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. Author manuscript; available in PMC: 2008 Oct 6.
Published in final edited form as: Viral Immunol. 2006;19(3):536–545. doi: 10.1089/vim.2006.19.536

Gene Expression Contributing to Recruitment of Circulating Cells in Response to Vesicular Stomatitis Virus Infection of the CNS

DEREK DC IRELAND 1,2, CAROL SHOSHKES REISS 1,3,4,5,6
PMCID: PMC2562241  NIHMSID: NIHMS66994  PMID: 16987071

Abstract

During acute Vesicular Stomatitis Virus (VSV) infection of the mouse central nervous system, neutrophils, natural killer (NK) cells, macrophages, and CD4+ and CD8+ T cells are recruited from the circulation in response to chemokines and cytokines. This study elucidated the production of these factors and infiltration of these peripheral cells. Chemokines that were observed included CCL1, CXCL10 (IP-10), CCL5 (RANTES), CCL3 (MIP-1α), CCL4 (MIP-1β), CXCL1 (MIP-2), CCL2 (MCP-1), and CCL11 (eotaxin). Cytokines produced in response to the infection include IL-1 and interferon-γ, but not type I interferons. Neutrophils are the first recruited cell type, appearing as early as 24 h after intranasal application of the virus. NK cells follow, but T cells are not detected until 6 days postinfection.

INTRODUCTION

Vesicular Stomatitis Virus (VSV) is an enveloped, single-stranded negative sense RNA virus of the family Rhabdoviridae. When infection of mice occurs by intranasal administration, VSV infects olfactory receptor neurons of the neuroepithelium and travels caudally through the cribriform plate to the olfactory bulbs. The virus then spreads caudally throughout the CNS, infecting many different cell types, including ependymal cells (23,29,30,47-49,51,53-56). In the absence of a vigorous early innate immune response, VSV infection can cause acute encephalitis resulting in hindlimb paralysis, with half the mice succumbing to the disease within 10 days post-infection. Mice that survive the disease recover and appear normal. Peripheral routes of infection of immunocompetent mice are rapidly cleared. In the CNS, the immune response is initiated by the innate arm of the immune response, with neutrophils infiltrating the CNS within 36 h postinfection (10,11). These neutrophils are quickly followed by natural killer (NK) cells (4,16), peaking 72 h postinfection; this innate response is quickly followed by the entry of T cells and macrophages, beginning day 6 post-infection, which is also required for clearance of the virus (29,51).

Cells are recruited from the peripheral circulation to the CNS in response to distinct chemoattractant signals. We had previously investigated, using genetic and pharmacologic approaches, the roles of leukotriene B4 (LTB4) and the small fragment of complement component C5a in contributing to the recruitment of neutrophils in response to VSV infection (10,11).

Chemokines are a family of small, soluble proteins that have been shown to play critical roles in the chemoattraction, maturation, and activation of immune cells to a site of infection, including into the CNS. Chemokines and their receptors are characterized by conserved N-terminal cysteine sequences, including the C, CC, CXC, and CX3C subfamilies. Unlike cytokines, the majority of which bind to only a single receptor, chemokines are much more promiscuous, with a single ligand binding multiple receptors. The function of a chemokine depends, in part, on the receptor expressed by the target cell. Therefore, a single chemokine may have multiple functions in a single target cell (3,57).

Many chemokines and chemokine receptors have been shown to be expressed by most resident cell types in the CNS, including microglia, astrocytes, and neurons (1,3,45,58,67). These chemokines are expressed both in the normal CNS and during times of immune stress. During immune responses to infection in the CNS, the cells that are attracted to the CNS also produce a large number of chemokines. Therefore, during an inflammatory response, the expression of both CNS intrinsic and extrinsic chemokines is present. Understanding the mechanisms governing the chemoattraction and subsequent infiltration of immune cells across the blood–brain barrier (BBB) and into the CNS during viral encephalitis or as a result of autoimmune disease is critical to our overall understanding of immune regulation in the CNS and the derivation of clinical methods to protect the CNS from inflammatory damage. In this study we have examined the expression of chemokines, inflammatory cytokines, and T cell-specific genes in brain tissue homogenates during the response of both BALB/c and B6 mice to VSV infection of the CNS.

