Nuffield Department of Medicine1 and Nuffield Department of Surgery2, John Radcliffe Hospital, Oxford, UK
Author for correspondence: Philip Stevenson. Present address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK. Fax +44 1223 336926. e-mail pgs27{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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The prolonged survival of allogeneic tissue grafts in the brain implies a degree of immune privilege (Brent, 1990 ). The rejection of an intracerebral graft can be triggered by a subsequent extracerebral graft from the same donor, so this privilege probably reflects a lack of immune priming by antigens sequestered in the brain (Medawar, 1948
). Mycobacterial antigens also appear to be much less inflammatory in the brain parenchyma than in extracerebral sites, probably due to a lack of immune priming (Matyszak & Perry, 1998
). We have previously used a stereotactically guided, small volume inoculation of a non-neuroadapted strain of influenza virus to confine virus infection to the parenchymal substance of the brain and thus to demonstrate that such an infection does not lead to immune priming (Stevenson et al., 1997a
). Infection with the neuroadapted influenza virus A/WSN in the same site also appears not to prime efficiently but, nevertheless, elicits an intracerebral inflammatory response (Stevenson et al., 1997b
). However, because this virus spreads throughout the brain (Stuart-Harris, 1939
; Takahashi et al., 1995
) and reaches the cerebrospinal fluid (CSF) (Stevenson et al., 1997b
), it is unclear whether such inflammation represents the early signs of an immune response consequent upon an extracerebral seeding of infection. Here, we have analysed the local response to infection with a non-neuroadapted influenza virus, which does not produce infectious virions in the brain and thus does not spread. This has allowed us to exclude the possibility of extracerebral infection.
Soluble protein antigens injected into the brain parenchyma can reach the deep cervical lymph nodes (Cserr & Knopf, 1992 ) and stimulate immune responses (Gordon et al., 1992
). The lack of immune priming by cell-associated antigens such as influenza virus thus suggests that deficient antigen-presenting cell function in the brain is an important factor in immune privilege. While the brain parenchyma lack classical dendritic cells (Hart & Fabre, 1981
), the bone marrow-derived perivascular microglial cells (Hickey & Kimura, 1988
) resemble immature antigen-presenting cells (Carson et al., 1998
) and can be induced to differentiate towards a dendritic cell phenotype in vitro (Aloisi et al., 1999
; Santambrogio et al., 2001
). CD11c+ microglia (Fischer et al., 2000
; Serafini et al., 2000
) also share some features with dendritic cells and can prime naive T cells in vitro (Fischer & Reichmann, 2001
). However, there is clearly a functional deficit in vivo, which presumably applies both to the carriage of cell-associated antigens from the brain parenchyma to lymph nodes and to the priming of intracerebral T cells in situ. Either should suffice to overcome immune privilege.
Two possibilities, not mutually exclusive, are that bone marrow-derived microglia are not functionally equivalent to dendritic cells in vivo and that the microenvironment of the brain parenchyma does not support immune priming by any cell type. Influenza virus-infected dendritic cells are known to stimulate potent immune responses in vitro (Macatonia et al., 1989 ; Nonacs et al., 1992
) and in vivo (Hamilton-Easton & Eichelberger, 1995
; Lopez et al., 2000
). Thus, we have used ex vivo dendritic cells infected with influenza virus to explore the possibility that professional antigen-presenting cells delivered to the brain parenchyma might overcome the barrier to immune priming.
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Methods |
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Preparation of dendritic cells.
Dendritic cells were purified from mouse spleens by standard methods (Austyn et al., 1983 ; Kupiec-Weglinski et al., 1988
). Briefly, low-density cells were enriched from splenocytes by centrifugation onto a cushion of dense (1·08 g/ml) BSA. These cells were adhered to plastic for 3 h at 37 °C. Non-adherent cells were removed by pipetting and discarded. After overnight culture at 37 °C, cells were detached by pipetting and readhered to plastic for 2 h to remove macrophages. The proportion of dendritic cells [major histocompatibility complex (MHC) class II+, B220-, Mac-1-, CD3- and Fc receptor-] in the final non-adherent cell population was 5070%. These cells were infected for 2 h with influenza virus A/NT/60/68 (2 p.f.u. per cell) and washed three times prior to intracerebral microinjection, as described above. Cell-free virus was undetectable by plaque assay in the intracerebral inoculum.
