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The Journal of Immunology, 2006, 177: 6871-6879.
Copyright © 2006 by The American Association of Immunologists, Inc.

A Novel Model of Demyelinating Encephalomyelitis Induced by Monocytes and Dendritic Cells1

Glaucia C. Furtado2,*, Beatrice Piña*, Frank Tacke3,{dagger}, Stefanie Gaupp§, Nico van Rooijen, Thomas M. Moran{ddagger}, Gwendalyn J. Randolph{dagger}, Richard M. Ransohoff#, Stephen W. Chensue||, Cedric S. Raine§ and Sergio A. Lira*

* Immunobiology Center, {dagger} Department of Gene and Cell Medicine, {ddagger} Department of Microbiolology, Mount Sinai School of Medicine, New York, NY 10029; § Department of Pathology (Neuropathology), Albert Einstein College of Medicine, New York, NY 10461; Department of Molecular Cell Biology, Free University Medical Center, Amsterdam, The Netherlands; || Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; and # University of Michigan Medical School, Ann Arbor, MI 48105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Local inflammation may be a precipitating event in autoimmune processes. In this study, we demonstrate that regulated influx of monocytes and dendritic cells (DC) into the CNS causes an acute neurological syndrome that results in a demyelinating encephalomyelitis. Expansion of monocytes and DC by conditional expression of Flt3 ligand in animals expressing CCL2 in the CNS promoted parenchymal cell infiltration and ascending paralysis in 100% of the mice within 9 days of Flt3 ligand induction. Depletion of circulating monocytes and DC reduced disease incidence and severity. Unlike the classical models of experimental autoimmune encephalomyelitis, depletion of CD4+ and CD8+ T cells did not affect disease induction. T cells and demyelinating lesions were observed in the CNS at a later stage as a result of organ-specific inflammation. We propose that alterations in the numbers or function of monocytes and DC coupled to dysregulated expression of chemokines in the neural tissues, favors development of CNS autoimmune disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neuroinflammation is caused by the recruitment of immune cells into the CNS in response to trauma, infection, or as a consequence of inappropriate immune responses against CNS Ags (1). One of the most widely studied models of neuroinflammation is experimental autoimmune encephalomyelitis (EAE)4. EAE is a demyelinating autoimmune disease that displays some of the clinical manifestations observed in patients with multiple sclerosis (2, 3). EAE is generally induced in susceptible strains of animals by immunization with CNS Ags such as myelin basic protein (MBP), proteolipid protein, or myelin oligodendrocyte glial protein, along with immunogenic adjuvants and pertussis toxin. EAE can also be induced by adoptive transfer of in vitro-activated encephalitogenic CD4+ T cells (2). EAE also develops spontaneously in transgenic animals carrying TCRs directed to CNS Ags (4, 5). In all of these models, migration of activated encephalitogenic T cells from the blood circulation into the CNS leads to the development of perivascular and parenchymal inflammation that causes demyelination and neuronal degeneration. However, in some models the presence of T cells directed against CNS Ags is not sufficient for development of neuroinflammation. For instance, mice expressing a transgenic TCR specific for the encephalitogenic proteolipid protein peptide 139–151 on an EAE-resistant background rarely develop spontaneous disease. However, in these animals, stimulation of APCs through TLR4 or TLR9 triggers fulminant disease (6). In concordance with these results, there is evidence (7) that multiple sclerosis (MS) may be secondary to microbial infection. Thus, activation of innate immune responses may be required for development of neuroinflammation.

Macrophages and microglial cells influence neuroinflammation by stimulating the production of complement proteins, cytokines, and chemokines (8). Indeed, many neurological processes are linked to recruitment into and activation of mononuclear cells in the CNS. Transgenic mice expressing IL-3 under the astrocyte-specific glial fibrillary acidic protein promoter develop a spontaneous progressive neurodegenerative disease at 5–7 mo of age. Persistent production of IL-3 in the CNS promotes the recruitment, proliferation, and activation of macrophage/microglial cells with evidence of demyelination in the white matter (9). In HIV dementia, for example, activated monocytes traffic to the CNS and play a role in neuronal injury in macaques depleted of CD8 lymphocytes (10). There is also evidence that DC isolated from scrapie-infected mice migrate to the CNS and induce disease when transferred to RAG–/– mice (11).

Trafficking of innate immune cells into tissues is dependent on chemokines, low-molecular mass proteins that interact with G-coupled chemokine receptors (12). Migration of macrophages and dendritic cells (DC) is controlled by multiple chemokines, including CCL2 (MCP-1) and its receptor CCR2 (13). Our group and others (14, 15) have shown that targeted overexpression of CCL2 to the CNS drives monocyte/macrophage accumulation in these tissues. CCL2 expression in CNS parenchymal cells is tightly linked to clinical manifestations of many neurological disorders and to experimental models of neuroinflammation such as EAE (15). Conversely, animals that lack CCL2 and its major receptor CCR2 have a mild EAE phenotype associated with impaired migration of macrophages to the CNS (15, 16, 17, 18). However, CCL2-driven migration of mononuclear cells to the CNS, by itself, is not sufficient to trigger acute inflammatory disease (14).

