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The Journal of Immunology, 2003, 171: 3102-3109.
Copyright © 2003 by The American Association of Immunologists

Measles Virus Infection Induces Chemokine Synthesis by Neurons 1

Catherine E. Patterson*, John K. Daley*, Lisa A. Echols*, Thomas E. Lane{dagger} and Glenn F. Rall2,*

* Fox Chase Cancer Center, Philadelphia, PA 19111; and {dagger} Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role that neurons play in the induction of the immune response following CNS viral infection is poorly understood, largely owing to the belief that these cells are immunologically quiescent. In this report, we show that virus infection of neurons results in the synthesis of proinflammatory chemokines, which are early and important mediators of leukocyte recruitment to sites of viral infection. For these studies, a transgenic mouse model of neuron-restricted measles virus (MV) infection was used. Inoculation of immunocompetent and immunodeficient transgenic adult mice resulted in CNS induction of the mRNAs encoding IFN-{gamma} inducible protein of 10 kD, monokine inducible by {gamma} and RANTES. Colocalization of chemokine proteins with MV-infected neurons was detected by immunofluorescence in infected brain sections. Both IFN-{gamma} inducible protein 10 kD and RANTES were also induced in MV-infected primary hippocampal neurons cultured from transgenic embryos, as shown by RNase protection assay, confocal microscopy, and ELISA. Interestingly, neuronal infection with another RNA virus (lymphocytic choriomeningitis virus) was not associated with induction of these chemokines. In immunocompetent mice, chemokine synthesis preceded the infiltration of T lymphocytes, and chemokine ablation by neutralizing Abs resulted in a 20–50% reduction in the number of infiltrating lymphocytes. Collectively, these data indicate that neurons play an important role in the recruitment of a protective antiviral response to the CNS following viral infection, although such a role may be virus type-dependent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CNS is considered an immune-privileged organ and neurons within the CNS, despite their importance to the host, are often described as immunologically inert (1). Although neurons can be induced in vitro to express class I MHC determinants under certain circumstances (2, 3), they are generally not MHC-positive in vivo, and therefore cannot be targets for CTL recognition via TCR-class I MHC interactions. Moreover, the fact that most CNS neurons are nonrenewable has lead to the notion that a cytolytic antiviral immune response within the brain could be as deleterious to the host as the infection itself (1). Nevertheless, antiviral immune responses do occur in the infected CNS. However, how immune cells are recruited to the infected parenchyma, especially under conditions of neuronal infection, is not fully understood.

Neuron-specific enolase (NSE) 3-CD46 transgenic mice express one of the human measles virus (MV) receptors, CD46 (4, 5, 6), under the transcriptional control of a neuron-specific promoter, NSE (7, 8, 9, 10). Previous work from our laboratory demonstrated that inoculation of NSE-CD46 transgenic adults and neonates with a vaccine strain of MV (MV-Edmonston) resulted in a neuron-restricted infection followed by recruitment of CD4+ and CD8+ T cells to the infected brain parenchyma within 6–9 days after inoculation (8). The pathogenic consequences of these infections, however, were age-dependent. In adults, resolution of the infection by the host immune response was crucial for survival, because adult NSE-CD46+ mice on an immunodeficient background could not resolve the infection and died of subsequent CNS disease (8). In contrast, MV-inoculated neonates developed unrestricted neuronal infection and fatal CNS disease (10), despite the recruitment of T cells to the parenchyma. Although this immune response did not afford protection in newborn mice, it did not contribute to neuropathology either, because CD46+ and CD46+/recombinase-activating gene (RAG)-2 knockout (KO) neonates showed identical kinetics of infection and illness (8).

Because only neurons were susceptible to infection in NSE-CD46+ neonates and adults, it was somewhat surprising that such a rapid and robust induction of the host T lymphocyte response occurred. It was previously shown that CNS infection of neonatal NSE-CD46+ mice resulted in induction of proinflammatory molecules, including class I and II MHC, glial cell activation markers, and chemokines (11). Cytokines and chemokines were also shown to be induced during CNS infection with MV mutants lacking the putative virulence factors, V and C (12). In these reports, the cellular source of chemokines was not evaluated. We therefore wished to explore the role of chemokines in the anti-MV immune and the potential contribution of neurons to their own survival, through the synthesis of such molecules.

