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The Journal of Immunology, 2007, 178, 8175 -8182
Copyright © 2007 by The American Association of Immunologists, Inc.
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IFN-{gamma}-Induced Chemokines Synergize with Pertussis Toxin to Promote T Cell Entry to the Central Nervous System1

Jason M. Millward*,{dagger}, Maria Caruso*, Iain L. Campbell{ddagger}, Jack Gauldie§ and Trevor Owens2,*,{dagger}

* Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada; {dagger} Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark; {ddagger} School of Molecular and Microbial Biosciences, University of Sydney, Sydney, Australia; and § Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Inflammation of the CNS, which occurs during multiple sclerosis and experimental autoimmune encephalomyelitis, is characterized by increased levels of IFN-{gamma}, a cytokine not normally expressed in the CNS. To investigate the role of IFN-{gamma} in CNS, we used intrathecal injection of a replication-defective adenovirus encoding murine IFN-{gamma} (AdIFN{gamma}) to IFN-{gamma}-deficient (GKO) mice. This method resulted in stable, long-lived expression of IFN-{gamma} that could be detected in cerebrospinal fluid using ELISA and Luminex bead immunoassay. IFN-{gamma} induced expression in the CNS of message and protein for the chemokines CXCL10 and CCL5, to levels comparable to those seen during experimental autoimmune encephalomyelitis. Other chemokines (CXCL2, CCL2, CCL3) were not induced. Mice lacking the IFN-{gamma}R showed no response, and a control viral vector did not induce chemokine expression. Chemokine expression was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. IFN-{gamma}-induced chemokine expression did not lead to inflammation. However, when pertussis toxin was given i.p. to mice infected with the IFN-{gamma} vector, there was a dramatic increase in the number of T lymphocytes detected in the CNS by flow cytometry. This increase in blood-derived immune cells in the CNS did not occur with pertussis toxin alone, and did not manifest as histologically detectable inflammatory pathology. These results show that IFN-{gamma} induces a characteristic glial chemokine response that by itself is insufficient to promote inflammation, and that IFN-{gamma}-induced CNS chemoattractant signals can synergize with a peripheral infectious stimulus to drive T cell entry into the CNS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Entry of lymphocytes to tissues is a prerequisite for immune surveillance, and also for organ-specific autoimmune disease. There is evidence that peripheral infections may contribute to immune cell entry to tissue. Infectious agents may play a role in the etiology of multiple sclerosis (MS),3 a disease presumed to be mediated by autoreactive T cells specific for myelin Ags in the CNS (1, 2, 3). Experimental autoimmune encephalomyelitis (EAE) is an animal model for MS. Infectious agents or their products are frequently used as adjuvants in EAE induction protocols. The toxin produced by Bordetella pertussis toxin (Ptx) is one such agent, used in EAE induction since 1955 (4). The generally held view has been that Ptx acts to "open up the blood-brain barrier," based on evidence that Ptx increases vascular permeability, in part by enhancing vascular sensitivity to vasoactive amines (5, 6). However, the role of Ptx in promoting inflammation is likely much more complex, influencing many facets of the immune response.

In EAE and MS, inflammation of the CNS is accompanied by elevated levels of the cytokine IFN-{gamma}. IFN-{gamma} is produced by activated T cells and NK cells (7), and is not detectable in the CNS except in the context of inflammation, when immune cells cross the blood-brain barrier and enter the CNS. IFN-{gamma} is a prototypic proinflammatory cytokine that can induce cascades of cytokine and chemokine expression. Although deleterious effects have been associated with its administration to an otherwise unmanipulated CNS by direct injection (8, 9), or by transgenic expression (10, 11, 12, 13), there is compelling evidence that it has a protective effect during CNS inflammation. Intracerebroventricular injection of recombinant, or intrathecally administered virus-encoded IFN-{gamma} suppressed the onset of EAE (14, 15), and depletion of IFN-{gamma} with anti-IFN-{gamma} Abs exacerbated EAE (16, 17, 18) and rendered resistant mouse strains susceptible (19). EAE in IFN-{gamma} knockout (GKO) mice, or mice lacking the IFN-{gamma}R, is more severe than in wild-type (WT) mice (20, 21, 22), with disseminated neutrophilia. Expression of chemokines in the CNS differs during disease in GKO and WT mice (23). The T cell and monocyte/macrophage-attracting chemokines CXCL10 (IFN/IP-10) and CCL5 (regulated upon activation T cell expressed and secreted, RANTES) are increased in WT EAE, but are not prominent in GKO EAE. Conversely, expression of the neutrophil-attracting CXCL2 (MIP-2) is elevated in GKO but not WT EAE (23, 24, 25). We have proposed that IFN-{gamma} influences chemokine profiles in the CNS, although direct cause and effect have not been established.

