN-{gamma} mRNA in CNS tissues from CVS-F3 RV-infected wild-type mice. In contrast, no correlation is evident between BBB permeability and the levels of expression of mRNAs specific for TNF-{alpha}, iNOS, or IL-6 in the same tissues (Fig. 9A, inset table). To examine whether IFN-{gamma} may have the capacity to directly induce BBB permeability, we added this cytokine to monolayers of disaggregated primary murine CNS microvessel cells with barrier function in vitro. Significantly enhanced permeability to Na-F rapidly develops following the addition of IFN-{gamma} but not TNF-{alpha} (Fig. 9B). As shown in Fig. 9C, over the same time frame IFN-{gamma} treatment stimulates the release of ONOO–, detected by the conversion of DHR123 to rhodoamine123, by primary neurovascular endothelial cells prepared from TNF–/– mice. ONOO–, which has previously been implicated in the induction of enhanced BBB permeability (5, 6, 7, 37), is likely to mediate the elevated permeability triggered by IFN-{gamma} as UA, an inhibitor of certain ONOO– chemistries such as tyrosine nitration (6), prevents IFN-{gamma}-mediated BBB permeability changes in vitro (Fig. 9D).


Figure 9
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  		FIGURE 9. IFN-{gamma} induces BBB permeability through a ONOO–-dependent pathway. The extent of BBB permeability changes and the expression of IFN-{gamma}, TNF-{alpha}, iNOS, and IL-6 mRNA in the cerebellum were assessed in 129/SvEv mice infected i.n. with CVS-F3 RV at 6, 8, 10, 12, 18, and 24 days postinfection as detailed in Materials and Methods. In A, the average increase in Na-F accumulation is plotted against the average IFN-{gamma} mRNA copy number in each tissue sample. Numbers above the symbols indicate time points where BBB permeability was significantly higher than controls by the Mann-Whitney U test (p < 0.001). Regression analysis shows that the relationship between Na-F uptake and IFN-{gamma} mRNA levels is linear (r2 = 0.98). The inset table in A shows the results of linear regression analysis of the relationship between Na-F uptake and TNF-{alpha}, iNOS, as well as IL-6 mRNAs. The data expressed in B and D were obtained with primary 129SvEv mouse CNS microvessel cells cultured as described in Materials and Methods. B, The results of TNF-{alpha} and IFN-{gamma} treatment on Na-F diffusion across cells grown with barrier function. Statistically significant differences in the mean amount of Na-F leakage between untreated and treated cells by the unpaired t test are denoted by *** (p < 0.001). In C, evidence of ONOO– production by primary microvessel cells prepared from TNF–/– mice treated with IFN-{gamma} is shown. The conversion of DHR123 to fluorescent rhodamine 123 was used as an indicator of ONOO– activity as detailed in Materials and Methods and data are expressed as the mean ± SEM fluorescence of rhodamine 123. A significant difference in the mean fluorescence between treated and untreated cells by the unpaired t test is denoted by *** (p < 0.001). In D, the effects of UA treatment on IFN-{gamma}-induced permeability of cultures prepared as described for B is shown. Cultures were treated with IFN-{gamma} with and without UA. Significant differences determined by the unpaired t test in the mean amount of Na-F leakage between untreated and IFN-{gamma}-treated cultures are denoted by *** (p < 0.001) and between the latter in the presence and absence of UA as ## (p < 0.005).

 
CD4 T cells and NT colocalize in regions of enhanced neurovascular permeability

If enhanced BBB permeability in the cerebellum of CVS-F3 RV-infected mice is a consequence of CD4 T cell-induced ONOO– production by neurovascular endothelial cells, it is expected that NT, a product of the reaction of ONOO– with tyrosine (44), would accumulate in the vicinity of invading CD4 T cells. As shown in Fig. 10, this is indeed the case. Visible amounts of Na-F do not leak from the circulation into the cerebellum of normal mice but Na-F accumulation can readily be seen in sections from the CVS-F3 RV-infected cerebellum (Fig. 10, A and B). CD4-positive T cells and NT staining is evident in sections prepared from permeable areas of the CVS-F3 RV-infected cerebellum, however, not in tissues from normal mice (Fig. 10, C–F). When photographs of consecutive sections stained for CD4 and NT are superimposed, it is evident that these markers colocalize, predominantly around blood vessels (Fig. 10F, inset).