ELISA.
Influenza virions were purified from infectious allantoic fluid by sucrose density gradient centrifugation, washed in PBS, lysed with detergent and adsorbed overnight onto Nunc Polysorb immunoplates (Life Technologies). Virus-specific IgG, IgA and IgM were assayed as described previously (Stevenson et al., 1997a ).
Restimulation and assay of CD8+ cytotoxic T lymphocytes (CTL).
Single-cell suspensions from lymph nodes (5x105 cells/ml) or cells from infected brains (5x104 cells/ml) were restimulated with virus-infected feeder cells (1x106 cells/ml) in 2 ml cultures. Spleen cells (1x106 cells/ml) were restimulated with virus-infected feeder cells (3x105 cells/ml) in 15 ml cultures. Syngeneic feeder spleen cells were incubated with 400 µl influenza virus A/NT/60/68-infected allantoic fluid per 1x107 cells at 37 °C for 1 h, irradiated (20 Gy) and washed twice in complete medium before use. Cytotoxicity was tested after 5 days of culture in complete medium at 37 °C with 5% CO2. 51Cr-labelled EL-4 cells (100 µCi/1x106 cells) were either incubated with complete medium alone, pulsed with an H-2Db-restricted influenza virus nucleoprotein epitope ( 1 µM ASNENMDAM) or infected with influenza virus A/NT/60/68 (5 p.f.u. per cell). After 1 h at 37 °C, the target cells were washed twice in complete medium and incubated with CTL for 4 h before harvesting supernatants for scintillation counting. Chromium release from the targets alone was 515% of the release with Triton.
Proliferation assay.
Lymphocytes were aliquotted into triplicate cultures (2x103 cells per well) in Terasaki plates (20 µl per well) or 96-well plates (2x105 cells per well) with irradiated (20 Gy) naive feeder spleen cells (2x104 or 2x105 per well, respectively). Feeder cells were either untreated or infected with influenza virus, as for CTL restimulation. After culture for 72 h at 37 °C with 5% CO2 using complete medium but with 1% normal mouse serum substituted for 10% foetal calf serum, 0·11·0 µCi [3H]thymidine was added to each well and the cells were harvested for scintillation counting 18 h later.
Flow cytometry.
Lymphocytes were incubated for 15 min on ice with 5% normal mouse serum and 5% normal rat serum followed by a 30 min incubation with rat anti-mouse antibodies as follows: anti-CD4, KT6-phycoerythrin (Serotec); anti-CD8, KT15-FITC (Serotec); anti-CD62L, biotinylated MEL-14; anti-CD44, biotinylated IM7.8.1; anti-CD49d, biotinylated PS/2; or anti-CD25, Quantum Red-3C7 (Sigma). After washing in PBS/FCS (1%), streptavidinQuantum Red (Sigma) was added for 15 min followed by a further wash. Samples were analysed on a FACSort instrument using CELLQUEST, version 1.1 (Becton-Dickinson).
Immunohistochemistry.
Acetone-fixed frozen sections (710 µm) were preincubated in 10% rabbit serum, and stained with the following rat anti-mouse antibodies: anti-CD8, YTS169.4; anti-CD4, GK1.5; anti-MHC class I, M1-42.3.9.8; anti-MHC class II, M5/114.15.2; and anti-B220, RA3-6B2 (Serotec). A mouse-absorbed, biotinylated, rabbit anti-rat serum (Dako) was used as the secondary antibody, followed by streptavidinbiotinperoxidase complexes (Vector Laboratories). For anti-virus staining, the sections were preincubated in 10% goat serum and then incubated with an anti-influenza virus ribonucleoprotein rabbit serum (Reinacher et al., 1983 ) followed by a peroxidase-coupled goat anti-rabbit serum (Vector Laboratories). All antibody incubations were for 1 h at room temperature and sections were washed three times in PBS after each incubation. Endogenous peroxidase activity was blocked with 0·3% hydrogen peroxide in methanol after the primary antibody incubation. Diaminobenzidine was used as the peroxidase substrate and haematoxylin was used as the counterstain.