MATERIALS AND METHODS

Virus

VSV, Indiana strain, San Juan serotype was propagated in Chinese hamster ovary (CHO) cells and purified over a sucrose gradient as previously described (4).

Animals and infection

Male C57BL/6J and BALB/cJ mice, 6 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in the New York University (New York, NY) animal facility under specific pathogen-free (SPF) conditions and in accordance with the Institutional Animal Care and Use Committee of New York University. Animals were given food and water ad libitum.

For infection, mice were anesthetized with 3% isoflurane (Fisher Scientific, Pittsburgh, PA) mixed with O2 (3 L/min). Mice were infected intranasally with 1.0 × 102 plaque-forming units (PFU) of VSV, Indiana strain, by placing 5 μL of VSV (1.0 × 104 PFU/mL) on each nostril for inhalation.

Tissue RNA extraction

VSV-infected mice were anesthetized by intraperitoneal injection of a lethal dose of Avertin. Each mouse was then perfused with 10 mL of sterile, RNase-free Hanks’ balanced salt solution (HBSS) and the brain was removed and placed in RNAlater RNA stabilizer (Ambion, Austin, TX). The tissue was homogenized in 1× RNA lysis buffer and total RNA was isolated with an RNAqueous-Midi kit, as per the manufacturer’s instructions (Ambion). The isolated RNA was stored in RNase-free double-distilled H2O in aliquots at −80°C to minimize freeze–thaw cycles.

Ribonuclease protection assay

Commercial multitemplate probe sets (mCD-1, mCK-2b, and mCK-5c; BD Biosciences Pharmingen, San Diego, CA) were transcribed with a MAXIscript T7 kit as per the manufacturer’s protocol (Ambion, Austin, TX), using [α-32P]dUTP (Perkin-Elmer, Norwalk, CT) to radiolabel single-stranded RNA probes for the ribonuclease protection assay (RPA). The probes were diluted to 3.2 × 105 and 2.5 × 105 cpm/μL for the mCD-1, mCK-2b, and mCK-5c probe sets, respectively, as per the manufacturer’s instructions (BD Biosciences).

The RPA was performed with an RPA III kit (Ambion), in accordance with the manufacturer’s recommended protocol for the BD Biosciences probe sets. Briefly, 40 μg of total RNA isolated from the brains of C57BL/6J and BALB/c mice (n = 4 per time point) killed on days 1, 2, 3, 6, 7, and 8 postinfection was concentrated by ammonium acetate–ethyl alcohol precipitation and resuspended in 8 μL of hybridization buffer. Sample RNA was mixed with 2 μL of diluted radiolabeled RNA probes. This mixture was incubated overnight at 50°C to allow the probes to hybridize with the sample mRNA. After hybridization, the samples were treated with RNase A/T1 for 30 min at 30°C to digest any single-stranded RNA remaining in the tube. The protected double-stranded RNA (dsRNA) hybrids were concentrated by precipitation and resuspended in 10 μL of gelloading buffer and denatured by heating at 95°C for 5 min.

The resulting RNA fragments were resolved by gel electrophoresis, using an 8 M urea–5% polyacrylamide gel (16 cm, 1-mm spacer) run at 180 V for approximately 3 h. The gels were dried onto Whatman filter paper (Whatman, Florham Park, NJ) and exposed to Kodak MS film in cassettes equipped with Kodak MS intensifying screens (Eastman Kodak, Rochester, NY). The radiographs were digitized with an HP Scanjet 6200Cxi flatbed scanner (Hewlett-Packard, Palo Alto, CA). The intensity of the bands was measured with Un-Scan-It gel densitometry software (Silk Scientific, Orem, UT) and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal loading control. The mean values of four mice were calculated and plotted, using MS Excel software (Microsoft, Redmond, WA).

Staining of neutrophils

Tissue was prepared for hematoxylin and eosin staining as previously described (23). At least three sections from three individuals were examined in three replicate experiments.

RESULTS AND DISCUSSION

Chemokine expression in the CNS of mice during VSV infection

Preliminary experiments using the ribonuclease protection assay (RPA) performed every other day during the course of VSV encephalitis (data not shown) indicated periods of change in chemokine expression. Significant changes in chemokine mRNA expression were observed during two periods. The first period of chemokine expression occurred in the first 3 days after infection. The second period occurred at the peak of disease (days 6–8 postinfection), independent of mouse strain. In contrast, naive age- and sex-matched controls tested negative for expression of all chemokines tested.