Figure 10
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  		FIGURE 10. CD4 T cells and NT in the neurovasculature of CVS-F3 RV-infected mice. Consecutive sections from the cerebellum of Na-F-treated mice either uninfected (A, C, and E) or infected with CVS-F3 8 days previously (B, D, and F) were stained for CD4 (C and D) and NT (E and F) as detailed in Materials and Methods. In A and B, the accumulation of Na-F in the cerebellum is viewed using a fluorescent microscope. Staining for CD4 and NT was generally detected around blood vessels from CVS-F3 RV-infected animals (D and F) and absent in uninfected animals (C and E). To demonstrate colocalization, photomicrographs of CD4 and NT were superimposed and presented as an inset in F. Photomicrographs are presented at a magnification of ~x30 (A and B) or ~x200 (C–F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TNF-{alpha} can contribute to CNS inflammation through a variety of mechanisms. Either directly or indirectly, this proinflammatory cytokine induces the expression of a number of chemokines and other cytokines (45, 46), the activation of matrix metalloproteinases (47, 48), and adhesion molecule expression on the neurovasculature (20, 21). Although many of these activities are likely to influence BBB function, there is controversy regarding whether or not TNF-{alpha} has a direct impact on BBB integrity (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). For the most part, the effects of TNF-{alpha} on BBB integrity have been examined in experiments involving TNF-{alpha} addition to in vitro endothelial cell monolayers or in vivo delivery using diverse recipient species and methodologies to assess BBB permeability (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). To resolve the issue of TNF-{alpha}’s involvement in the loss of BBB integrity, we have adopted a different approach: studies in animal models where enhanced BBB permeability occurs in association with protective or pathogenic CNS immunity.

Mice lacking TNF-{alpha} show no deficit in the enhancement of BBB permeability following CVS-F3 RV infection or immunization with MOG. From the opposite perspective, the expression of TNF-{alpha} becomes elevated in CVS-F3 RV-infected RAG-2–/– mice in the absence of any change in BBB integrity. We conclude that TNF-{alpha} is neither required for the loss of BBB integrity that occurs in CNS immune responses nor directly induces enhanced BBB permeability. These conclusions are supported by the observation that TNF-{alpha} expression in the CNS tissues does not differ between CVS-F3 RV-infected RAG-2–/– mice and similar animals with enhanced BBB permeability following T cell transfer.

TNF-{alpha} evidently does not play an essential role in the loss of BBB integrity in CVS-F3 clearance or EAE. However, while TNF-{alpha} is not required for a protective immune response in CVS-F3 RV-infected mice, the elevation in BBB permeability is prolonged in CVS-F3-infected TNF–/– mice and a somewhat higher level of RV nucleoprotein mRNA is seen in their CNS tissues at 18–24 days postinfection by comparison with wild-type mice. More strikingly, in the absence of TNF-{alpha} the onset of EAE is delayed and the severity of the disease reduced despite there being no difference in the extent of BBB permeability between MOG-immunized TNF–/– and wild-type mice. A number of prior studies have demonstrated that the onset or severity of EAE in TNF–/– mice can be altered (49, 50, 51, 52, 53, 54, 55). The reduced expression of certain chemokines and chemokine receptors in TNF–/– mice with EAE has led to the suggestion that a reduction in the severity of EAE in these animals may be the result of limitations in the CNS proinflammatory response (54). A reduced proinflammatory response may in turn be the cause of a deficit in the invasion of immune/inflammatory cells into the CNS parenchyma seen in TNF–/– mice (51, 53). This possibility is supported by the more extensive CNS inflammation seen when MOG-immunized TNF–/– mice are infected with CVS-F3, which independently triggers the expression of proinflammatory mediators.