In the present study, we asked whether increased numbers and/or dysregulation of monocytes/macrophages and DCs would lead to neurological disorder in animals expressing CCL2 in the CNS. To promote these changes, we crossed animals expressing CCL2 in the CNS (MBPCCL2 mice) with mice expressing Fms-like tyrosine kinase 3 ligand (Flt3L) conditionally (19). Flt3L is a known growth factor for pluripotent hemopoietic stem cells and progenitor cells (20, 21).

In this study, we show that simultaneous expression of CCL2 in the CNS and Flt3L in the periphery results in neuroinflammation and a striking neurological phenotype in 100% of the mice within 9 days of doxycycline (DOX) treatment. This study suggests that aberrant expression of chemokines concomitant with changes in the number or activation of circulating innate immune cells is sufficient to cause massive neuroinflammatory disease that culminates with the development a demyelinating encephalomyelitis.


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

A transgenic line expressing the CCL2 chemokine under the MBP promoter similar to that described by Fuentes et al. (14) was used in this study. The Flt3L-transgenic mice expressing Flt3L conditionally have been described by Manfra et al. (19). MBPCCL2/Flt3L mice were derived from a cross between MBPCCL2 with Flt3L mice. Mice were housed under specific pathogen-free conditions in individually ventilated cages (Thoren) at the Mount Sinai School of Medicine. All experimental protocols were approved by the Mount Sinai School of Medicine Institutional Animal Care and Use Committee.

Abs and FACS analysis

Anti-CD4 (RM5-5), CD3e (145-2C11), CD115 (MCSFR-AFS98), CD11b (M1/70), CD11c (N418), and F4/80 (BM8) were obtained from eBioscience. Anti-B220 (RA3-6B2) and Gr-1 (Ly6C/G-RB6-8C5) were obtained from BD Pharmingen. Single-cell suspensions were stained for 30 min at 4°C with Ab mixtures and analyzed in a FACS CANTO instrument (BD Biosciences).

DOX treatment

Flt3L expression in Flt3L and MBPCCL2/Flt3L mice was promoted by administration of DOX (1000 ppm) in the food chow once animals were 30–40 days old. The expansion of DC in the blood was monitored by FACS analysis using CD11c as a marker.

Disease evaluation

Neurological disease was scored as previously described (22): level 1, limp tail; level 2, hind leg weakness or partial paralysis; level 3, total hind leg paralysis; level 4, hind leg paralysis and front leg weakness or partial paralysis; and level 5, moribund.

Cell suspensions

Mice were anesthetized (with a mixture containing 12.5 mg/ml ketamine and 2.5 mg/ml xylazine diluted in PBS i.p., 100 µl/mouse) and perfused with 80 ml of PBS containing 5 mM EDTA through the left ventricle of the heart. The success of the perfusion was assessed by the white color of the lungs and liver. The CNS (spinal cord, brain stem, and cerebellum) was dissected and incubated in 5 mg/ml collagenase D (Boehringer Ingelheim) at 37°C for 45 min. Samples were strained through a 70-µm diameter nylon mesh to obtain a single-cell suspension, centrifuged, and washed in PBS. The CNS preparation was applied to a 38% Percoll solution (Amersham Biosciences), centrifuged at 1000 x g for 30 min, and the pellet was washed twice in PBS. All samples were resuspended in PBS containing 2% FCS and 0.1% sodium azide for FACS analysis.

In vivo depletion of T cell subsets

Depletion of CD4+ and CD8+ T cell subsets in MBPCCL2/Flt3L mice was achieved by i.p. injections of depleting anti-CD4 (GK1.5) and anti-CD8 (2.43) mAb on days –3, –2, and –1. A total of 100 mg of anti-CD4 and 500 mg of anti-CD8 mAb was injected per animal. T cell depletion was assessed by FACS analysis of peripheral blood.

Depletion of circulating monocytes and DC

Depletion of peripheral blood monocytes was achieved using liposomes loaded with the biphosphonate clodronate (23). Liposome-encapsulated clodronate and control liposomes (containing PBS only) were prepared as previously described (24). Clodronate was a gift from Roche Diagnostics. We used the dose of clodronate-liposomes shown to fully eliminate blood monocytes (23, 25). MBPCCL2/Flt3L mice were treated with DOX for 4 days before injection of 300 µl of PBS-liposomes or clodronate-liposomes into the tail vein. Injections were repeated on days 5 and 6. Efficient peripheral blood depletion of monocytes and circulating DC was assessed by FACS analysis.