Chemokines are a superfamily of small, secreted proteins that promote recruitment of leukocytes to the site of a pathogenic challenge by signaling through G protein-coupled seven transmembrane receptors (13, 14). Such engagement results in leukocyte activation (enhanced cytokine production, cell shape changes, and proliferation) and increased adhesion to capillary endothelia (13, 14, 15). In the brain, a rapid induction of chemokines by astrocytes and microglia has been observed in multiple mouse models of RNA virus infection, including mouse hepatitis virus, Theiler’s murine encephalomyelitis virus, and lymphocytic choriomeningitis virus (LCMV) (16, 17, 18). Moreover, depletion of chemokines with specific neutralizing Abs was shown to markedly reduce immune cell entry and alter the pathogenic outcome of infection (19, 20, 21). Although neurons have been shown to produce chemokines under severe CNS insults such as ischemia and chronic inflammation (22, 23), virus infections have, to date, not been associated with neuronal chemokine synthesis. Because many viruses can infect CNS neurons, in some cases exclusively, neurons may play a crucial and early role in the induction of a protective antiviral response by producing proinflammatory mediators such as chemokines.

To ascertain whether virus-infected neurons contributed to the recruitment of immune T cells to the brain, we evaluated the expression of chemokines in MV-infected adult NSE-CD46+ mice and in primary hippocampal neurons explanted from transgenic embryos. MV infection of adult immunocompetent and immunodeficient mice and susceptible primary neurons resulted in the induction of the chemokines IFN-{gamma} inducible protein 10 kD (IP-10), monokine inducible by {gamma} (Mig), and RANTES. Moreover, inoculation of infected adults with neutralizing Abs to these chemokines substantially reduced the magnitude of the protective T cell response within the brain parenchyma. Interestingly, infection of primary neurons with the arenavirus, LCMV, did not result in the induction of any of the chemokines analyzed. These data suggest that neurons play a crucial and early role in the induction of the immune response to certain viruses within the CNS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and virus

Vero fibroblasts were maintained in DMEM (Life Technologies, Grand Island, NY), supplemented with 5% FCS, 2 mM L-glutamine, 100 U penicillin, and 100 ng streptomycin per milliliter. Primary hippocampal neurons were prepared from day 15.5 embryonic mice, as previously described (24, 25, 26), although cells were maintained in neurobasal media (Life Technologies) containing 4 µg/ml glutamate, in the absence of an astrocyte feeder layer. These cultures are >95% mitogen-activated protein 2-positive (data not shown). All cells were maintained at 37°C in a humidified incubator with 5% CO2 and were routinely tested for mycoplasma contamination. All studies were performed with mycoplasma-free cells.

MV, Edmonston strain, was purchased from American Type Cell Collection (Manassas, VA), and passaged and titered in Vero fibroblasts. LCMV Armstrong strain, a gift from M. Oldstone, Scripps Research Institute, was passaged on BHK-21 fibroblasts, and plaque purified and titered on Vero fibroblasts.

Mice

Inbred C57BL/6 (H-2b) and homozygous NSE-CD46 transgenic mice (line 18; H-2b) (10) were maintained in the closed animal colony of the Fox Chase Cancer Center. All experimental protocols were approved by the Institutional Animal Care and Use Committee. Homozygous NSE-CD46+ and homozygous RAG-2 KO mice (27) were intercrossed for at least two generations. To verify genotype, peripheral blood from these progeny was immunostained with fluorophore-conjugated Abs to mouse CD4 and CD8 Ags and analyzed by flow cytometry (28).

Mice were infected with 104 PFU MV-Ed. The inoculum was diluted in PBS, and delivered intracerebrally to Metofane-anesthetized (Janssen-Cilag, Neuss, Germany) mice along the midline, in a volume of 20 µl using a sterile 27-gauge needle.

RNase protection assays (RPA)

Brains were removed from infected mice at the indicated days postinfection (dpi), quick frozen in liquid nitrogen, and homogenized in Tri-Reagent (Sigma-Aldrich, St. Louis, MO) using a tissue homogenizer. Total RNA was isolated according to the manufacturer’s instructions and resuspended in diethyl pyrocarbonate-treated water.