In EAE, the CNS proinflammatory response follows the loss of BBB integrity (43). This contrasts with CVS-F3 infection where a strong proinflammatory response occurs in the CNS tissues in the absence of BBB permeability changes as evidenced by our studies of RAG-2–/– mice. Moreover, mice lacking TNF-{alpha} show no apparent defect in the proinflammatory response to CVS-F3 RV infection or in the resulting accumulation of immune/inflammatory cells in CNS tissues. This suggests that the response of CNS-resident cells to the virus infection overcomes any limitation in the CNS proinflammatory response due to the absence of TNF-{alpha} and that CVS-F3 infection can substitute for TNF-{alpha} in this regard. As expected, if the reduction in the severity of EAE in TNF–/– mice is due to the absence of the proinflammatory activities of TNF-{alpha}, CVS-F3 infection of MOG-immunized TNF–/– mice resulted in the development of severe EAE resembling that of MOG-immunized wild-type animals. We speculate that through the induction of proinflammatory mediators, RV infection targets the invasion of MOG-induced inflammatory cells deeper into the CNS parenchyma of TNF–/– mice resulting in more severe disease. Also noteworthy from these studies is that CVS-F3 infection does not increase the magnitude of BBB leakage in MOG-immunized TNF–/– mice. Together with the fact that BBB permeability does not differ between MOG-immunized TNF–/– and wild-type mice, this leads us to conclude that while the loss of BBB integrity is required for the development of EAE, the severity of the disease is not merely a direct consequence of the extent of BBB permeability changes. The degree of lesion formation is evidently more important.

If TNF-{alpha} is not a direct trigger of enhanced BBB permeability in CVS-F3 infection and EAE, what is? The fact that CVS-F3 infection of RAG-2–/– mice fails to trigger the loss of BBB integrity indicates that, in addition to TNF-{alpha}, the chemokines and other factors that are produced by CNS-resident cells in the innate response to the infection do not directly contribute to BBB permeability changes. These include IP-10, MCP-1, RANTES, MIP-1{alpha}, and MIP-1beta. T but not B cells are necessary as enhanced BBB permeability develops in CVS-F3-infected JHD–/– mice. The T cells involved are identified as CD4+8– T lymphocytes because the transfer of CD4 but not CD8 T cells is sufficient to confer the capacity to mediate BBB permeability changes on CVS-F3 RV-infected RAG-2–/– mice. The development of enhanced BBB permeability in CVS-F3 RV-infected RAG-2–/– mice following the adoptive transfer of CD4 T cells also argues against the possibility that these mice have a developmental defect in the capacity to open the BBB apart from the absence of the T cells.

Regional differences in the extent of BBB permeability changes observed during the clearance of CVS-F3 are paralleled by disparate levels of CD4 and IFN-{gamma} mRNA (8). This led us to suspect that IFN-{gamma} may be the trigger of enhanced BBB permeability in these animals. A nearly 1:1 correlation between the extent of Na-F accumulation and the level of IFN-{gamma} mRNA in the cerebellum of CVS-F3 RV-infected mice supports this hypothesis. To test this possibility, we added IFN-{gamma} to an in vitro BBB prepared from primary cells derived from CNS microvessels. Permeability to Na-F developed within 8 min of IFN-{gamma} addition while TNF-{alpha} treatment had no effect. The rapid onset of enhanced permeability suggests that the mechanism may be mediated through the modification of pre-existing components of the BBB. One possibility is through a ONOO–-dependent chemical reaction, which is known to contribute to enhanced BBB permeability in several models of CNS inflammation (5, 6, 7, 37). We speculated that IFN-{gamma} might trigger neurovascular endothelial cells to produce ONOO– through the activity of constitutively expressed endothelial NOS. Indeed, IFN-{gamma} induces primary neurovascular endothelial cells to rapidly produce ONOO– and the ONOO–-dependent radical scavenger UA inhibits IFN-{gamma}-triggered BBB permeability changes in vitro. The likelihood that a CD4 T cell-dependent, ONOO–-mediated mechanism is operative in CVS-F3 RV-infected mice is supported by the fact that CD4 T cells and NT residues, evidence of ONOO– formation, are colocalized in permeable areas of the neurovasculature.