Histology

Tissues for light microscopy were fixed by immersion in 10% phosphate-buffered formalin and then processed for paraffin sections. Routinely, 5-µm sections were cut and stained with H&E. For immunohistochemical analysis, the spinal cords were fixed in 4% paraformaldehyde (EMS) containing 30% sucrose for 20 h at 4°C. Tissue samples were embedded in OCT (Tissue Tek) and snap-frozen in 2-methylbutane (Merck), chilled with dry ice. Cryostat sections (8 µm) were incubated with primary Abs in a humidified atmosphere for 1 h at room temperature. After washing, conjugated secondary Abs were added for 35 min, washed, and the sections were mounted with Fluoromount-G (Electron Microscopy Sciences). Analysis was performed in an Eclipse E600 fluorescence microscope (Nikon). For neuropathological analysis, age-matched mice were anesthetized and perfused with cold 2.5% glutaraldehyde in PO4 buffer (pH 7.4). Slices of brain and spinal cord were postfixed for 90 min in 1% osmium tetroxide, dehydrated through a graded series of ethyl alcohol, and embedded in Epon 812. One-micrometer epoxy sections were cut and stained with 1% toluidine blue (TB) for light microscopy. The presence of infiltrating cells in the spinal cord was scored on a scale of 1–5 by an investigator blinded to the code.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MBPCCL2/Flt3L mice develop severe neurological disease when treated with DOX

We have previously shown that CCL2 expression under the MBP promoter induces recruitment of mononuclear cells to the perivascular area of the CNS of MPBCCL2 mice (14), but no neurological abnormalities or neuropathology. The CNS infiltrates were composed of monocytes and DC with scattered T cells (14). We hypothesized that expansion and activation of innate immune functions could induce the development of an inflammatory process in the CNS of MBPCCL2 mice. To test this hypothesis, we crossed MBPCCL2 mice with animals that express conditionally the myeloid growth factor Flt3L (19 ; see Fig. 1A). Animals containing the CCL2 and Flt3L transgenes are referred to as MBPCCL2/Flt3L mice. Untreated MBPCCL2/Flt3L mice had a monocytic CNS infiltration similar to MBPCCL2 animals, and did not show any signs of neuropathology (Fig. 1B). In dramatic contrast, treatment of MBPCCL2/Flt3L mice with DOX for 8 days led to rapid development of ascending progressive paralysis, that began with limp tail (level 1), progressed to hind limb weakness (level 2), hind limb paralysis (level 3), forelimb weakness and paralysis (level 4), and eventually death (level 5) (Fig. 1C). The affected mice exhibited typical signs of bilateral, symmetrical hind limb weakness when grade 3 severity was established. On the day of onset of significant hind limb weakness (day 7 after DOX treatment), mice demonstrated clonic spasms when tested for righting reflex. These spasms affected all four limbs in coordinate fashion and lasted for several seconds. By the following day, hind limb weakness was fixed and spasms were no longer observed during the righting reflex test. This motor disorder resembled classical EAE (22), and was observed in 90% of the animals by day 8 and in 100% of the animals by day 9 after DOX treatment (Fig. 1B). No disease was observed in control (wild type), Flt3L mice (>3 mo of treatment), or MBPCCL2/Flt3L (DOX–) mice (>12 mo of age, data not shown). Thus, DOX treatment of MPBCCL2/Flt3L mice resulted in the rapid development of a severe neurological condition.


Figure 1
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  		FIGURE 1. MBPCCL2/Flt3L mice develop spontaneous neurological disease upon DOX treatment. A, Sketches of the transgene used to drive CCL2 expression under control of the MBP (MBPCCL2 mice) and of the activator and responder transgenes in the Flt3L mice. In the Flt3L mice, the activator transgene (top) the hCMV enhancer (hCMVe) was juxtaposed to the chicken β-actin promoter to control the expression of rtTA. The responder transgene encodes β-galactosidase and mFlt3L in opposite orientation. Transcription of the β-gal and mFlt3L genes is strongly induced when DOX and rtTA are present. TRE, Tetracycline-responsive element; CMV, minimal CMV promoter; rβglob, rabbit β-globin. MBPCCL2 animals were crossed with Flt3L mice to generate the MBPCCL2/Flt3L mice. Double-transgenic mice without DOX treatment (MBPCCL2/Flt3L (DOX–)) were healthy and did not develop any neurological disorder, whereas MBPCCL2/Flt3L mice treated with DOX for 15 days (MBPCCL2/Flt3L (DOX+)) developed neurological disease with high incidence (B) and with severe clinical manifestations (B; n = 60 ± SE).