Total brain RNA (10 µg) or ex vivo neuronal RNA (3 µg) was analyzed for chemokine expression by RNase protection analysis. Probes were transcribed from a multitemplate set (Riboquant; BD PharMingen, San Diego, CA) with 32P-UTP (DuPont NEN, Boston, MA). The custom-ordered mouse template set, in descending probe size, included: TNF-{beta}, RANTES, TNF-{alpha}, macrophage-inflammatory protein-1{beta}, -1{alpha}, -2, IP-10, monocyte chemoattractant protein-1, IFN-{gamma}, L32, and GAPDH. The freshly labeled radioactive probe set was hybridized overnight with the indicated RNA samples, digested with RNase A and T1 followed by proteinase K, as described by the manufacturer. Digested RNA samples were extracted, precipitated and separated on 6% polyacrylamide-urea gels. Gels were dried, exposed to film, and placed on an imaging screen for quantitation. Pixel values of both L32 and GAPDH were quantitated to normalize the signal intensity of the expressed chemokines and cytokines, and to allow for intersample comparisons. Based on these normalized samples, mean adjusted pixel values and SDs were determined. Yeast tRNA and mouse control RNA were used as controls in all experiments. Statistical analysis was performed using Student’s t test, and significance was determined based on the indicated controls. At least three independent trials were analyzed for each experiment.

RNA dot blots

RNA was prepared as previously described. Total RNA samples (5 µg) were mixed to a final concentration of 4.8% formamide and 6.6% formaldehyde, and incubated at 68°C for 15 min. Samples were then chilled on ice and diluted 2-fold with 20x SSC. The RNA samples were applied to a nylon membrane using a dot blot apparatus; the membrane was UV cross-linked and hybridized with a radioactive probe at 68° for 1 h in hybridization solution (QuikHyb; Stratagene, La Jolla, CA). DNA probes were labeled by random priming (Prime-It II; Stratagene) using 32P-dCTP (DuPont NEN). Mig and GAPDH DNA fragments were isolated from cDNA clones (29). Signal intensity of radioactive slots was quantified by phosphor imager analysis (Fuji). The GAPDH values were used to normalize Mig signals and these are shown as adjusted pixel values.

Immunofluorescence

MV-infected neurons grown on polylysine-treated glass coverslips were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS for 5–10 min, washed with PBS, and blocked with 2% donkey serum (Sigma-Aldrich) in PBS with 0.1% Triton X-100 and 0.05% Tween 20 (Fisher, Pittsburgh, PA). MV hemagglutinin was detected with a mouse mAb (1:3000; Chemicon International, Temecula, CA) and Alexa Fluor 488 donkey anti-mouse as a secondary (1:1000; Molecular Probes, Eugene, OR). The murine chemokines RANTES and chemokine responsive to {gamma}-2/IP-10 were labeled with goat antisera (on separate coverslips, 1:75; R&D Systems, Minneapolis, MN) followed by incubation with Alexa Fluor 594 donkey anti-goat secondary (1:1000; Molecular Probes). The coverslips were mounted in VectaShield (Vector Laboratories, Burlingame, CA) and sealed with nail polish. Fluorescent images were obtained with a BioRad Radiance 2000 laser scanning confocal microscope (Bio-Rad, Hercules, CA) paired to a Nikon Optishot II (Melville, NY). The images obtained were composites of 0.5-µm thick optical sections taken through a 100x objective.

For immunofluorescence on brain sections, brains from MV-infected CD46+/RAG-2 KO adult mice were removed, immersed in tissue embedding compound (Fisher), snap-frozen in a dry ice/isopentane bath, and stored at -70°C. Horizontal cryosections (10 µm) were air dried and stored at -70°C. On the day of staining, sections were fixed in ice-cold 95% ethanol, rehydrated in PBS and blocked for 20 min with 2% goat and 2% donkey serum in PBS (Vector). To detect MV-infected cells, a human immune serum (a gift from M. Oldstone) was used at a dilution of 1:2000, followed by a fluorescein anti-human secondary Ab (1:300; Vector). IP-10 protein was identified as previously described as well as with a mouse mAb followed by a rhodamine red anti-mouse secondary (Vector). Slides were visualized at 100x with a Nikon TE300 microscope equipped with a mercury lamp paired to a Nikon CoolPix 995. Uninfected tissues or omission of the primary Ab served as negative controls.