In summary, we have demonstrated that TNF-{alpha} is not required for the development of enhanced BBB permeability in CNS autoimmunity or RV clearance. We conclude that a ONOO–-dependent mechanism is common to immune-mediated changes in BBB integrity. However, unlike EAE and other models of CNS inflammation where the production of ONOO–-dependent radicals is associated with disease, ONOO–-mediated BBB permeability changes in CVS-F3 RV-infected mice contribute to virus clearance and are therapeutic. We expect that this is a result of the production of ONOO– by vascular endothelial cells rather than by invasive iNOS-positive macrophages and neutrophils.


   Acknowledgments
 
We thank Christine Soto and Tatiana Mikheeva for excellent technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI09706 and a grant to the Biotechnology Foundation Laboratories from the Commonwealth of Pennsylvania. Back

2 Address correspondence and reprint request to Dr. D. Craig Hooper, Department of Cancer Biology, Thomas Jefferson University, Jefferson Alumni Hall 454, 1020 Locust Street, Philadelphia, PA 19107. E-mail address: c_hooper{at}mail.jci.tju.edu Back

3 Abbreviations used in this paper: BBB, blood-brain barrier; RV, rabies virus; EAE, experimental allergic encephalomyelitis; iNOS, inducible NO synthase; i.n., intranasally; MOG, myelin oligodendrocyte glycoprotein; Na-F, sodium-fluorescein; NT, nitrotyrosine; UA, uric acid; DHR, dihydrorhodamine; IP-10, IFN-{gamma}-inducible protein-10; ONOO–, peroxynitrite. Back