 
MBPCCL2/Flt3L mice develop severe meningoencephalomyelitis after DOX treatment

To determine whether coexpression of CCL2 and Flt3L caused neuropathology, we examined the CNS of Flt3L and MBPCCL2/Flt3L mice (Fig. 2). Analysis of H&E-stained sections demonstrated that mononuclear cells were present in perivascular locations in the MBPCCL2/Flt3L (DOX–) mice, with little parenchymal infiltration (Fig. 2, A and B). The most extensive pathology was encountered in the MBPCCL2/Flt3L (DOX+) mice treated with DOX for 10 days. These displayed widespread leptomeningeal and perivascular inflammation (Fig. 2A). Large infiltrating mononuclear cells were present in the vicinity of the meninges and blood vessels and penetrated into the neuropil (Fig. 2, A and C). Dorsal and ventral tracts, as well as surrounding nerve roots, were infiltrated by these mononuclear cells. We used an arbitrary histological grade to quantify the spinal cord infiltrates in different transgenic lines (Fig. 2B). MBPCCL2/Flt3L (DOX+) mice had the highest degree of infiltration in the spinal cord meninges and parenchyma when compared with MBPCCL2/Flt3L (DOX–) mice. Flt3L (DOX–) and (DOX+) mice did not have CNS infiltration (Fig. 2, A and B). Immunostaining of spinal cord sections with anti-CD11c Abs revealed an increase in the number of DC in the CNS meninges and parenchyma of MBPCCL2/Flt3L (DOX+) mice (Fig. 2C). F4/80+ monocytes/macrophages were also increased in the CNS of these animals (data not shown). TB-stained semithin sections of the spinal cord of MBPCCL2/Flt3L (DOX+) mice did not show evidence of demyelinating axons at this phase of the disease. In addition, strong TUNEL staining of apoptotic cells was observed in the spinal cord of MBPCCL2/Flt3L mice treated with DOX (Fig. 2). This was particularly evident within the myelinated white matter, where small round dead cells were common (Fig. 2C, arrows and inset). Electron microscopy (EM) analysis showed that these apoptotic cells were inflammatory cells and oligodendrocytes (data not shown). These results show that coexpression of CCL2 and Flt3L induces a massive mononuclear cell infiltration in the perivascular and parenchymal areas of the CNS in MBPCCL2/Flt3L (DOX+) mice. Despite the severe inflammation and neurological impairment, no demyelinating lesions were observed in mice treated with DOX for 10 days.


Figure 2
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  		FIGURE 2. Severe mononuclear cell infiltration in the spinal cord of MBPCCL2/Flt3L mice treated with DOX. A, H&E-stained paraffin sections of the spinal cord of untreated (DOX–) and animals treated with DOX for 10 days (DOX+) (original magnification, x40). B, Arbitrary histological grades of the infiltrates in different areas of the CNS of transgenic lines (n = 5/group/±SD). C, H&E-stained paraffin section of the spinal cord of transgenic lines (original magnification, x200); immunostaining of spinal cord section of MBPCCL2/Flt3L (DOX+) mice with anti-CD11c Ab (original magnification, x200); TB-stained semithin section of the spinal cord of MBPCCL2/Flt3L (DOX–) and MBPCCL2/Flt3L (DOX+) mice (original magnification, x400). The presence of apoptotic bodies in the spinal cord parenchyma of MBPCCL2/Flt3L (DOX+) is shown (arrows and inset). TUNEL staining of spinal cord sections of Flt3L (DOX+) and MBPCCL2/Flt3L (DOX– and DOX+). Significant increase of apoptotic cells in the spinal cord parenchyma of MBPCCL2/Flt3L (DOX+) when compared with MBPCCL2/Flt3L (DOX–) mice.

 
Having demonstrated a synergistic effect of CCL2 and Flt3L in the recruitment of inflammatory cells to the CNS, we analyzed the phenotype of these cells by flow cytometry. The CNS was isolated from transgenic lines and stained with different combinations of Abs to various leukocyte surface markers. We first determined the absolute number of CD45+ cells in the CNS of Flt3L and MBCCL2/Flt3L mice untreated or treated with DOX for 10 days (Fig. 3A). The numbers of CD45+ cells were significantly increased in MBPCCL2/Flt3L (DOX–) mice and in MBPCCL2/Flt3L (DOX+) mice when compared with Flt3L mice untreated or treated with DOX (Fig. 3A). We next determined the relative numbers of monocytes/macrophages, DC, T, and B cells in the CNS of these mice (Fig. 3B). The CNS preparations of Flt3L mice (DOX– and DOX+) were primarily composed of microglial cells (CD45low, CD115+, CD11b+, CD11c–, and GR-1low). The CD11c+ population in these animals represented only 2% of the total CD45+ cells even after DOX treatment (Fig. 3B). In contrast, the CD11c+ cell population in the CNS of MBPCCL2/Flt3L mice without DOX treatment represented 21% of the infiltrates. The vast majority of these cells were CD11b+CD115+CD11c+GR-1low monocytes or monocyte-derived DC (Fig. 3B). These infiltrates increased to ~47% in the MBPCCL2/Flt3L mice that received DOX for 10 days (Fig. 3B). A 17- and 145-fold increase in the number of CD11c+ cells over Flt3L (DOX+) was observed in the CNS of MBPCCL2/Flt3L (DOX–) and MBPCCL2/Flt3L (DOX+) mice, respectively (Fig. 3A). Lymphoid DC (CD11c+/CD8+) and plasmacytoid DC (CD11c+/B220+) were not present in significant numbers in the CNS of MBPCCL2/Flt3L mice with or without DOX treatment (data not shown). An increase in the CD115+CD11b+GR-1+ cells was also observed in the CNS of MBPCCL2/Flt3L (DOX–) and (DOX+). In summary, DOX treatment induced a significant increase in the number of monocytes and myeloid DC in the CNS of MBPCCL2/Flt3L mice. Interestingly, unlike the EAE models, the lymphocyte population was not a significant component of the CNS infiltrate during the acute phase of the neuroinflammatory disease (Fig. 3B).


