RESEARCH ARTICLE

Lymphoid Chemokines CCL19 and CCL21 are Expressed in the Central Nervous System During Experimental Autoimmune Encephalomyelitis: Implications for the Maintenance of Chronic Neuroinflammation Sandra Columba-Cabezas, Barbara Serafini, Elena Ambrosini, Francesca Aloisi Laboratory of Organ and System Pathophysiology, Istituto Superiore di Sanità, Roma, Italy S. C.-C. and B. S. equally contributed to this work.

The simultaneous presence of dendritic, T- and Bcells in the central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, suggests that interactions among these cell types might be instrumental in the local induction and maintenance of autoimmune reactions. In this study, we explored the possibility that such aberrant leukocyte recruitment in the CNS could be sustained by “lymphoid” chemokines which orchestrate dendritic cell and lymphocyte homing to lymphoid organs. Transcripts for CCL19 and CCL21 and their common receptor CCR7 were induced in the CNS of mice undergoing relapsing-remitting and chronic-relapsing EAE. While CCL21 immunoreactivity was confined to the endothelium of some inflamed blood vessels, CCL19 was expressed by many infiltrating leukocytes and some astrocytes and microglia in the CNS parenchyma. CCR7+ cells accumulated in inflammatory lesions during EAE progression, when abundant infiltration of the CNS by mature dendritic cells, B-cells and cells expressing naive Tcell markers also occurred. These findings suggest that CCL19 and CCL21 produced in the EAE-affected CNS may be critical for the homing of antigen presenting cells and lymphocytes, resulting in continuous local antigenic stimulation and maintenance of chronic neuroinflammation. Brain Pathol 2003;13:38-51.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by focal myelin destruction, varying degree of axonal

pathology and progressive neurological dysfunction (35, 44). Perivascular infiltrates of T-cells, B-cells and monocytes/macrophages, are a prominent histological feature of the discrete lesions that develop in the brain and spinal cord in typical cases of MS (35). Studies in patients and in the animal model, experimental autoimmune encephalomyelitis (EAE), indicate that a Th1mediated immune response to various myelin antigens and myelin-specific autoantibodies might be implicated in the pathogenesis of MS (52, 60). However, the initial events triggering autoimmune responses and the mechanisms sustaining CNS inflammation in MS remain to be elucidated. In recent years, dendritic cells (DCs) have received increasing attention as key players in the pathogenesis of autoimmunity (18, 37). DCs have the unique ability to activate resting T-cells and play an important role in the initiation and regulation of immune responses (5, 28). DCs capturing self-antigen in the target organ and migrating to draining lymph nodes or spleen may initiate stimulation of autoreactive T-cells and sustain new waves of priming of naive T-cells. In several organ-specific autoimmune diseases and related animal models, DCs have been shown to infiltrate the target organ and to interact with T and B lymphocytes to form lymphoidlike structures (25, 33, 36, 55). This process, which is also referred to as lymphoid neogenesis, is thought to be crucial for continuous immune activation in the target tissue itself (33). Recent studies have documented that cells expressing DC-specific markers accumulate in the CNS tissue and cerebrospinal fluid during EAE (39, 51, 53) and MS (47), respectively, suggesting that DC trafficking to the CNS (a site where DCs are normally confined to the meninges and choroid plexuses) could contribute to tolerance breakdown and sustain the inflammatory self-recognition process. The intracerebral expression of maturation signals for DCs and chemokines/chemokine receptors regulating trafficking

Corresponding author: Dr Francesca Aloisi, Laboratory of Organ and System Pathophysiology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy (e-mail: [email protected])

and positioning of DCs and lymphocytes may thus represent critical elements in the progression of CNS autoimmunity. A recently identified set of chemokines, referred to as “lymphoid,” have emerged as key signals for the migration of lymphocyte subsets into T- and B-cell zones of secondary lymphoid organs and for the development of lymphoid tissue compartments (15, 41, 50, 64). Among these, CCL19, also known as macrophage inflammatory protein-3
or EBV-induced molecule 1 ligand, and CCL21, also known as 6Ckine, secondary lymphoid tissue chemokine, Exodus-2 and thymus-derived chemotactic agent-4, are expressed in T-cell zones by stromal cells and interdigitating DCs and by HEV endothelia and stromal cells, respectively (24, 38, 43). CCL19 and CCL21 are structurally related chemokines and share a common receptor, CCR7 (62, 63), that is expressed on mature DCs, B-cells, naive and central memory T-cells (7, 30, 50, 59, 61). CCR7-deficient mice and paucity of lymph node T-cell (plt) mice lacking CCL19 and CCL21 expression show defective migration of lymphocytes and DCs into T-cell zones, decreased B-cell homing, and profound morphological alterations of secondary lymphoid organs (20, 23, 38, 58). Recent studies in chronic infectious and autoimmune diseases have demonstrated expression of lymphoid chemokines, including CCL19 and CCL21, in the target organ, indicating that these molecules may orchestrate leukocyte microenvironmental homing resulting in the formation of ectopic lymphoid tissue (25, 26, 40, 55). In this study, we explored the hypothesis that CNS autoimmunity could be sustained by aberrant lymphoid chemokine expression favoring cell trafficking and interactions between DCs and lymphocytes in the neural tissue. Using RT-PCR and immunohistochemical techniques, we show that expression of CCL19, CCL21 and their cognate receptor CCR7 is induced in the CNS of mice developing EAE, particularly during disease progression. These findings provide novel insights into the signals regulating leukocyte homing to the inflamed CNS and suggest novel approaches to treat CNS autoimmune diseases by targeting the lymphoid chemokine system. Materials and Methods