Received for publication September 7, 2006. Accepted for publication March 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Gloor, S. M., M. Wachtel, M. F. Bolliger, H. Ishihara, R. Landmann, K. Frei. 2001. Molecular and cellular permeability control at the blood-brain barrier. Brain Res. Rev. 36: 258-264. [Medline]
  2. Vajkoczy, P., M. Menger. 2000. Vascular microenvironment in gliomas. J. Neurooncol. 50: 99-108. [Medline]
  3. Silver, N., C. Good, M. Sormani, D. MacManus, A. Thompson, M. Filippi, D. Miller. 2001. A modified protocol to improve the detection of enhancing brain and spinal cord lesions in multiple sclerosis. J. Neurol. 248: 215-224. [Medline]
  4. Andersen, I., O. Marker, A. Thomsen. 1991. Breakdown of blood-brain barrier function in the murine lymphocytic choriomeningitis virus infection mediated by virus-specific CD8+ T cells. J. Neuroimmunol. 31: 155-163. [Medline]
  5. Hooper, D. C., G. Scott, A. Zborek, T. Mikheeva, R. B. Kean, H. Koprowski, S. Spitsin. 2000. Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis. FASEB J. 14: 691-698. [Abstract/Free Full Text]
  6. Kean, R. B., S. Spitsin, T. Mikheeva, G. Scott, D. C. Hooper. 2000. The peroxynitrite scavenger uric acid prevents inflammatory cell invasion into the CNS in experimental allergic encephalomyelitis through maintenance of blood-CNS barrier integrity. J. Immunol. 165: 6511-6518. [Abstract/Free Full Text]
  7. Hooper, D. C., R. B. Kean, G. Scott, S. Spitsin, T. Mikheeva, K. Morimoto, M. Bette, A. Rohrenbeck, B. Dietzschold, E. Weihe. 2001. The central nervous system inflammatory response to neurotrophic virus infection is peroxynitrite dependent. J. Immunol. 167: 3470-3477. [Abstract/Free Full Text]
  8. Phares, T., R. B. Kean, T. Mikheeva, D. C. Hooper. 2006. Regional differences in blood-brain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system. J. Immunol. 176: 7666-7675. [Abstract/Free Full Text]
  9. Megyeri, P., C. Abraham, P. Temesvari, J. Kovas, T. Vas, C. Speer. 1992. Recombinant human tumor necrosis factor {alpha} constricts pial arterioles and increases blood-brain barrier permeability in newborn piglets. Neurosci. Lett. 148: 137-140. [Medline]
  10. Kim, K., C. Wass, A. Cross, S. Opal. 1992. Modulation of blood-brain barrier permeability by tumor necrosis factor and antibody to tumor necrosis factor in the rat. Lymphokine Cytokine Res. 11: 293-298. [Medline]
  11. Wright, J., R. Merchant. 1994. Blood-brain barrier changes following intracerebral injection of human recombinant tumor necrosis factor-{alpha} in the rat. J. Neurooncol. 20: 17-25. [Medline]
  12. Rosenberg, G., E. Estrada, J. Dencoff, W. Stetler-Stevenson. 1995. Tumor necrosis factor-{alpha}-induced gelatinase B causes delayed opening of the blood-brain barrier: an expanded therapeutic window. Brain Res. 703: 151-155. [Medline]
  13. Abraham, C., M. Deli, F. Joo, P. Megyeri, G. Torpier. 1996. Intracarotid tumor necrosis factor-{alpha} administration increases the blood-brain barrier permeability in cerebral cortex of the newborn pig: quantitative aspects of double-labeling studies and confocal laser scanning analysis. Neurosci. Lett. 208: 85-88. [Medline]
  14. Schnell, L., S. Fearn, M. Schwab, V. Perry, D. Anthony. 1999. Cytokine-induced acute inflammation in the brain and spinal cord. J. Neuropathol. Exp. Neurol. 58: 245-254. [Medline]
  15. Trembovler, V., E. Beit-Yannai, F. Younis, R. Gallily, M. Horowitz, E. Shohami. 1999. Antioxidants attenuate acute toxicity of tumor necrosis factor-{alpha} by brain injury in rat. J. Interferon Cytokine Res. 19: 791-795. [Medline]
  16. Megyeri, P., L. Nemeth, K. Pabst, M. Pabst, M. Deli, C. Abraham. 1999. 4-(2-Aminoethyl)benzenesulfonyl fluoride attenuates tumor-necrosis-factor-{alpha}-induced blood-brain barrier opening. Eur. J. Pharmacol. 374: 207-211. [Medline]
  17. Yang, G., C. Gong, Z. Qin, X. Liu, A. Betz. 1999. Tumor necrosis factor-{alpha} expression produces increased blood-brain barrier permeability following temporary focal cerebral ischemia in mice. Mol. Brain Res. 69: 135-143. [Medline]