At 24 h postinfection, significant expression of two chemokines was observed in the CNS: CCL1 (TCA-3) and CXCL10 (IP-10) (Fig. 1, top). CCL1 is a known chemoattractant of neutrophils (20,22) and macrophages. Expression of CCL1 by microglia has been observed in experimental autoimmune encephalomyelitis (EAE) (43,62), supporting the early expression of CCL1 by CNS-resident cells to attract innate immune factors into the CNS.

FIG. 1.

FIG. 1

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Chemokine expression. Representative radiographs demonstrate the expression of chemokine mRNAs in the CNS during VSV encephalitis during early and late VSV encephalitis. Each lane represents an individual mouse, killed on the indicated day postinfection (day 0, uninfected controls). Lane D, digested probe control set; lane Un, undigested probe set, with the chemokine represented by each undigested band listed on the right. Arrows on the left mark the indicated chemokine in each lane of the radiograph (digested size). These data are representative of three individual experiments.

The expression of CXCL10, which recruits macrophages and monocytes, has been observed early in the course of other viral encephalitis models, including Theiler’s murine encephalitis virus (TMEV), lymphocytic choriomeningitis virus (LCMV), human immunodeficiency virus (HIV), and JHM strain of mouse hepatitis virus (JHMV) infections (2,3,8,9,21,25,26,28,42,44,45,50,66,68); as well as in EAE, the mouse model for multiple sclerosis (MS). In these models, it has been shown to be expressed by the CNS-resident cell population, including microglia, astrocytes, and neurons.

At the peak of VSV encephalitis (days 6–8 postinfection), we observed the expression of many other chemokines in the CNS (Fig. 1, bottom) in addition to CCL1 and CXCL10, including the following: CCL5 (RANTES), CCL3 (MIP-1α), CCL4 (MIP-1β), CXCL1 (MIP-2), CCL2 (MCP-1), and CCL11 (eotaxin).

CCL1, CCL2, CCL5, and CXCL10 are expressed at the most significant levels in the CNS of VSV-infected mice at the peak of inflammation. CCL1, CCL2, and CXCL10 all appear to be expressed in a biphasic pattern, with CCL2 being expressed only at lower levels in the early phase (days 1–3 postinfection) and at much greater levels at the peak of inflammation. CCL5 expression increases throughout the course of infection. This profile of chemokine expression is consistent with the chemoattraction and activation of multiple cell types into the CNS, including CD4- and CD8-positive T cells, which are important for the clearance of VSV infection, but may also cause significant immune-mediated damage to CNS-resident cells.

The chemokines expressed during VSV encephalitis have also been observed in other models of viral encephalitis, and in EAE and ischemia–reperfusion injury (2,3,8,9,21,25,26,28,42,44,45,50,66,68). This commonality underlines the importance of understanding the role chemokines play in the regulation of immune response in the CNS and potentially their use as therapeutic targets to control disease (67).

During acute TMEV, West Nile virus (WNV), mouse hepatitis virus (MHV), HIV, and LCMV infections (2,3,9,25,26,28,42,45,66,68), several chemokines are expressed by brain-resident cells. Common to many of these models are the following: CCL1, CCL2, CCL5, and CXCL10. In our intranasal VSV model, we confirm and expand on previous findings. In the brain of naive mice, we did not detect the expression of any chemokine tested. However, within 24 h postinfection, CXCL10 and CCL1 mRNAs were expressed.

CCL1 is a known chemotactic factor of neutrophils but not of lymphocytes in mice (20,22). The kinetics of CCL1 correlate well with the observed infiltration of neutrophils into periventricular regions of the VSV-infected brain. Expression of CCL1 and the presence of neutrophils may play important roles in BBB regulation, as research has indicated that neutrophils in the CNS act to disrupt BBB permeability (24,31). CXCL10 is a potent chemotactic factor of monocytes and NK cells to a site of infection. Expression of this chemokine has been shown to be in neurons, microglia, and astrocytes in other models of brain inflammation (25,42,45,58,67). The exact role of this chemokine is still a matter of intense study in several inflammatory models. The function of CXCL10 depends in part on the model in which it is studied (66). The rapidity with which the RNAs for CCL1 and CXCL10 are expressed implies that brain-resident cells are the cellular source, as cells from the peripheral immune response are not observed in significant numbers until at least 24 h postinfection, when neutrophils are present at periventricular locations. After day 3 post-infection, expression of many more chemokines is observed in the brain, including CCL5, CCL2, CCL3, and CCL4. This expression could also be the result of the activation of microglial cells, or expression by immune cells that are infiltrating into the CNS, including neutrophils and macrophages.