Chemokine ELISA

Murine RANTES was measured from the supernatants of mock or MV-infected neurons. Tissue culture medium was collected from the indicated samples and frozen at -70°C. The concentration of RANTES in the samples was assayed directly by a quantitative sandwich enzyme immunoassay (Quantikine M; R&D Systems). The optical densities of the standard samples, measured at 450 and 570 nm, fell between 2.5 and 0.123 (500 and 7.8 pg/ml, respectively). The concentrations of triplicate samples were determined from the standard graph and reported here as means with SD in picograms per milliliter.

Neutralizing Ab assays

Homozygous NSE-CD46+ mice were infected as described and at 1 dpi dosed i.p. with 250 µl of anti-chemokine sera or normal goat serum (NGS). Additional doses of 250 µl were given 3, 5, and 7 dpi. Mice were sacrificed at 9 dpi by perfusion with PBS, and the brains removed. Brains were either frozen for immunohistochemistry as previously described (8) or lymphocytes were removed for subsequent flow cytometric analysis. To obtain lymphocytes from perfused brain tissues, brains were diced, mashed, and washed with DMEM. Debris was allowed to settle, supernatant was removed to a fresh 15-ml conical tube and the volume was brought up to 7 ml with DMEM. Three milliliters of 90% Percoll (in PBS; Amersham Pharmacia Biotech, Piscataway, NJ) were added, mixed, and the full 10-ml sample underlaid with 1 ml 70% Percoll (in DMEM). Tubes were spun at 4°C, 1300 g, for 30 min. Each sample was aspirated down to the 5 ml demarcation, and the remaining layer was pipetted off to the interphase, transferred to a new tube, and brought up to 12 ml with DMEM. These were spun, washed, and used for immunofluorescence staining with anti-mouse CD4 and CD8 Abs directly conjugated to fluors (BD PharMingen). Percentages of CD4+ and CD8+ lymphocytes were determined using a Becton Dickinson FACScan running CellQuest software (BD Biosciences, Mountain View, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MV CNS infection of susceptible NSE-CD46+ mice results in chemokine induction

We previously reported that intracerebral MV inoculation of NSE-CD46+ adult mice resulted in a rapid and robust T cell infiltration into the brain parenchyma (8, 10). To establish whether chemokines play a role in attracting this T cell response to the infected CNS, expression profiles of selected chemokines were determined in a time course of infection. Following MV inoculation of 6-wk-old, immunocompetent NSE-CD46+ mice or nontransgenic littermates, total RNA was isolated from brains at dpi 0, 1, 3, 5, 7, 9, 12, 18, and 24. The induction profiles for a panel of genes were analyzed by RPA (Fig. 1A, representative autoradiograph). The quantified expression results indicate that both RANTES and IP-10 mRNA were induced in infected NSE-CD46 mice as early as 3 dpi, well before the appearance of T cells in the CNS (8). No induction of chemokines was noted in nontransgenic mice (data not shown). Other transcripts detected by the probe set showed less than a 2-fold induction at any time post infection in infected NSE-CD46 mice. Expression of both RANTES and IP-10 peaked from 7–12 dpi and declined thereafter (Fig. 1B), coincident with the clearance of virus from these mice. Despite viral clearance from all infected NSE-CD46+ mice, individual chemokine expression values were highly variable: the scatter plots of chemokine RPA pixel values for individual mice are shown for both RANTES (Fig. 1C) and IP-10 (Fig. 1D). The 7 dpi values for RANTES expression ranged from 1.8- to 21.2-fold induction over the day 0 baseline value. IP-10 expression at 7 dpi was elevated from 3.2- to 25.0-fold over baseline. The levels of induction of these chemokines, despite the broad range, resulted in p values <0.05 on 3, 5, 7, 12, 18, and 24 dpi for both chemokines compared with day 0. Only the variance in the 9 dpi values was too great to be statistically convincing. We considered that the broad range of chemokine expression observed in mice within groups may be due to variable infection levels; however, low expression of chemokine mRNA in a given sample did not necessarily correlate with less viral RNA from the brain homogenates (data not shown).