To examine the role of IFN-{gamma} on chemokine expression in the CNS, we have delivered the IFN-{gamma} gene to GKO mice using a replication-defective adenovirus. This allows expression of IFN-{gamma} in the CNS without the complex assortment of signals present during immune inflammation. Using the ependymal route, described by Martino et al. (26), allows vectors to distribute throughout cerebrospinal fluid (CSF) and infect ependymal and leptomeningeal cells, which in turn secrete the product of the transgene into the CSF. This route avoids the traumatic effects of tissue injury associated with intraparenchymal and intracerebroventricular injections, which can include chemokine production and disruption of the blood-brain barrier (27, 28, 29, 30).

Our findings demonstrate that IFN-{gamma} induces expression of a specific restricted pattern of chemokines in meningeal and ependymal cells. When intrathecal production of IFN-{gamma} was accompanied by i.p. administration of Ptx, there was a dramatic increase in the number of blood-derived leukocytes, mostly T cells, in the CNS. This increase was not accompanied by inflammatory pathology or up-regulation of inflammatory cytokines IL-1beta or TNF-{alpha}. These results show that IFN-{gamma} induces specific chemokine profiles in the CNS and that the resulting tissue microenvironment synergizes with pathogen-associated signals to promote leukocyte entry.


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

BALB/c mice homozygous for a disrupted IFN-{gamma} gene (originally developed by D. K. Dalton and colleagues; Genentech) were crossed to SJL/J mice (The Jackson Laboratory). The F1 progeny were backcrossed for five generations to yield F5 SJL GKO mice (31). WT F5 SJL littermates were used as controls. In some experiments, C57BL/6-backcrossed GKO mice with B6 controls (The Jackson Laboratory) were used. We confirmed that results were identical on both backgrounds. EAE was induced in WT mice by s.c. immunization at the base of the tail with 100 µl of an emulsion containing 200 µg of proteolipid protein 139–151 (Sheldon Technologies) and CFA (Fisher). SJL/J-backcrossed mice lacking the IFN-{gamma}R (32) were bred from mice donated by Dr. D. Willenborg (Neuroscience Research Unit, The Canberra Hospital, Garran, Australia). All animals were kept in microisolator cages in a pathogen-free facility. Animal maintenance and experimental protocols were in accordance with guidelines of the Canadian Council for Animal Care, as approved by the McGill University Animal Care Committee. Additional experiments were done at the University of Southern Denmark (Odense, Demnark). These procedures were conducted according to the Danish Laboratory Animal Inspectorate (J. no. 2004/561-920). All experiments conformed to international guidelines on the ethical use of animals.

Intrathecal injection

All procedures were conducted under aseptic conditions. Mice were i.p. anesthestized with 0.05 ml of a ketamine/xylazine/acepromazine mixture (50/5/1 mg/kg), and the back of the head shaved. A 30-gauge needle (bent at a 40° angle 3 mm from the tip) attached to a 50-µl Hamilton syringe was inserted into the intrathecal (subarachnoid) space of the cisterna magna (cerebellomedullary cistern) as described previously (33). This space is located at the back of the head between the skull and the cervical vertebra. Adenovirus (10 µl) in sterile PBS (109 PFU/ml) was injected over 30 s. This adenovirus was type 5 E1-E3-deleted encoding murine IFN-{gamma} gene (AdIFN{gamma}), driven by the CMV immediate-early promoter (34). Adenovirus encoding the beta-galactosidase gene (AdLacZ), provided by Dr. J. Nalbantoglu (Montreal Neurological Institute, Montreal, Quebec, Canada), or an equal volume of sterile PBS alone was used as control. Some mice received 300 ng of Ptx (List Biochemicals) by i.p. injection 1 day postintrathecal injection. After receiving AdIFN{gamma} and Ptx, some mice were treated with monoclonal anti-mouse CCL5 (clone R6G9) or polyclonal rabbit anti-mouse CXCL10, provided by Dr. T. Lane (University of California, Irvine, CA). Treatments of 0.5 ml of anti-CXCL10 antiserum (~0.5 mg/ml), 250 µg of R6G9, or 250 µg of IgG were i.p. administered for 5 days (35, 36).