CCL5 is a potent chemotactic factor drawing T cells into sites of infection. It is expressed by endothelial cells as well as macrophages and microglia. CCL5 has also been shown to increase macrophage adherence to endothelial cells at inflammatory sites, causing blood-borne cells to stop and enter sites of inflammation (44). The absence of CCR5 (the receptor for CCL5) results in increased virus load and decreased lymphocyte infiltration during West Nile virus infection of the brain (26).

Mice deficient in the CX3C chemokine, which is expressed by neurons, and whose receptor is expressed by microglia and NK cells, were examined for their response to VSV infection. No difference between Fractalkine-deficient and wild-type mice was noted (C.S. Reiss and S. Lira, unpublished observation).

Neutrophil infiltration

On the basis of previous studies these time periods correspond to the observation of infiltration of innate cells (neutrophils, natural killer cells, and macrophages) and lymphocytes into the brain parenchyma (4,10,11,16,29). The first phase of expression corresponds to observed positive staining for neutrophils (peak, 36 h postinfection; Fig. 2) and infiltrating NK cells (peak, approximately 3–4 days postinfection). The second phase of expression corresponds to the infiltration of macrophages and CD4+ and CD8+ T cells (Figs. 3 and 4) (29,36).

FIG. 2.

FIG. 2

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Infiltration of neutrophils into the CNS. (a) Uninfected; (b) 24 h postinfection; (c) 36 h postinfection; (d) 48 h postinfection. Neutrophils (solid arrows) were counted in the periventricular region (open arrows mark the lumen of the lateral ventricle). (e) Quantification of neutrophils. Three individual mice per time point were used for immunohistochemical staining to determine the influx of neutrophils into the CNS. Cells were counted in four high-power fields (×400), three sections per donor, and average counts ±S.E. are shown.

FIG. 3.

FIG. 3

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Inflammatory cytokine expression. Representative radiographs demonstrate the expression of inflammatory mRNAs in the CNS during VSV encephalitis. Each lane represents an individual mouse, killed on the indicated day post-infection. Lane D, digested probe control; lane Un, undigested probe set, with the chemokine represented by each undigested band listed on the right. Arrows on the left mark the indicated cytokine in each lane of the radiograph (digested size). These data are representative of three individual experiments.

FIG. 4.

FIG. 4

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T cell marker expression. Representative radiographs demonstrate the expression of T cell mRNAs in the CNS during VSV encephalitis. Each lane represents an individual mouse, killed on the indicated day postinfection. Lane Un, undigested probe set, with the chemokine represented by each undigested band listed on the right. The arrows on the left mark the indicated cell marker in each lane of the radiograph (digested size). These data are representative of three individual experiments.

This rapid expression of CCL1 in the CNS (Fig. 1) correlates well with the infiltration of neutrophils, which peaks at periventricular locations within the infected CNS at 36 h postinfection, as indicated by both hematoxylin and eosin staining (Fig. 2) as well as immunohistochemistry for myeloperoxidase (data not shown). In other experiments, we have depleted neutrophils with the monoclonal antibody (mAb) RB6-8C5 (the generous gift of R. Coffman, DNAX/Schering-Plough Biopharma, Palo Alto, CA). These unpublished data are consistent with a critical early contribution of neutrophils in the immune response to VSV encephalitis. Similarly, the increased morbidity and mortality observed in infected mice that received zileuton treatment highlights the contribution of these cells (10).

Expression of inflammatory cytokines during VSV infection

We also examined the expression of inflammatory cytokines in RNA samples obtained from VSV-infected brains (Fig. 3), as well. As we have previously observed (27), interleukin (IL)-18, and caspase-1 (also known as ICE, the IL-1-converting enzyme), which activates the IL-18 zymogen, are constitutively expressed by mice and their levels of expression are not regulated by infection. Macrophage migration inhibitory factor (MIF) is also constitutively expressed.