  18. Dickstein, J. B., H. Moldofsky, J. Hay. 2000. Brain-blood permeability: TNF-{alpha} promotes escape of protein tracer from CSF to blood. Am. J. Physiol. 279: R148-R151.
  19. Tsoa, N., P. Hsu, C. Wu, C. Liu, H. Lei. 2001. Tumor necrosis factor-{alpha} causes an increase in blood-brain barrier permeability during sepsis. J. Med. Microbiol. 50: 812-821. [Abstract/Free Full Text]
  20. Dobbie, M. S., R. Hurst, N. Klein, R. Surtees. 1999. Upregulation of intracellular adhesion molecule-1 on human endothelial cells by tumor necrosis factor-{alpha} in an in vitro model of the blood-brain barrier. Brain Res. 830: 330-336. [Medline]
  21. Wong, D., K. Dorovini-Zis. 1992. Upregulation of intracellular adhesion molecule-1 (ICAM-1) expression in primary cultures of human brain microvessel endothelial cells by cytokines and lipopolysaccharide. J. Neuroimmunol. 39: 11-22. [Medline]
  22. Lou, J., Y. Gasche, L. Zheng, B. Critico, C. Monso-Hinard, P. Juillard, P. Morel, W. Buurman, G. Grau. 1998. Differential reactivity of brain microvascular endothelial cells to TNF reflects the genetic susceptibility to cerebral malaria. Eur. J. Immunol. 28: 3989-4000. [Medline]
  23. Dietrich, J.. 2002. The adhesion molecule ICAM-1 and its regulation in relation with the blood-brain barrier. J. Neuroimmunol. 128: 58-68. [Medline]
  24. Kido, G., J. Wright, R. Merchant. 1991. Acute effects of human recombinant tumor necrosis factor-{alpha} on the cerebral vasculature of the rat in both normal brain and in an experimental glioma model. J. Neuroonocol. 10: 95-109.
  25. Gutierrez, E., W. Banks, A. Kastin. 1993. Murine tumor necrosis factor {alpha} is transported from blood to brain in the mouse. J. Neuroimmunol. 47: 169-176. [Medline]
  26. Saija, A., P. Princi, M. Lanza, M. Scalese, E. Aramnejad, A. De Sarro. 1995. Systemic cytokine administration can affect blood-brain barrier permeability in the rat. Life Sci. 56: 775-784. [Medline]
  27. Anthony, D. C., S. Bolton, S. Fearn, V. Perry. 1997. Age-related effects of interleukin-1beta on polymorphonuclear neutrophil-dependent increases in blood-brain barrier permeability in rats. Brain 120: 435-444. [Abstract/Free Full Text]
  28. Deli, M., L. Descamps, M. Dehouck, R. Cecchelli, F. Joo, C. Abraham, G. Torpier. 1995. Exposure of tumor necrosis factor-{alpha} to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation. J. Neurosci. Res. 41: 717-726. [Medline]
  29. de Vries, H., M. Blom-Roosemalen, M. Oosten, A. Boer, T. Berkel, D. Breimer, J. Kuiper. 1996. The influence of cytokines on the integrity of the blood-brain barrier in vitro. J. Neuroimmunol. 64: 37-43. [Medline]
  30. Anda, T., H. Yamashita, H. Khalid, K. Tsutsumi, H. Fujita, Y. Tokunaga, S. Shibata. 1997. Effect of tumor necrosis factor-{alpha} on the permeability of bovine brain microvessel endothelial cell monolayers. Neurol. Res. 4: 369-376.
  31. Descamps, L., R. Cecchelli, G. Torpier. 1997. Effects of tumor necrosis factor on receptor-mediated endocytosis and barrier functions of bovine brain capillary endothelial cell monolayers. J. Neuroimmunol. 74: 173-184. [Medline]
  32. Mark, K., D. Miller. 1999. Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-{alpha} exposure. Life Sci. 64: 1941-1953. [Medline]
  33. Trickler, W., W. Mayhan, D. Miller. 2005. Brain microvessel endothelial cell responses to tumor necrosis factor-{alpha} involve a nuclear factor {kappa}B (NF-{kappa}B) signal transduction pathway. Brain Res. 1048: 24-31. [Medline]
  34. Sharief, M., E. Thompson. 1992. In vivo relationship of tumor necrosis factor-{alpha} to blood-brain barrier damage in patients with active multiple sclerosis. J. Neuroimmunol. 38: 27-34. [Medline]
  35. Sharief, M., M. Ciardi, E. Thompson. 1992. Blood-brain barrier damage in patients with bacterial meningitis: association with tumor necrosis factor-{alpha} but not interleukin-1beta. J. Infect. Dis. 166: 350-358. [Medline]
  36. Thiel, V., K. Audus. 2001. Nitric oxide and blood-brain barrier integrity. Antioxid. Redox. Signal 3: 273-278. [Medline]
  37. Kastenbauer, S., U. Koedel, H. Pfister. 1999. A role of peroxynitrite as a mediator of pathophysiological alterations in experimental pneumococcal meningitis. J. Infect. Dis. 180: 1164-1170. [Medline]