IFN-{gamma} and chemokine detection in CSF

Anesthetized mice (Somnotol; MTC Pharmaceuticals) were perfused intracardially with 20 ml of cold PBS. The musculature was dissected to reveal the dura covering the cisterna magna. A fine glass capillary tube was inserted through the dura, and CSF was aspirated. Samples visibly contaminated with blood were discarded. IFN-{gamma} protein was detected is individual CSF samples (approximate volume 10 µl) by ELISA (BD Biosciences). In addition IFN-{gamma}, CXCL10, and CCL5 protein was measured simultaneously in a multiplex Luminex assay (BioSource International). Individual CSF samples were diluted to 25 µl of volume and incubated with a suspension of analyte capture Ab-conjugated microspheres, according to the manufacturer’s instructions. After further incubation with biotinylated detection Abs and PE-conjugated streptavidin, fluorescent signal was read on a Luminex 100 system (Applied Cytometry Systems). A five-parameter logistic curve generated from standards of known concentration was used to convert median fluorescent intensity to concentration values, with STarStation software (Applied Cytometry Systems). Concentration values were adjusted for sample dilution. The limits of detection were <1 pg/ml for IFN-{gamma}, <40 pg/ml for CXCL10, and <60 pg/ml for CCL5.

Histology

After anesthesia and perfusion, brain and spinal cord samples were removed, embedded in OCT (Canemco-Marivac) and frozen in methylbutane and dry ice, then cut into 10-µm cryostat sections. Sections were stained with H&E. Three brain and three spinal cord samples for each treatment group (two sections per sample) were examined at high power. For TCR-beta immunohistochemistry, sections were fixed with 1% paraformaldehyde in TBS for 15 min at room temperature. Sections were incubated with 10% normal mouse serum for 1 h at room temperature. Primary mAb (hamster anti-mouse TCR-beta; BD Pharmingen) was applied overnight at 4°C, followed by secondary Ab (biotinylated mouse anti-hamster Ig) for 1 h at room temperature then PE-conjugated streptavidin (BD Pharmingen) for 1 h at room temperature. Sections were stained with 0.01% Hoechst dye, mounted and visualized on a Leica DMIRE2 fluorescent microscope. Control sections were incubated with isotype-matched primary Abs or with secondary alone.

Flow cytometry

Brain and spinal cord homogenates were prepared as described previously (37). Cell suspensions were stained with FITC-conjugated rat anti-mouse CD11b, PE-conjugated rat anti-mouse CD45, and PerCP-conjugated hamster anti-mouse TCR-beta (BD Pharmingen) for 20 min at 4°C, washed, and analyzed with a BD Biosciences FACScan flow cytometer.

Quantitative real-time PCR

RNA was extracted from brain and spinal cord homogenates using the TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. The RNA was then reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) with random hexamer primers as described previously (23). Quantitative real-time PCR was performed with 1 µl of sample cDNA in a 25-µl reaction volume containing 12.5 µl of TaqMan PCR Master mix (Applied Biosystems), forward and reverse primers (900 nM; Sigma-Aldrich), and TaqMan probe (200 nM; Applied Biosystems). Primer and probe sequences were as follows: IFN-{gamma} (forward) CAT TGA AAG CCT AGA AAG TCT GAA TAA C, (reverse) TGC TCT GCA GGA TTT TCA TG, (probe) TCA CCA TCC TTT TGC CAG TTC CTC CAG-MGB; CXCL10 (forward) GCC GTC ATT TTC TGC CTC AT, (reverse) GGC CCG TCA TCG ATA TGG, (probe) GGA CTC AAG GGA TCC-MGB; CCL5 (forward) GGA GTA TTT CTA CAC CAG CAG CAA, (reverse) CAC ACA CTT GGC GGT TCC TT, (probe) TGC AGT CGT GTT TGT C-MGB; IL-1beta (forward) CTT GGG CCT CAA AGG AAA GAA, (reverse) AAG ACA AAC CGT TTT TCC ATC TTC, (probe) AGC TGG AGA GTG TGG AT-MGB; and TNF-{alpha} (forward) CCA AAT GGC CTC CCT CTC AT, (reverse) TCC TCC ACT TGG TGG TTT GC, (probe) CTC ACA CTC AGA TCA T-MGB. The quantitative real-time PCR was done on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Cycle threshold values were converted to arbitrary units using standard curves generated by serial dilutions of cDNA from one sample for each gene analyzed. Data are reported as the ratio of target gene expression over expression of 18S rRNA, which served as the endogenous reference. cDNA was diluted 1/1000 for 18S rRNA analysis.