Figure 3
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  		FIGURE 3. Increased number of monocytes and DC in the CNS of MBPCCL2/Flt3L (DOX+) mice. A, Absolute numbers of CD45+, CD45+CD11b+CD11c–, and CD45+CD11c+ cells in the CNS of different transgenic lines (n = 3/group/±SD). B, Relative number of DC, monocytes, and macrophages in the CNS of Flt3L and MBPCCL2/Flt3L mice untreated (DOX–) and treated with DOX (DOX+) for 10 days. Cells were stained with the indicated Abs. Dead cells were excluded by propidium iodine. Plots are representative of one animal in each group (n = 4/group).

 
The acute phase of the neuroinflammatory disease is not mediated by CD4+ or CD8+ T cells

DOX-treated MBPCCL2/Flt3L mice had clinical manifestations similar to those observed in animals with EAE. EAE is a Th1-mediated autoimmune process induced by activated encephalitogenic CD4+ T cells (2). To investigate whether CD4+ and/or CD8+ T cells were also required in this neuroinflammatory process, we injected MBPCCL2/Flt3L mice with 100 mg of anti-CD4 (clone GK1.5) and 500 mg of anti-CD8 (clone 2.43) or isotype control Abs at days –3, –2, and –1 and started DOX treatment on day 0. Three days after DOX treatment, the animals received a fourth dose of T cell-depleting Abs. We analyzed the presence of CD4+ and CD8+ T cells in the peripheral blood on days 0, 6, and 8 after DOX treatment. Before treatment, T cells represented ~30% of the circulating cells. CD4+ and CD8+ T cells were virtually depleted from circulation and CNS after Ab treatment (Fig. 4A), and represented at best 0.1% of total cells at day 8. Significantly, 8 days after the start of DOX treatment, controls and T cell-depleted MBPCCL2/Flt3L animals developed disease of comparable severity with similar kinetics (Fig. 4B). Thus, mature CD4+ and CD8+ T cells do not play a role in the induction of disease in MBPCCL2/Flt3L mice treated with DOX.


Figure 4
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  		FIGURE 4. Neurological disease in MBPCCL2/Flt3L mice is not dependent on CD4+ and CD8+ T cells. MBPCCL2/Flt3L mice were treated with anti-CD4 and anti-CD8 Abs before and during DOX treatment. A, An aliquot of the blood of animals treated with isotype control Ab or with T cell-depleting Abs was analyzed by flow cytometry using anti-CD8 and anti-CD4 Abs at day 6 after DOX treatment. B, MBPCCL2/Flt3L mice with (n = 4) or without T cells (n = 4) treated with DOX developed neuroinflammatory disease with similar incidence and severity (±SD).

 
Flt3L-transgenic animals have increased numbers of monocytes and DC in circulation

Monocytes give origin to macrophages and DC (26, 27). To determine whether there was a correlation between the number of DC in the tissue and levels of monocytes and DC in the periphery, we analyzed by FACS the phenotype of the circulating cells in Flt3L and MBPCCL2/Flt3L +/– DOX treatment (Fig. 5). We used the M-CSFR (CD115) marker in combination with CD11b, CD11c, and GR-1 (Ly6C/G) to characterize the monocyte and DC populations in the blood (23, 28, 29). Because monocytes and DC contain lower granular content, we used a low side scatter gate to analyze the blood populations. CD11b+ monocytes in the peripheral blood of untreated Flt3L mice represented 4% of the gated cells. The majority of these cells expressed the CD115 marker. After 8 days of DOX treatment, monocyte numbers increased to 16% in the Flt3L mice. Similarly, the number of circulating CD11b+CD115+ monocytes in MBPCCL2/Flt3L increased dramatically after DOX treatment. The majority of these cells were inflammatory monocytes (CD115+GR-1high) (Ref. 29 ; Fig. 5). A marked increase in the number of CD115+CD11c+ cells was also observed in the blood of Flt3L and MBPCCL2/Flt3L mice treated with DOX. These cells expressed DC and monocyte markers and could represent a DC precursor subset expanded by Flt3L. These results indicate that Flt3L expands all circulating monocyte subsets and suggest that recruitment of monocytes from the periphery into the CNS leads to the generation of DC and to the development of the severe neurological disorder observed in MBPCCL2/Flt3L mice.