  38. Imaizumi, S., T. Kondo, M. Deli, G. Gobbel, F. Joo, C. Epstein, T. Yoshimoto, P. Chan. 1996. The influence of oxygen free radicals on the permeability of the monolayers of cultured brain endothelial cells. Neurochem. Int. 29: 205-211. [Medline]
  39. Hooper, D. C., K. Morimoto, M. Bette, M. Weihe, H. Koprowski, B. Dietzschold. 1998. Collaboration of antibody and inflammation in the clearance of rabies virus from the CNS. J. Virol. 72: 3711-3719. [Abstract/Free Full Text]
  40. Dietzschold, B., W. Wunner, T. Wiktor, A. Lopes, M. Lafon, C. Smith, H. Koprowski. 1983. Characterization of an antigenic determinant of the glycoprotein which correlates with pathogenicity of rabies virus. Proc. Natl. Acad. Sci. USA 80: 70-74. [Abstract/Free Full Text]
  41. Dietzschold, B., T. Wiktor, J. Trojanowski, R. Macfarlan, W. Wunner, M. Torres-Anjel, H. Koprowski. 1985. Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro. J. Virol. 56: 12-18. [Abstract/Free Full Text]
  42. Wu, Z., F. Hofman, B. Zlokovic. 2003. A simple method for isolation and characterization of mouse brain microvascular endothelial cells. J. Neurosci. Methods 130: 53-63. [Medline]
  43. Scott, G. S., R. Kean, M. Fabis, T. Mikheeva, C. Brimer, T. Phares, S. Spitsin, D. C. Hooper. 2004. ICAM-1 upregulation in the spinal cords of PLSJL mice with experimental allergic encephalomyelitis is dependent upon TNF-{alpha} production triggered by the loss of blood-brain barrier integrity. J. Neuroimmunol. 155: 32-42. [Medline]
  44. Ischiropoulos, H., L. Zhu, J. Chen, M. Tsai, J. Martin, C. Smith, J. Beckman. 1992. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophs. 298: 431-437.
  45. Hofman, F., D. Hinton. 1991. Cytokine interactions in the central nervous system. Reg. Immunol. 3: 268-278.
  46. McManus, C., C. Brosnan, J. Berman. 1998. Cytokine induction of MIP-1{alpha} and MIP-1beta in human fetal microglia. J. Immunol. 160: 1449-1455. [Abstract/Free Full Text]
  47. Gottschall, P., X. Yu. 1995. Cytokines regulate gelatinase A and B (matrix metalloproteinase 2 and 9) activity in cultured rat astrocytes. J. Neurochem. 64: 1513-1520. [Medline]
  48. Muir, E., K. Adcock, D. Morgenstern, R. Clayton, N. von Stillfried, K. Rhodes, C. Ellis, J. Fawcett, J. Rogers. 2002. Matrix metalloproteases and their inhibitors are produced by overlapping populations of activated astrocytes. Mol. Brain Res. 100: 103-117. [Medline]
  49. Korner, H., D. Riminton, D. Strickland, F. Lemckert, J. Pollard, J. Sedgwick. 1997. Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting. J. Exp. Med. 186: 1585-1590. [Abstract/Free Full Text]
  50. Liu, J., M. Marino, G. Wong, D. Grail, A. Dunn, J. Bettadapura, A. Slavin, L. Old, C. Bernard. 1998. TNF is a potent anti-inflammatory cytokine in autoimmune-mediated demyelination. Nat. Med. 4: 78-83. [Medline]
  51. Riminton, D., H. Korner, D. Strickland, F. Lemckert, J. Pollard, J. Sedgwick. 1998. Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice. J. Exp. Med. 187: 1517-1528. [Abstract/Free Full Text]
  52. Kassiotis, G., M. Pasparakis, G. Kollias, L. Probert. 1999. TNF accelerates the onset but does not alter the incidence and severity of myelin basic protein-induced experimental autoimmune encephalomyelitis. Eur. J. Immunol. 29: 774-780. [Medline]
  53. Ruuls, S., R. Hoek, V. Ngo, T. McNeil, L. Lucian, M. Janatpour, H. Korner, H. Scheerens, E. Hessel, J. Cyster, et al 2001. Membrane-bound TNF supports secondary lymphoid organ structure but is subservient to secreted TNF in driving autoimmune inflammation. Immunity 15: 533-543. [Medline]
  54. Matejuk, A., J. Dwyer, A. Ito, Z. Bruender, A. Vandenbark, H. Offner. 2002. Effects of cytokine deficiency on chemokine expression in CNS of mice with EAE. J. Neurosci. Res. 67: 680-688. [Medline]
  55. Murphy, C., R. Hoek, M. Wiekowski, S. Lira, J. Sedgwick. 2002. Interactions between hemopoietically derived TNF and central nervous system-resident glial chemokines underlie initiation of autoimmune inflammation in the brain. J. Immunol. 169: 7054-7062. [Abstract/Free Full Text]



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