In situ hybridization (ISH)

Mice were anesthetized with Somnotol and perfused intracardially with 20 ml of PBS followed by 20 ml of 4% paraformaldehyde. Brain and spinal cord samples were dissected and postfixed in 4% paraformaldehyde for 24 h at 4°C, then dehydrated and embedded in paraffin. ISH for CXCL10/IP-10 was conducted as described previously (38). Briefly, 10-µm sagittal sections were dewaxed and rehydrated. After pretreatment with 4% buffered formaldehyde, proteinase, and acetic anhydride, slides were dehydrated and dried. 33P-labeled antisense CXCL10 probes were hybridized to the tissue at 55–59°C overnight. After digestion with RNase A, slides were washed in SSC. Sections were immunostained for glial fibrillary acidic protein and tomato lectin. Slides were dipped in Kodak NTB-2 emulsion, exposed at 4°C for 6–21 days, and developed. Control sense CXCL10 probes did not show any signal (38).

Statistics

Results were analyzed using Student’s t test or ANOVA (with Tukey’s multiple comparisons). Values for p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Intrathecal IFN-{gamma} induces selective chemokine expression in CNS

AdIFN{gamma} (107 PFU) was injected intrathecally via the cisterna magna into GKO mice. Efficacy of gene transfer was confirmed by detection of IFN-{gamma} mRNA in perfused brain and spinal cord of recipient GKO mice by quantitative real-time PCR up to 30 days postinjection (data not shown) and of IFN-{gamma} protein in CSF by ELISA and Luminex (Fig. 1A). IFN-{gamma} mRNA was also detected in cervical lymph nodes of GKO mice that received AdIFN{gamma} (Fig. 1A, inset). IFN-{gamma} significantly increased expression of CXCL10 and CCL5 in the CNS, as shown by quantitative real-time PCR 7 days postinfection (Fig. 1, B and C). Chemokine expression in CNS of AdLacZ- or sham-injected mice was no different from expression in CNS of unmanipulated controls. The magnitude of expression of both CXCL10 and CCL5 was equivalent to that seen in the CNS of WT mice with EAE. Importantly, mice lacking the IFN-{gamma}R (IFN-{gamma}RKO) did not show induction of CXCL10 or CCL5 expression by AdIFN{gamma} (Fig. 1, B and C).


Figure 1
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  		FIGURE 1. Intrathecal injection of AdIFN{gamma} induced IFN-{gamma} and chemokine expression in the CNS. A, IFN-{gamma} protein (pg/ml) was detected by Luminex in CSF of GKO mice 7 days postinjection of AdIFN{gamma} and in CSF of WT mice with severe EAE. IFN-{gamma} was not detected (nd) in CSF of GKO or WT mice receiving sham or AdLacZ injections, or in unmanipulated mice. Each data point represents one mouse. IFN-{gamma} message was detected in cervical lymph nodes of GKO mice after receiving AdIFN{gamma} (inset). Real-time PCR detection of message for CXCL10/IP-10 (B) and CCL5/RANTES (C) in spinal cord of GKO mice or IFN-{gamma}R-deficient ({gamma}RKO) mice 7 days posttreatment with AdIFN{gamma} or AdLacZ, or in sham-injected or unmanipulated animals. Target expression is relative to 18S RNA (1/1000 dilution) for each sample. The horizontal bar represents the mean value.

 
CXCL10 and CCL5 proteins were substantially up-regulated in CSF of GKO mice 5 days after receiving AdIFN{gamma} (Fig. 2). The magnitude of chemokine protein in the CSF was greater than that detected in CSF from WT mice with EAE, demonstrating that AdIFN{gamma}-induced chemokines were present in biologically relevant quantities. However, AdIFN{gamma} did not induce expression beyond baseline levels of other chemokines, including CXCL2, CCL2/MCP-1, or CCL3/MIP-1{alpha} (data not shown). These chemokines have been implicated in CNS inflammatory responses (25, 30, 39, 40), and their absence in this system suggests that other signals in addition to IFN-{gamma} are required for immune pathology.


Figure 2
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  		FIGURE 2. Chemokine protein present in CSF of mice given AdIFN{gamma} plus Ptx. CXCL10 (A) and CCL5 (B) were measured simultaneously in CSF samples using a Luminex cytometric bead assay system. Limit of detection was 40 pg/ml for CXCL10 and 60 pg/ml for CCL5. Each data point represents one mouse. The horizontal bar represents the mean value.