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  		FIGURE 1. Time course of RNA expression of RANTES and IP-10 in the brains of MV-infected NSE-CD46+ mice. Homozygous line 18 mice were infected intracerebrally as described and at least three mice per timepoint were sacrificed at 0, 1, 3, 5, 7, 9, 12, 18, and 24 dpi. RNA was prepared from total brain tissue and analyzed by RPA as described in Materials and Methods. A, A representative autoradiograph from a total of five independent experiments is shown. RANTES (R) and IP-10 (IP) probe fragments are indicated on the left side, as well as the loading controls L32 and GAPDH (G). Digested, protected RNA fragments are indicated on the right. The reduced size of the protected fragment ensures complete RNase digestion of the radiolabeled probe. P, Undigested probe, 0–18, brain samples indicated as dpi. B, Mean values from at least three experiments of adjusted Fuji pixels for both RANTES mRNA () and IP-10 mRNA (). C and D, Scatter plot of individual mice as RANTES (C) and IP-10 (D) values (respectively). Bars represent the mean value, gray diamonds represent values for individual mice.

 
The IFN-{gamma} responsive CXC chemokines IP-10 and Mig are often coinduced under diverse in vivo conditions (30, 31, 32). Therefore, given the pronounced IP-10 gene induction within the CNS of MV infected mice, we next determined whether Mig expression was also induced. Mig was not in our probe set for the RPA; therefore, we determined the transcript levels in our tissue collection time course by RNA dot blot analysis. A representative experiment is shown in Fig. 2, in which the levels for Mig RNA peaked between 9 and 14 dpi in MV-infected adult transgenic mice brains, similar to the profile of RANTES expression (Fig. 1B). This experiment was repeated twice more with similar results.



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  		FIGURE 2. Mig transcript level induction in brains of MV-infected mice. Homozygous line 18 mice were infected and at least three mice per timepoint were sacrificed at 0, 1, 3, 5, 7, 9, 14, 18, and 24 dpi. Total RNA was prepared from brain tissue and analyzed by RNA dot blot as described in Materials and Methods. The mean adjusted pixel value is shown.

 
Because lymphocytes that enter the MV-infected CNS are likely a major source of chemokines, we next determined the extent of chemokine expression in brains of mice lacking an adaptive immune response. NSE-CD46+/RAG-2 KO adult mice and CD46-/RAG-2 KO littermates (8) were inoculated with MV and brain tissues were harvested over a similar time course as described for immunocompetent mice. RPA showed that expression of RANTES and IP-10 was induced in NSE-CD46+/RAG-2 KO following infection (Fig. 3, representative experiment), but not in CD46-/RAG-2 KO animals. RNA levels of both mediators peaked at 13 dpi, but did not decline thereafter as seen in immunocompetent adults (Fig. 1), consistent with the unrestricted spread of MV in these immunodeficient mice. The induction at day 13 in NSE-CD46+/RAG-2 KO mice was 4.1-fold for RANTES and 14.0-fold for IP-10 transcripts as compared with baseline. These values are lower than those observed in immunocompetent mice (Fig. 1) but remained elevated later in the time course, correlating with the continued MV spread (8, 28).



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  		FIGURE 3. Chemokine induction in MV-infected immunodeficient NSE-CD46+ mice. NSE-CD46+/RAG-2 KO mice were infected with MV and at least four mice per timepoint were sacrificed at 0, 1, 5, 9, 13, and 18 dpi. Total RNA was prepared from brain tissue and analyzed by RPA. Mean values and error bars for adjusted Fuji pixels for both RANTES mRNA () and IP-10 mRNA () are shown.

 
Neurons are a source of chemokines

The sustained expression of chemokines in the CNS of MV-infected NSE-CD46+/RAG-2 KO mice suggested that neurons may be a source of chemokines following viral infection. Although neurons are known to produce chemokines following traumatic injury (22, 23), to date, viral infection has not been shown to trigger neuronal chemokine induction and secretion. To address this possibility, we employed primary hippocampal neuron cultures established from embryonic NSE-CD46+ mice. These neuron cultures are routinely >95% microtubule-associated protein 2-positive (9), and given the restricted expression of CD46 in neurons, the small percentage of contaminating non-neuronal cells in these cultures are not susceptible to MV infection and therefore are not likely a major source of chemokines. Four days postplating, neurons were infected with MV (multiplicity of infection; MOI = 3), an equivalent number of UV-inactivated MV particles, conditioned media, or LCMV (MOI = 3). Productive MV and LCMV infection was verified by immunostaining of infected neurons with viral Ag-specific Abs. Consistently, LCMV infection resulted in at least 40% of the primary neurons staining as viral Ag positive, whereas MV infection typically resulted in 25% Ag-positive neurons by 2 dpi. As shown in Fig. 4, transcripts for both RANTES and IP-10 were strongly induced in MV-infected primary neurons compared with signals obtained from conditioned media controls. No induction of chemokine synthesis in cultures treated with conditioned media or PBS was observed. Incubation of neurons with either LCMV (MOI = 3) or UV-inactivated MV (one dose of an MOI = 3 equivalent) minimally induced IP-10 and RANTES production. Thus, induction of RANTES and IP-10 in primary neurons is dependent on virus type, and although MV binding to neuronal receptors may stimulate basal expression of some chemokines, productive MV infection appears to be required for maximal induction.