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Results |
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Analysis of infiltrating lymphocytes
After parenchymal virus infection, the presence of infiltrating lymphocytes in the absence of detectable anti-viral serum antibody suggested that these cells had been recruited without an antigen-specific immune response and, thus, that they were not virus-specific. To test this possibility, T cells were recovered from infected brains 10 days after ventricular or parenchymal influenza virus infection or from infected lungs 10 days after intranasal virus inoculation and were assayed for virus specificity in vitro (Table 1). The cells recovered after ventricular or intranasal, but not after parenchymal, virus infections showed virus-specific proliferation and cytotoxicity. Virus-specific CTL precursors could be recovered from the brain, spleen and deep cervical lymph nodes after ventricular or intranasal infections but not after parenchymal infection (Table 1
). All mice given intracerebral virus were checked for the presence of virus-specific serum antibody. As observed previously (Stevenson et al., 1997a
), virus-specific antibody was undetectable in the serum or the CSF after parenchymal virus inoculation (Fig. 3
). Thus, there was no evidence of an antigen-specific immune response to parenchymal influenza virus infection, either systemically or locally, within the CNS.
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Discussion |
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The brain has historically been viewed as a site of decreased immune reactivity and inflammation. However, there is still controversy over the factors involved; for example, whether a lack of immune priming or a defect in intracerebral T cell function is the crucial influence (Streilein, 1993 ). We have investigated immune privilege using intracerebral challenge with viral antigens, since immune evolution has probably been driven by the requirement to detect and eliminate such parasites. While intracerebral autoimmunity may be downregulated by lymphocyte apoptosis (Ford et al., 1996
; Gold et al., 1997
), this does not seem to be a major feature of influenza virus infection. Indeed viable, activated, virus-specific CD8+ T cells can persist long-term in the CNS (Hawke et al., 1998
). Brain gangliosides have been reported to inhibit T cell proliferation (Irani et al., 1996
) but again we have found no evidence for this during influenza virus infection: virus-specific intracerebral T cells proliferate in vivo (Stevenson et al., 1997c
) and in vitro (Stevenson et al., 1997a
; Hawke et al., 1998
). Overall, anti-viral T cells appear to function very effectively in the brain. Here, intracerebral virus infection recruited primed T cells even when they were not virus-specific (Table 1
, Fig. 4
). The focus in understanding CNS immune privilege is thus shifted onto the generation of primed T cells and onto the antigen-presenting cell.
Cells with a dendritic cell-like surface phenotype accumulate in the brain during chronic toxoplasma infection (Fischer et al., 2000 ) and experimental allergic encephalomyelitis (Serafini et al., 2000
). The precise function of these cells in vivo remains unclear. Even though influenza virus infection stimulated a local inflammatory response in the brain parenchyma (Fig. 1
), it did not stimulate any resident dendritic cell-like antigen-presenting cells to generate detectable immunity. And, while influenza virus-infected dendritic cells are normally highly efficient at presenting viral antigens in vivo, these were ineffective when inoculated into the brain parenchyma (Fig. 5
). This suggested that it is not the lack of dendritic cells but an inappropriate environment for their function that prevents immune priming. It may be that the brain microenvironment fails to support normal dendritic cell migration. A lack of suitable anatomical pathways, chemokines or adhesion molecules could all contribute. The end result is that inflammation can be driven locally but that priming requires extracerebral antigen.
What are the potential implications of the uncoupling of inflammation and immunity in the brain parenchyma? Congenital infections and the retrograde transport of virions along peripheral neurons are both mechanisms by which a virus may reach the brain while avoiding extracerebral immune priming. Viruses may also express genes uniquely in the brain during a period of latency in this site. A lack of immune priming could then contribute to the persistence of an intracerebral, inflammatory focus. The immune response to unrelated extracerebral infections could have significant effects on antigen non-specific intracerebral inflammation by increasing the circulating pool of activated cells capable of entering the brain. Clearly, a priority with any intracerebral inflammatory disease is to consider not only antigen-specific immunity but also the potential role of non-antigen-specific responses to cryptic infections.
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Acknowledgments |
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Footnotes |
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References |
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Received 12 November 2001;
accepted 19 February 2002.
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