 
Cellular localization of IFN-{gamma}-induced chemokine response

ISH was used to localize IFN-{gamma}-induced chemokine expression. Chemokine message was predominantly localized to ependymal (Fig. 3A, CXCL10 message in fourth ventricle) and meningeal cells (Fig. 3B, cerebellar leptomeninges). This localization reflects the distribution of infected cells that was separately visualized by histochemical detection of beta-galactosidase after intrathecal injection of a LacZ-encoding vector (data not shown), and matches the previously reported distribution of intrathecally injected vectors (15). Chemokine message was also seen in glial cells, by colocalization with immunohistochemistry for glial fibrillary acidic protein (Fig. 3C) and tomato lectin (Fig. 3D), indicating that astrocytes and microglia, respectively, also responded to intrathecally produced IFN-{gamma} to induce chemokine expression. Chemokine message was not detected in tissue from AdLacZ- or sham-injected controls (data not shown).


Figure 3
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  		FIGURE 3. Cellular localization of AdIFN{gamma}-induced chemokine expression in CNS and absence of inflammation. A–D, ISH detection of CXCL10/IP-10 message in CNS of mice 7 days after infection with AdIFN{gamma}. Chemokine-expressing cells are identified by foci of silver grains. Ependyma of the fourth ventricle (A) and cerebellar meningeal region (B) are represented. Parenchymal ISH signal colocalized with immunohistochemically detected glial fibrillary acidic protein (astrocytes) (C) and tomato lectin (microglia) (D). E–G, Histology showing that chemokine expression was not accompanied by inflammation in regions adjacent to ISH signals, examined 7 days after infection with AdIFN{gamma}. H&E staining of fourth ventricle (E), immunofluorescent staining for TCR-beta (F) showing lack of positive cells in cerebellar meninges (Hoechst stained nuclei in blue), H&E staining in thoracic spinal cord (G), and H&E staining showing extensive inflammation in spinal cord (H) of a GKO mouse with severe EAE. I–K, Histology showing absence of inflammation in mice receiving both AdIFN{gamma} and Ptx. H&E staining of fourth ventricle (I), immunofluorescent staining for TCR-beta (J) showing lack of positive cells in cerebellar meninges, and H&E staining of thoracic spinal cord (K). L, Positive staining (red) is shown for TCR-beta in EAE spinal cord. In situ was done on two mice from each group; remaining panels are representative of three mice per group (two sections per mouse). Original magnification at x1000 (A–D), x400 (E, F, I, and J), and x200 (G, H, K, and L).

 
IFN-{gamma}-induced chemokines plus peripheral PTx promote T cell entry into CNS

Local sites of chemokine expression were not accompanied by inflammation (Fig. 3, A–D), nor was inflammation observed in other CNS regions. H&E stained sections were examined at high power to screen for foci of inflammation. Brain and spinal cord samples from AdIFN{gamma}, AdLacZ, or sham-injected mice showed neither IFN-{gamma}- nor adenovirus-induced inflammation. Representative sections of brain (Fig. 3E, fourth ventricle) and thoracic spinal cord (Fig. 3G) 7 days posttreatment with AdIFN{gamma} are shown. A comparable section from spinal cord of a GKO mouse with EAE is shown in Fig. 3H as a positive example of CNS inflammation. Additionally, immunofluorescent staining for TCR-beta was used to screen for T cells in the CNS that might have eluded detection on H&E stained sections. No TCR-beta-positive cells were observed in brain or spinal cord samples of AdIFN{gamma}-treated mice (Fig. 3F, cerebellar meninges), in contrast to readily detectable TCR-beta-positive cells observed in EAE lesions (Fig. 3L).

Entry of blood-derived cells to the CNS can be quantitated by detection of CD45high cells in cell suspensions of perfused CNS tissue by flow cytometry. CNS resident microglia, which express low levels of CD45, can be distinguished from blood-derived immune cells (41). CNS inflammation, such as occurs during EAE, leads to a dramatic increase in the number of CD45high cells (31). CD45high cells in unmanipulated CNS (Fig. 4) likely represent a combination of perivascular microglia/macrophages (41) and a baseline level of T cells associated with immune surveillance (42). There was a dramatic increase in the number of CD45high cells in the CNS of mice that received 300 ng of Ptx following intrathecal AdIFN{gamma} (Fig. 4). This increase did not occur in mice given AdIFN{gamma} alone (although a slight elevation in CD45high proportions was seen, it was not statistically significantly greater than the proportion in unmanipulated mice), and was not seen in CNS of mice that received Ptx after sham intrathecal injection or after injection with the control AdLacZ vector (Fig. 4, combined results of four repetitions of the experiment).