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  		FIGURE 4. Chemokine expression in MV-infected primary neuron cultures. Embryonic transgenic primary hippocampal neurons were incubated with MV (MOI = 3), UV-inactivated MV (UV-MV; equivalent of MOI = 3), LCMV (MOI = 3), or conditioned medium. Total RNA was prepared from cells at 48 h postinfection and analyzed by RPA. The expression of RANTES, IP-10, L32, and GAPDH were quantitated and used for plotting. No expression of the other target RNAs in the probe set was observed. L32 and GAPDH-adjusted RANTES and IP-10 values from infected samples were compared with media, and the mean fold induction and SE obtained from three independent experiments is shown.

 
To determine whether the protein products of these transcripts were also produced in neurons, immunofluorescence was used to identify cells producing chemokines in NSE-CD46+/RAG-2 KO MV-infected mouse brain sections as well as in infected primary neuron cultures. Brain sections from infected NSE-CD46+/RAG-2 KO mice were stained by immunofluorescence to visualize IP-10 and MV-positive neurons, and to determine whether these proteins colocalize. Two different anti-IP-10 Abs showed that IP-10 expression colocalized with MV infected neurons (Fig. 5). A polyclonal Ab to IP-10 (Fig. 5, B and C, in red) as well as a mAb to IP-10 (Fig. 5, E and F) revealed IP-10 positive cells in the same regions as MV-infected neurons. MV Ag (Fig. 5, A and D, in green) overlaps with the IP-10 staining shown as yellow (white arrows, Fig. 5, C and E).



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  		FIGURE 5. IP-10 protein expression in the brains of MV-infected mice. NSE-CD46+/RAG-2 KO mice were infected with MV and the brains removed at 15 dpi. Sections were stained for MV Ag (A and D) and IP-10 protein with either a polyclonal antiserum (B) or monoclonal supernatant (E). Secondary Ab conjugates show the MV proteins as green and IP-10 as red. Overlaid images are shown in C and F, and were captured at x100. White arrows denote examples of colocalization.

 
Similarly, in primary neuron cultures, both IP-10 and RANTES proteins could be detected following MV infection. In the images shown in Fig. 6, B and C, RANTES staining (in red) appeared to be perinuclear with less staining observed in the neuronal processes. RANTES was exclusively found in virus-infected neurons. Consistent with our RNA analysis, IP-10 was heavily expressed in many infected neurons (composite scan, Fig. 6, E and F, in red), and expression was seen throughout the neuron including within the processes. Moreover, cells early in the infection cycle (showing less MV staining) appeared to be producing the greatest amount of IP-10 protein (data not shown).



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  		FIGURE 6. Chemokine protein expression in MV-infected primary neurons. Primary neurons were infected with MV and harvested at 48 h postinfection. Neurons were immunostained for MV-hemagglutinin (H) protein (A and D) and either RANTES (B) or IP-10 (E). Secondary Ab conjugates show the MV-hemagglutinin protein as green and chemokines as red. Merged images are shown in C and F, taken with an x100 objective.

 
To quantify the amount of chemokines secreted by infected neurons, supernatants from MV-infected primary neuron cultures were analyzed by ELISA. By this approach, RANTES expression in the supernatants of MV-infected neurons was greater than 20 pg/ml at 48 h postinfection (Fig. 7). Mock-infected samples yielded no chemokine production and the positive control sample consistently assayed as >100 pg/ml (Fig. 7). To date, we have not been able to quantify murine IP-10 by ELISA. Nevertheless, as noted in Discussion and shown in Fig. 5, other resident CNS cells also contribute to chemokine pools in vivo, and thus quantification of chemokines in vitro likely underestimates the amount made in infected brains.



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  		FIGURE 7. RANTES protein is secreted from MV-infected primary neurons in culture. Primary neurons were infected with MV or conditioned media and harvested at 48 h postinfection. Standard quantitative ELISA was used to calculate RANTES protein levels.