Figure 5
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  		FIGURE 5. Increased number of monocytes and DC in the blood of Flt3L and MBPCCL2/Flt3L mice treated with DOX. Peripheral blood of Flt3L and MBPCCL2/Flt3L untreated or treated with DOX for 8 days was stained with the indicated Abs. All cells were gated in a lower side scatter. Monocyte subsets were identified using the markers CD115, CD11b, CD11c, and GR-1. Data are representative of one mouse in each group (n = >30 mice/group).

 
Monocytes/DC precursors and circulating DC are necessary for the induction of neuroinflammation

To determine the role of monocytes and myeloid DC in this disease model, we used the phagocyte "suicide" method that involves liposome-mediated intracellular delivery of clodronate. Once ingested by phagocytic cells, the cells die by apoptosis. Systemic administration of clodronate-loaded liposomes fully depletes circulating monocytes as well as macrophages in wild-type mice (23, 25). Four days after the initiation of DOX treatment, MBPCCL2/Flt3L mice received three consecutive doses of PBS-liposomes or clodronate-liposomes (300 µg i.v.) at 24-h intervals (Fig. 6). Flow cytometric analysis of the blood of the animals treated with two doses of clodronate-liposomes (day 6 after initiation of DOX treatment) showed a drastic decrease in the number of monocytes and CD11c+ cells relative to the PBS-liposome control (Fig. 6A). Injection of PBS-liposomes had no effect on blood monocytes or CD11c+ cells or on the induction of disease (Fig. 6B). In stark contrast, only 25% of mice that received clodronate-liposomes developed disease by day 10, reaching 33% by day 12. Furthermore, the disease was delayed and less severe when compared with that observed in the PBS control group. Thus, the decrease in disease incidence and severity observed in the animals treated with clodronate-liposomes was caused by depletion or dysfunction of circulating monocytes and DC. After day 12, monocytes and CD11c+ cells reappeared in the blood of MBPCCL2/Flt3L mice and disease incidence and severity increased in these animals (data not shown). These results indicate that circulating monocytes and DC have a critical role in the development of the neuroinflammatory disease.


Figure 6
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  		FIGURE 6. Depletion of circulating monocytes and DC decreases neuroinflammatory disease incidence and severity. MBPCCL2/Flt3L mice were treated with DOX and after 4, 5, and 6 days of treatment the animals received an injection of PBS-liposomes (n = 7) or clodronate-liposomes (n = 8) in the tail vein. A, An aliquot of blood of animals treated with PBS or clodronate-liposomes was analyzed by flow cytometry for the presence of monocytes and DC 2 days after clodronate injection (day 6 after initiation of DOX treatment). B, Disease incidence and severity (±SD) was analyzed on the indicated days.

 
MBPCCL2/Flt3L mice develop chronic inflammatory disease after DOX withdrawal

To examine whether the neuroinflammation observed in MBPCCL2/Flt3L mice depended on the constant administration of DOX, we treated animals for 10 days and suspended treatment thereafter (Fig. 7). To our surprise, 66% of the mice developed a chronic neuroinflammatory disease (Fig. 7A). To determine the composition of the CNS infiltrates, spinal cords were examined histologically 40 days after DOX cessation. TB-stained sections and electron micrographs of the spinal cord of animals with chronic disease showed dense leptomeningeal and perivascular infiltration throughout the neuraxis (Fig. 7B). Lymphocytes, as well as monocytes, occasional plasma cells, dendritic-appearing cells, lipid-laden macrophages, and mast cells were present in these infiltrates (Fig. 7, B and E). Within the levels of spinal cord examined (C7, Th2, L2, L5, L6, and S1), scattered nerve fibers displayed axonal dystrophy and/or a vacuolar myelinopathy (Fig. 7G). Myelin sheaths were highly distended and axons generally preserved (Fig. 7, C and G). At the level of the thoracic spinal cord, extensive pathology involving 20% of the white matter showed vacuolation of nerve fibers, some of which lacked axons. In addition, demyelinating lesions were present, with naked axons surrounding perivascular cuffs of debris-laden macrophages and mononuclear cells (Fig. 7D). Myelin droplets were scattered throughout the neighboring parenchyma and there was a mild reactive astrogliosis (Fig. 7F). Thus, the pathology in MBPCCL2/Flt3L animals with chronic neuroinflammatory disease was marked by the presence demyelinating lesions in the CNS.