Figure 4
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  		FIGURE 4. Treatment with AdIFN{gamma} combined with peripheral Ptx elicits immune cell entry to the CNS. The number of CD45high (blood-derived) cells in suspensions from perfused CNS was measured by flow cytometry, as described in Fig. 5. Results are the percentage of total live cells (by forward and side scatter gating). Samples were analyzed 5 days posttreatment with AdIFN{gamma} plus Ptx. ns, Not significant. The horizontal bar represents the mean value.

 
The great majority of CD45high cells detected in the CNS were T cells, as indicated by staining with anti-TCR-beta and shown as representative flow cytometry profiles (Fig. 5). It is likely that the T cells detected in the CNS did not have an activated phenotype because expression of the activation markers CD44, CD49a, and CD62 on T cells isolated from lymph nodes did not differ between unmanipulated mice and mice that received Ptx and AdIFN{gamma}, as measured by flow cytometry (data not shown). It is also unlikely that Ptx affected T cells by up-regulating chemokine receptors because expression of CXCR3 and CCR5 did not differ between mice given AdIFN{gamma} with Ptx and mice given AdIFN{gamma} alone, as measured by quantitative real-time PCR of lymph nodes and brain (data not shown).


Figure 5
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  		FIGURE 5. Flow cytometric detection of T cells in CNS. Flow cytometry profiles of CNS cell suspensions showing staining for CD45 (y-axis, all profiles) and CD11b or TCR-beta (x-axis, as indicated) for samples analyzed 5 days posttreatment.

 
To evaluate the role of chemokines, mice given AdIFN{gamma} and Ptx were treated with chemokine blocking Abs. Mice were given monoclonal anti-CCL5 Ab or polyclonal anti-CXCL10 antiserum i.p. for 5 days following administration of AdIFN{gamma} and Ptx. Efficacy of these reagents has been validated in studies involving blocking of chemokine-driven immune events in mouse CNS inflammation (35, 36). In neither case did the blocking Ab affect infiltration of CD45high leukocytes, as assessed by flow cytometry (data not shown).

Mice infected with AdIFN{gamma} alone or in combination with Ptx remained healthy, with no clinical signs of CNS inflammation or disease (i.e., no signs of neurological dysfunction, no loss of body weight, no change in grooming or behavior). Consistent with this finding, the increase in blood-derived CD45high cells in the CNS in response to the combination of AdIFN{gamma} and Ptx did not manifest as histopathology. No foci of inflammation were seen on H&E sections and no TCR-beta-positive cells were observed by immunofluorescence (Fig. 3, I–K). Furthermore, addition of Ptx to AdIFN{gamma}-treated mice did not cause up-regulation of message for proinflammatory cytokines IL-1beta or TNF-{alpha} in CNS, despite robust detection in EAE by quantitative real-time PCR (Fig. 6).


Figure 6
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  		FIGURE 6. AdIFN{gamma} and Ptx do not up-regulate proinflammatory cytokines in CNS. Quantitative real-time PCR detection of message for IL-1beta (A) and TNF-{alpha} (B) in CNS of mice that received AdIFN{gamma} or AdIFN{gamma} with Ptx, or mice with EAE. The horizontal bar represents the mean value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Two principal findings emerge from this study. First IFN-{gamma}, in the absence of any accompanying stimulus, induced astrocytes, microglia, meningeal, and ependymal cells to express a restricted profile of chemokines. Second, this IFN-{gamma}-induced inflammatory microenvironment synergized with peripherally administered Ptx to elicit an influx of T cells into the CNS that did not occur when either AdIFN{gamma} or Ptx were given alone.

This study offers an in vivo examination of the specific role of IFN-{gamma} on chemokine induction in the CNS, distinct from the multitude of other mediators normally present during inflammation. Our results confirm that IFN-{gamma} is not present in the uninflamed CNS, as IFN-{gamma} mRNA was undetectable by quantitative real-time PCR in brain and spinal cord of unmanipulated WT mice, and IFN-{gamma} protein was undetectable in unmanipulated WT CSF. As a further experimental control over IFN-{gamma} expression, we administered AdIFN{gamma} to the CNS of GKO mice, which have no endogenous IFN-{gamma} expression. Other studies that have applied IFN-{gamma} to mouse CNS, either by transgenic expression or direct injection into CNS parenchyma (8, 9, 11, 12, 13), or intrathecal injection of a viral vector encoding IFN-{gamma} to WT mice (15), did not include analysis of chemokine expression in the CNS. Our findings show that IFN-{gamma} induced a selective chemokine response. IFN-{gamma} regulates expression of CCL5 and CXCL10 (IP-10) via IFN-stimulated response elements that are present in their promoters. IFN-regulatory factor-1 is essential for IFN-{gamma}-induced CCL5 expression (43, 44). Chemokine induction was not due to nonspecific effects of the viral vector because chemokine expression in mice injected with the control LacZ virus did not differ from expression in unmanipulated controls. Furthermore, AdIFN{gamma} did not induce chemokine expression in mice lacking the IFN-{gamma}R.