 
Anti-chemokine treatment results in decreased T cell infiltration to the CNS

It has been shown in other viral model systems of CNS disease that chemokine depletion by neutralizing Abs reduces the infiltration of the immune response into the CNS (19, 20, 21). We used this strategy to determine whether a similar effect was observed in MV-infected mice. Mice were inoculated with MV and dosed i.p. with either chemokine antisera or preimmune goat serum every other day for 9 days. On day 9, brains were removed and T cell levels were determined either by immunohistochemistry or flow cytometry analysis of enriched lymphocyte fractions. Each of the Ab treatments resulted in a marked reduction in T cell infiltration. Fig. 8 shows a representative experiment (of a total of four, all producing similar results) in which the number of brain lymphocytes from an enriched lymphocyte fraction were evaluated by FACScan. Shown in Fig. 8A are two dot plots of brain-derived, enriched lymphocytes stained for CD4 and CD8 Ags. The large number of double-negative cells likely includes non-T immune cells as well as resident brain cells. Administration of anti-Mig and anti-IP-10 Abs to infected mice substantially reduced the number of CD4+ and CD8+ T lymphocytes within the brain (20–40% reduction; Fig. 8B). Interestingly, however, anti-RANTES treatment appeared to have a selective effect on T cell entry, reducing CD8+ T cell levels by as much as 50%, but not appreciably influencing CD4+ T cell entry into the brain parenchyma.



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  		FIGURE 8. CNS T cell infiltration is reduced with anti-chemokine treatments of MV-infected mice. MV-infected, serum-treated mice were perfused with PBS at 9 dpi. Enriched brain lymphocytes from these mice were analyzed by flow cytometry for CD4+ and CD8+ cells and the percentage reduction (as compared with NGS) plotted. A, Two representative samples from flow analyzed brains. NGS is shown on the left and an anti-IP-10 treated sample is shown on the right. Y-axes are CD4+ cells and x-axes are CD8+ cells. Red lines show regions used to calculate percent reductions in T cell numbers. NGS and anti-IP-10 sample show 1565 and 1564 cells dot plotted (respectively). B, Reduction in the percentage of intraparenchymal CD4+ T cells and CD8+ T cells following specific anti-chemokine Ab treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we demonstrate that MV infection of susceptible transgenic mice and primary neurons cultured ex vivo resulted in an induction of chemokines. RNA transcripts of IP-10, Mig, and RANTES were detected in brains from both MV-infected immunocompetent and immunodeficient mice. Immunofluorescence was used to demonstrate that both IP-10 and RANTES protein were detectable in infected neuron cultures, but not in uninfected cultures. Interestingly, some MV Ag-negative cells in the infected cultures were positive for IP-10 chemokine staining, suggesting that uninfected neurons may also contribute to chemokine pools within infected tissues. Quantitation of RANTES in MV-infected neuron cultures showed that at least 20 pg/ml protein was produced in cultures that were 30–40% infected. A similar profile of chemokines was also observed in brains of MV-infected NSE-CD46+/RAG-2 KO mice, and expression levels coincided with infection levels. IP-10 expression colocalized with MV-infected neurons, supporting the hypothesis that neurons can synthesize chemokines in response to infection.

Depleting individual chemokines with specific neutralizing Abs reduced the entry of the protective T cell response into the infected parenchyma by as much as 50%, suggesting that chemokines contribute to immune cell entry into the infected CNS. Moreover, the chemokines that were induced in MV-infected NSE-CD46+ brains were similar to those induced by infection with other neurotropic viruses (16, 17, 18). In these other infections, glial cells are likely the major chemokine source. The similarity in chemokine profiles, despite the differences in virus and target cells within the CNS, suggests that IP-10 and RANTES may be especially relevant for recruitment of T cells into the brain parenchyma. Although these data do not prove that neuronally derived chemokines are functional, the amount produced in vitro and the expression patterns following infection strongly suggest that chemokines made by neurons play an important role in T cell recruitment to the infected brain. Together, these data indicate that under conditions of neuronal infection, proinflammatory chemokines are critically involved in the recruitment of the antiviral response, and that neurons can contribute to this process.