Figure 7
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  		FIGURE 7. MBPCCL2/Flt3L develop chronic demyelinating inflammatory disease upon cessation of DOX treatment. A, MBPCCL2/Flt3L mice were treated with DOX for 10 days and fed normal diet afterward. Disease score average is represented by red bars (n = 15). B–D, Histological analysis of TB-stained semithin sections of the spinal cord of MBPCCL2/Flt3L mice with chronic neuroinflammatory disease. B, The presence of infiltrating lymphocytes (arrows; original magnification, x200). C, Axon surrounded by dilated myelin sheets (a) (original magnification, x400). D, Perivascular cuffs (PVC) (original magnification, x200). E–G, Electron micrographs of the spinal cord of chronic MBPCCL2/Flt3L mice with demyelinating pathologies. E, The presence of infiltrating lymphocytes (L). F, Section showing demyelinating axon (dm) and remyelinating axons (rm), myelin debris (md), and a macrophage (m{phi}) dying in a droplet of myelin (dm). G, EM section displaying axon surrounded by dilated myelin sheets (a) and a monocyte (m) within a myelin tube (G). H, Increased number of CD8+ T cells in the CNS of MBPCCL2/Flt3L mice with chronic disease. The CNS of MBPCCL2/Flt3L mice with chronic disease for 60 days were stained with the indicated Abs. Dead cells were excluded by propidium iodine. Cells were gated on CD45+ cells and plots are representative of one animal in each group (n = 3/group).

 
The phenotype of the cells that infiltrated the CNS of MBPCCL2/Flt3L mice with chronic disease for 60 days was analyzed by flow cytometry (Fig. 7H). The relative numbers of CD11b+ and CD11c+ cells in the CNS of these animals were similar to those observed in MBPCCL2/Flt3L mice before DOX treatment (Figs. 3B and 7H). In agreement with studies done by EM, we detected an increased number of CD3+ T cells in the CNS of mice with chronic disease (Fig. 7H). The majority of these T cells were CD8+. These results indicate that animals with chronic neuroinflammation have an increased number of T lymphocytes in the CNS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we show that a severe neurological disease can be promoted by controlled infiltration of monocytes and myeloid DC in the CNS. This novel demyelinating neuroinflammatory model does not require immunization or TCR skewing.

Expression of CCL2 under the MBP promoter (MBPCCL2 mice) induces recruitment of monocytes/macrophages and DC to the perivascular space of the CNS (14). Despite the increased number of potential inflammatory cells in the CNS, these mice do not develop any neurological symptoms. In contrast, simultaneous expression of CCL2 in the CNS and Flt3L in the periphery leads to severe progressive paralysis, comparable to that observed in animals with EAE. The onset of disease occurs rapidly after Flt3L expression. By day 8, 90% of the animals had neurological signs and by day 9, 100% of the animals were severely sick. Thus, the combined effect of CCL2 and Flt3L leads to the development of a severe, rapidly progressing neuroinflammatory disease.

Although MBPCCL2/Flt3L and EAE animals display identical clinical signs of CNS impairment, there are striking differences between the model shown in this study and EAE. Induction of EAE through immunization protocols involves the injection of susceptible strains of mice with bacterial components as a source of danger signals, together with CNS-specific proteins. In the EAE model, activated APCs present self-Ag to T cells in the periphery, leading to autoimmunity (2). In contrast, the induction of neuroinflammatory disease in MBPCCL2/Flt3L mice does not require immunization and is not dependent on CD4+ or CD8+ T cells. T cell-depleted MBPCCL2/Flt3L develop disease with similar incidence and severity as T cell-competent MBPCCL2/Flt3L mice. The neuroinflammatory disease in the MBPCCL2/Flt3L mice is primarily driven by monocytes and/or DC, as clodronate-liposome depletion of these cells decreased both disease incidence and severity. The demonstration that disease induction is suppressed by depletion of circulating monocytes and DC underscores the importance of these cells in the initiation of CNS autoimmune responses.

Our data indicate that the combined effect of CCL2 and Flt3L induced the expansion and retention of monocyte-derived APCs into the CNS parenchyma. It is possible that Flt3L induced the expansion of a pathogenic subset of cells in the peripheral blood of MBPCCL2 mice; induced the expansion or differentiation of an inflammatory subset that is already present in the perivascular area of the brains of MBPCCL2 mice; and/or induced the differentiation of a hemopoietic progenitor cell present in the CNS of MPBCCL2 mice. We have shown that Flt3L induced the expansion of monocytes/DC precursors in the circulation and that these cells were highly sensitive to depletion by clodronate-liposomes. Clodronate-liposomes do not cross the blood-brain barrier (30); therefore, it is unlikely that cells present in the perivascular region of the CNS played a major role in the disease outcome. Thus, the migration of circulating monocytes and DC precursors into the CNS is likely to be a key event in the induction of this neurological disorder.