The magnitude of chemokine message in response to AdIFN{gamma} was equivalent to that seen in EAE, and is therefore physiologically relevant. The level of chemokine protein in CSF of mice given AdIFN{gamma} was even greater than the protein level seen in EAE, which suggests that chemokine protein was not in limited quantity to achieve a biological effect. The high level of chemokine protein may reflect the nature of the intrathecal route of administration, whereby the viral vector infects meningeal and ependymal cells and the gene product is secreted directly into the CSF. Nevertheless, ISH showed CXCL10 message in the parenchyma, indicating that IFN-{gamma} secreted into the CSF could penetrate into CNS tissue.

Despite the clear induction of CXCL10 and CCL5 expression, administration of AdIFN{gamma} did not increase CNS expression of other chemokines (CXCL2, CCL2, CCL3) that have been implicated in CNS inflammation. This result indicates selectivity in the chemokine response to IFN-{gamma} and underscores that mediators other than IFN-{gamma} contribute to establishing a proinflammatory milieu during CNS inflammation. This indication is supported by the finding that AdIFN{gamma} with or without Ptx did not induce expression in CNS of IL-1beta or TNF-{alpha}, whereas these proinflammatory cytokines were abundantly expressed in EAE CNS.

The chemokines we examined are of particular relevance to the differences between EAE in WT and GKO mice (23). Our data show that IFN-{gamma} induces CXCL10 and CCL5 expression in the CNS, independent of infiltrating cells, suggesting that these chemokines are a cause of the differences in cellular infiltration in those EAE models, not a consequence. The prior entry of the IFN-{gamma}-producing T cells that induce this chemokine expression is driven by other mechanisms (45).

These chemokines are implicated in driving leukocyte entry to the CNS. The lack of histopathology induced by IFN-{gamma}-induced expression of chemokines in the CNS in the present study stands in contrast to results obtained by transgenic overexpression. Mice expressing CXCL10 in astrocytes showed spontaneous leukocyte accumulation in perivascular, meningeal, and ventricular areas, but did not show astrocyte activation or other evidence of brain pathology (46). Similarly, myelin basic protein promoter-driven transgenic expression of CCL2 in the CNS led to perivascular accumulation of monocytes/macrophages that did not penetrate into the brain parenchyma and was not associated with brain pathology (47). The leukocyte extravasation reported in these transgenic mice may reflect differences in the cellular source and location of chemokine production, as well as the cumulative effects of lifetime expression, as contrasted with the present study in which the increase in chemokine expression resulted from de novo administration of IFN-{gamma} in adulthood. However, in both of the transgenic models mentioned, as well as another transgenic model with CCL2 expression in astrocytes, leukocyte recruitment to the CNS was substantially increased following peripheral administration of infection-related stimuli (LPS) (47) or Ptx and CFA (46, 48).

When Ptx was administered in conjunction with AdIFN{gamma}, there was a dramatic and significant increase in the proportion of CD45highTCR-beta+ cells in the CNS that did not occur with Ptx alone. This effect was not inhibited by peripheral administration of CXCL10 or CCL5 blocking Abs. It may be that the Abs did not access the critical CNS compartment to achieve effect, or that Ab levels were insufficient to block chemokine effects in our system. Other studies have used these same reagents to influence CNS inflammatory events (35, 36), but in different model systems. Alternatively, it may simply be that CXCL10 and CCL5 are not the critical mediators driving CNS T cell entry in response to IFN-{gamma} and Ptx, despite their clear up-regulation. Future studies should address these alternative interpretations.