Although our data support the hypothesis that neurons augment chemokine pools within the MV-infected parenchyma, it is likely that other resident CNS cells, such as astrocytes and microglia, as well as infiltrating cells of the innate response such as NK cells and perivascular macrophages, also contribute. It has been suggested that the otherwise quiescent brain must be "primed" to allow for the peripheral immune response to enter and initiate clearance (33). If time must elapse to convert the "immune privileged" parenchyma to one that favors the recruitment and activation of the protective host immune response, then it is likely that chemokines made by resident CNS cells play a crucial role in this process. Synthesis of such mediators by multiple cell types, including adjacent cells that are not infected by virus, would likely increase T cell entry into the CNS.

A major conclusion of this paper is that neurons play an important role in their own defense against viral infections. Although this challenges the long-held belief that neurons are immunologically quiescent, an emerging literature indicates that neurons can both serve as beacons for the immune response, as well as being targets for T cell recognition. For example, while neurons do not normally express class I MHC determinants, under certain conditions in vitro, class I MHC can be detected on individual neurons, especially when elimination of that neuron would be advantageous to the host (2, 3). Thus, while neurons may be quiescent under homeostatic conditions, it cannot be ruled out that neurons may be targets for T cell recognition under certain circumstances, such as viral infection or loss of function. Experiments are underway to determine whether MV infection is associated with MHC induction in vivo.

In contrast to MV infection of adult NSE-CD46 mice, MV-inoculated NSE-CD46 neonates become heavily infected and succumb to CNS disease within 7–10 dpi, despite the recruitment of T cells to the parenchyma (10). Infected neonates up-regulate proinflammatory molecules, including chemokines, soon after infection (11), suggesting that the basis of the dichotomy between neonatal susceptibility and adult protection is not likely due to an inability of the neonate to establish a primed environment within the brain parenchyma. Instead, the fatal neonatal outcome may be attributable to either possible functional incompetence of the infiltrating T cell response or differences in the rate of viral spread within the neonatal CNS compared with the adult CNS.

Depletion of chemokines from MV-infected NSE-CD46+ mice resulted in a reproducible but modest reduction in T cell levels within the brain. It should be noted that only individual chemokines were depleted from these infected mice, and because of the potential of the antisera to induce an allogeneic response, the time course was terminated at 9 dpi, well before the peak of T cell infiltration into the parenchyma, which occurs 12–15 dpi. Whether a 40–50% decrease in T cell number in infected brains is of pathogenic relevance depends on the long-term response of chemokine-depleted mice to infection; these studies are underway using monoclonal neutralizing Abs.

Finally, one of the surprising results we obtained in the course of these studies was the finding that seemingly MV-Ag negative neurons were able to express IP-10, albeit to levels lower than those observed in neighboring MV-Ag positive cells. Although these data suggest that uninfected neighboring neurons can be stimulated in a paracrine manner to make chemokines, we cannot rule out that these IP-10-positive neurons were infected at a level too low to be detected by immunocytochemistry. Regardless, the observation that uninfected neurons (or neurons at a very early stage of infection) can produce chemokines confirms that the neuronal response to infection is very rapid, and underscores the likely importance of neurons in the recruitment of the adaptive immune response. We have shown that this protective response clears the infection in a cytokine-dependent, noncytolytic manner, resulting in protection from virus-induced neurologic damage (28). Thus, by rapidly contributing to the intraparenchymal chemokine pool and by favoring noncytolytic mechanisms of immune protection, neurons play a central role in their own survival following viral challenge.


   Acknowledgments
 
We thank Diane Lawrence and Jonathan Boyd for assistance with the experiments, and Michael B. A. Oldstone and Roberto Cattaneo for reagents. We are also grateful to Drs. David Wiest, Luis Sigal, and Kerry Campbell for their invaluable comments regarding the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health RO1 MH56951 (to G.F.R.), F32 NS11100 (to C.E.P.), and a grant from the Kirby Foundation. Back

2 Address correspondence and reprint requests to: Dr. Glenn F. Rall, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail address: glenn.rall{at}fccc.edu Back

3 Abbreviations used in this paper: NSE, neuron-specific enolase; MV, measles virus; NGS, normal goat serum; RPA, RNase protection assay; dpi, day postinfection; IP-10, IFN-{gamma} inducible protein 10 kD; Mig, monokine inducible by {gamma}; RAG, recombinase-activating gene; MOI, multiplicity of infection; KO, knockout; LCMV, lymphocytic choriomeningitis virus. Back

Received for publication November 20, 2002. Accepted for publication July 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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