Expression of Flt3L promoted expansion of all monocyte subsets in the blood. The majority of the monocytes in the blood of DOX-treated MBPCCL2/Flt3L mice expressed high levels of the GR-1 marker (GR-1high). In contrast, GR-1low cells were the most abundant subset in the CNS. GR-1low cells are part of the steady-state pool of tissue macrophages and do not migrate to inflammatory sites (29). We propose that the GR-1low population in the CNS may have derived from the GR-1high circulating pool based on studies that show that GR-1int and GR-1high cells are recruited to inflammatory sites and give rise to DC (27, 31). Additional studies will be required to clarify the origin and function of the myeloid cells recruited into the CNS of MBPCCL2/Flt3L mice.

The acute phase of the neuroinflammatory disease of the MBPCCL2/Flt3L mice was marked not only by the presence of a significant myeloid cell infiltrate, but also by a significant number of apoptotic cells in the CNS parenchyma. It is unclear what mechanisms contribute to the increased number of apoptotic cells during this phase of the disease. Interestingly, apoptosis of oligodendrocyte and inflammatory cell also occurs in the CNS in MS patients, suggesting that this may be an important aspect of CNS autoimmunity (32). Defects in the clearance of apoptotic cells can contribute to the generation of autoreactive lymphocytes and the development of autoimmune processes (33). For instance, accelerated macrophage apoptosis increased Ab formation in lupus-prone mice and led to the development of autoantibodies in non-lupus prone animals (34). Therefore, increased apoptotic cell death can generate a local inflammatory response and autoimmunity.

EAE induction by immunization protocols requires microbial products to activate innate immunity via the TLR ligand. In the absence of microbial stimuli, endogenous TLR ligands can also activate TLRs on innate immune cells in a process called sterile inflammation (35). MyD88 and TLR9 were recently shown (36) to have an important role in adoptive EAE. In this case, it was suggested that endogenous molecules in the CNS could trigger TLR signaling during the effector phase of the disease. Indeed, there is evidence of altered expression of endogenous TLR ligands in acute and chronic relapsing models of EAE (37) and in MS (38), but the extent to which these endogenous TLR ligands contribute to the pathogenesis of neuroinflammation remains to be clarified. Since disease development in MBPCCL2/Flt3L mice does not require immunization, use of adjuvants, or infectious agents, these mice may represent a good animal model to study sterile inflammation and to investigate whether endogenous TLR ligands can activate innate immune cells and cause autoimmunity.

Demyelinating lesions were found in the chronic phase of the disease. Demyelinating lesions and presence of lymphocytes in the CNS are hallmarks of MS. Lymphocytes, particularly CD8+ T cells, were present in the CNS during the chronic phase and may have been important for the development of such lesions because they have been implicated in the pathogenesis of EAE (39, 40) and are found within MS lesions (41). Despite the presence of such cells, it is important to note that CNS demyelination can occur in the absence of T and B cells (42, 43). It was recently shown (43) that mice infected with a recombinant neurotropic coronavirus that express CCL2 develop demyelinating encephalitis in the absence of lymphocytes. RAG–/– mice infected with the CCL2-expressing recombinant virus showed a 3-fold increase in the number of macrophages/microglial cells in the spinal cord when compared with non-CCL2 expressing virus and 14% of the animals developed demyelinating lesions in the CNS. Our own results indicate that expression of CCL2 alone is not responsible for the inflammatory process observed in the MBPCCL2/Flt3L mice and that the initial phase of neuroinflammatory disease in MBPCCL2/Flt3L mice is independent of T cells. Thus, it remains to be determined whether the demyelination observed in the chronic phase is due to the activity of myeloid cells, T cells, or both.

In summary, we described the generation of a novel model of demyelinating encephalomyelitis. In this model, CNS expression of a chemokine (CCL2) generated a permissive environment for entry of monocytes/DC precursors and DC in the CNS. Changes in the number or activation status of these cells led to severe neuroinflammation and development of paralysis. We suggest that alterations in the numbers or function of monocytes and DC concomitant with aberrant expression of chemokines may be critical factors for development of autoimmune disease.


   Acknowledgments
 
We thank Claudia Canasto-Chibuque for expert technical assistance. We also thank Dr. Jay Unkeless for critical comments about the manuscript.


   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 National Multiple Sclerosis Society Grants (G 3479-A-2 (to G.C.F.) and RG 1001-k-11 (to C.S.R.), the Irene Diamond Fund (to S.A.L.), and National Institutes of Health Grants DK 067989 (to S.A.L.), and NS 11920 and NS 08952 (to C.S.R.). Back

2 Address correspondence to Dr. Glaucia C. Furtado, Immunobiology Center, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1630, New York, NY 10029-6574. E-mail address: glaucia.furtado{at}mssm.edu Back

3 Current address: University Hospital Aachen, Medical Clinic III, Aachen, Germany. Back

4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; DC, dendritic cell; MBP, myelin basic protein; Flt3L, Fms-like tyrosine kinase 3 ligand; DOX, doxycycline; TB, toluidine blue; MS, multiple sclerosis; EM, electron microscopy. Back

Received for publication June 13, 2006. Accepted for publication August 23, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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