The fact that EAE-equivalent levels of IFN-{gamma} and chemokines did not on their own lead to clinical or histopathological inflammation suggests that disease pathology requires additional stimuli, such as pathogen-derived signals. Laflamme and Rivest (49) used LPS to induce chemokine and cytokine expression in regions of the CNS devoid of blood-brain barrier. Interestingly they did not describe leukocyte entry to the CNS. Alternatively, injury-related signals, as produced during experimental brain lesions, have been shown to induce chemokines and cytokines as well as immune cell entry (28). The absence of detectable histopathology in both of these studies and our own may reflect insufficient frequency of CNS Ag-specific T cells. This possibility is supported by the fact that transgenic mice expressing myelin-specific T cells, which do not develop EAE when housed in pathogen-free conditions (50), develop spontaneous encephalomyelitis when injected with Ptx or when they are exposed to pathogens in conventional housing conditions (50). The fact that the T cells that entered the CNS in our study showed a nonactivated phenotype is further consistent with this possibility.

There are a multitude of mechanisms by which Ptx can influence CNS inflammation (51). The EAE enhancing effects of Ptx have been proposed to be mediated in part by binding to TLR4, which results in increased expression of P-selectin (52). This increases lymphocyte rolling and adhesion in the cerebrovasculature and leads to increased permeability of the blood-brain barrier (52). Ptx was shown to enhance activation of APCs via TLRs and to induce EAE in an antimyelin (proteolipid protein 139–151) TCR transgenic mouse model on a genetically resistant background (53). In addition to local effects on the cerebrovasculature, Ptx has been reported to have a variety of other effects on T cells in the periphery (54, 55, 56, 57, 58, 59). We examined CXCR3 and CCR5 expression in lymph nodes of mice that received AdIFN{gamma} or AdIFN{gamma} with Ptx and did not detect a significant difference. We also did not detect a difference in CXCR3 or CCR5 expression in the uninfiltrated (AdIFN{gamma}) or infiltrated (AdIFN{gamma} plus Ptx) CNS, suggesting that Ptx did not affect chemokine receptor induction either on the T cells in the periphery, from which infiltrating cells derive, or on the infiltrating T cells themselves.

Nevertheless, the role of Ptx is complex. Despite its extensive use as a critical component of EAE induction protocols, Ptx has a clearly established effect as an inhibitor of G protein-coupled receptors. Chemokines operate through these types of receptors, which complicates the interpretation of studies using Ptx to facilitate chemokine-dependent phenomena (such as EAE). Future studies should attempt to reconcile these disparate effects.

The complex array of inflammatory mediators present during EAE and MS make it difficult to establish regulatory relationships between specific mediators and chemokines. This highlights the advantage of the gene transfer approach used in the present study, in which a specific cytokine (in this case IFN-{gamma}) was introduced to the CNS without inducing inflammation. We show that IFN-{gamma} induced chemokine expression in the CNS. Chemokines contribute to a tissue microenvironment that synergizes with infection-related stimuli to induce T cell entry to the tissue. Other factors are required for these immune cells to provoke inflammation and pathology. Detailed understanding of the interplay between cytokines, chemokines, and infectious agents will be essential for understanding and treating CNS inflammatory diseases.


   Acknowledgments
 
We thank Dr. Simone Zehntner, Leah Remington, and other members of the Owens lab for input and assistance. In situ hybridization was conducted by Carrie Kincaid (Neuropharmacology Department, The Scripps Research Institute, La Jolla, CA). Dr. Tanja Kuhlmann (Neuropathology Department, University of Göttingen, Göttingen, Germany) assisted with histological assessment. Dr. Marianne Jakobsen (Department of Clinical Immunology, Odense University Hospital, Odense, Denmark) assisted with Luminex assays. We also thank Drs. Allan Thomsen and Jan Christensen (The Panum Institute, University of Copenhagen, Copenhagen, Denmark) for their assistance.


   Disclosures
Top
 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 operating grants from the Canadian Institutes of Health Research and the Multiple Sclerosis Society of Canada (to T.O.), and by Grants MH62231, NS36979, and NS44905 from the National Institutes of Health (to I.L.C.). J.M.M. is supported by a studentship from the Multiple Sclerosis Society of Canada. Back

2 Address correspondence and reprint requests to Dr. Trevor Owens, Medical Biotechnology Center, Syddansk Universitet, Winsløwparken 25, DK-5000 Odense, Denmark. E-mail address: towens{at}health.sdu.dk Back

3 Abbreviations used in this paper: MS, multiple sclerosis; AdIFN{gamma}, adenovirus encoding IFN-{gamma}; AdLacZ, adenovirus encoding beta-galactosidase; CSF, cerebrospinal fluid; EAE, experimental autoimmune encephalomyelitis; GKO, IFN-{gamma} knockout; ISH, in situ hybridization; Ptx, pertussis toxin; WT, wild type. Back

Received for publication April 18, 2006. Accepted for publication March 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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