IL-1α and IL-1β mRNAs are rapidly induced by infection and are expressed in waves consistent with production 24 h post-infection by a parenchymal cell, probably microglia. When macrophages are recruited from the circulation, starting on day 6, IL-1 mRNA levels rebound and peak on day 7 postinfection.

Although others have observed IL-12 and IL-23 mRNA production by microglia in response to infections and EAE (17,18,46,59-61), the mRNA levels for IL-12 p35 and IL-12 p40 were below the level of detection during VSV infection. We have observed beneficial effects of exogenous IL-12 administration (5,7,14,32,34,35,37,39,52), but failed to find a requirement for IL-12Rβ1 or IL-12Rβ2 during infection (32,33,34).

IFN-γ is not detected early, but is rarely seen in B6 mice on day 3 post-infection, possibly the product of inflammatory NK cells. IFN-γ expression is closely tied to infiltration of CD4+ and CD8+ T cells (Fig. 3), late in infection. IFN-γ has been shown to promote the clearance of virus from infected neurons via induction of nitric oxide synthase type 1 (NOS-1) (6,7,12-15,38,40, 41,54). Mice deficient in IFN-γR fared well (J.L. Hodges, N. Chen, and C.L. Reiss, unpublished observation), indicating that IFN-γ is not essential for host responses to infection. There was no difference between B6 and BALB/c mice in the helper T cell type 1 (Th1) response to infection (data not shown).

The anti-inflammatory Th2-derived cytokine IL-10 was observed first in a few BALB/c mice on day 6 postinfection, but both B6 and BALB/c mice downregulate the mRNA production by day 8 postinfection. The disease course does not differ between BALB/c and B6 mice in terms of clearance of infection, morbidity and mortality, infiltration of inflammatory cells, and the other mRNAs examined (Figs. 1 and 3); thus, it is unlikely that this IL-10 mRNA observation indicates a regulatory or essential contribution of Th2 cells to the pathology or resolution of the infection.

Type 1 IFNs are not detected in the brains of VSV-infected mice (65). VSV evades this powerful antiviral cytokine by shutting off host cell synthesis and nuclear export. VSV is exquisitely sensitive to the protective effects of IFN-β on neurons (64).

Expression of T cell markers in the CNS after VSV infection

To determine the kinetics of expression of T cell, B cell, and macrophage mRNAs during VSV infection, we examined the mRNAs isolated from infected brains. The earliest detected marker that was upregulated is F4/80, which is expressed primarily by macrophages and activated microglia (Fig. 4). B cells had not been detected in our immunohistochemical studies (4,16), and CD19 was also below the level of sensitivity of the RNase protection assay.

CD4+ and CD8+ T cells enter the CNS at the same time, first detected 6 days postinfection; both CD4+ and CD8+ T cells are essential for recovery (29,51). Furthermore, γδ T cells are not observed in the CNS at the same time points.

We have examined the induction and expression of essential cytokines and chemokines and associated them with recruitment of inflammatory cells during the course of acute VSV encephalitis in BALB/c and B6 mice. Although chemokines are unambiguously important for infiltration, we also recognize the essential roles of other chemoattracting molecules including the lipid mediator LTB4 and anaphylotoxins, and complement fragments such as C5a (10,11). Cytokines such as IL-12 can also have recruiting as well as activating roles for NK cells (19,63).

Acknowledgments

The authors are grateful to Jane A. McCutcheon, D.D.S., Ph.D. (NYU School of Dental Medicine) for thoughtful discussions and suggestions while this work was underway; to Sergio Lira, M.D., Ph.D. (then at Schering-Plough) for Fractalkine-deficient mice; to Robert Coffman (DNAX/Schering-Plough Biopharma) for mAb RB6-2C5; to Steve Zimmer for technical assistance; to Muneeb Samma and Eugene Friedman for performing the immunohistochemical experiments to identify neutrophils in the brains of VSV infected mice; and to Paul M. D’Agostino for assistance with manuscript preparation. This work was supported by grants from the NIH to C.S.R. (DC003536 and NS039746), and by a grant from NYU (N5385).

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