Animals. Female adult SJL mice and Biozzi AB/H mice, 6 to 8 weeks of age, were obtained from Charles River (Calco, Italy) and Harlan U.K. Ltd (Bicester, United Kingdom), respectively. Mice were housed in a controlled environment in accordance with the guidelines

of the European Community Council of the Welfare of Experimental Animals (86/609/EEC). All experimental procedures were approved by the Italian Ministry of Health. EAE induction. For the induction of relapsingremitting EAE, 42 female SJL mice were injected subcutaneously in the flank with 0.2 mg proteolipid protein (PLP) 139-151 peptide (Primm, Milano, Italy), in complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, Mich) on day 0 and 7, and with pertussis toxin (0.2 g/mouse; Sigma Chemical Co., St. Louis, Mo) intraperitoneally on day 0, 1, 7 and 8, as previously described (17). For the induction of chronic-relapsing EAE, 26 female Biozzi AB/H mice were injected subcutaneously in the flank with 1 mg of lyophilized, autologous whole spinal cord homogenate reconstituted in PBS and mixed with CFA (incomplete Freund’s adjuvant [IFA] supplemented with 320 g/ml of Mycobacterium Tuberculosis H37 RA and 40 g/ml of Mycobacterium Butyricum [Difco Laboratories]) on day 0 and 7, and with pertussis toxin intraperitoneally on day 0, 1, 7 and 8, following published procedures (21). Control mice (6 for each strain) were injected with PBS in CFA and pertussis toxin, according to the same schedule. Mice were weighed and examined daily for clinical signs of EAE, which was scored on the following scale: grade 0, no abnormality; grade 1, reduced tail tonus or slightly clumsy gait; grade 2, tail atony, moderately clumsy gait, impaired righting ability, or any combination of these signs; grade 3, additional hind limb weakness; grade 4, hind limb paralysis and fore limb weakness; grade 5, tetraplegia or moribund state. Mice were sacrificed during the preclinical stage (day 10-13 post-immunization [p.i.]) and at different times after appearance of clinical signs. SJL mice were sacrificed during the first peak of disease (day 15-16 p.i.; EAE grade 2-4), during the early remission phase (day 20-22 p.i., 24 hours after disappearance of clinical signs), and 24 to 48 hours after the onset of relapses that followed complete remissions (up to 2 months p.i.; EAE grade 1-4). Biozzi mice were sacrificed immediately after disease onset (day 15 p.i.; EAE grade 3-4), and after partial remissions (EAE grade 1) and relapses occurring during the chronic-relapsing phase (up to 3 months p.i.; EAE grade 3-4). For each EAE stage examined, at least 6 mice were sacrificed for molecular and immunohistochemical studies. Reverse transcription-polymerase chain reaction (RT-PCR). Following anesthesia, mice were perfused intracardiacally with cold PBS. Different CNS areas

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Figure 1. Disease patterns of EAE-affected mice. A. Time course of the clinical score of three SJL mice developing relapsing-remitting EAE after immunization with PLP 139-151 peptide in CFA; mice were sacrificed at day 26 (), 29 (
) and 63 (
) p.i. B. Time course of the clinical score of three Biozzi AB/H mice developing chronic-relapsing EAE after immunization with spinal cord homogenate in CFA; mice were sacrificed at day 42 (), 50 (
) and 86 (
) p.i.

(cerebrum, cerebellum/brain stem and spinal cord) and positive control tissues (spleen and thymus) were collected, immediately frozen in liquid nitrogen and kept at -80°C until RNA extraction. Total RNAs were extracted from normal mouse control tissues and from the CNS of control and EAE-affected SJL and Biozzi AB/H mice using the SV Total RNA Isolation System (Promega, Madison, Wis), according to the manufacturer's instructions. One g of purified RNA was reverse-transcribed using the Reverse Transcription System (Promega), with 15 U of Avian Myeloblastosis Virus Reverse Transcriptase. For detection of murine CCL19 transcripts, one tenth of this reaction was added to 40 l of PCR mix and amplified for 29 cycles (45 s/94°C, 45 s/60°C, 60 s/72°C) with 2.5 units of Taq polymerase (Promega), using the following primers: 5GCC TCA GAT TAT CTG CCA T-3 (sense) and 5-AGA CAC AGG GCT CCT TCT GGT-3 (antisense) (size of the amplified product of CCL19 cDNA was 332 bp). Due to the presence of one functional CCL19 gene and three CCL19 pseudogenes in the mouse genome, all of which are transcribed, the PCR primer pairs were chosen to

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detect expression of the functional CCL19 gene bearing the ATG start codon (38). For detection of murine CCL21 transcripts, cDNA was subjected to PCR amplification for 28 to 30 cycles (30 s/94°C, 60 s/60°C, 60 s/72°C), using the following primers: 5-GAA AAT TCC CTA CAG TAT TGT CCG AG-3 (sense) and 5GAC TTA GAG GTT CCC CGG TTC-3 (antisense) (size of the amplified product of CCL21 cDNA was 222 bp). CCL21-specific primers were designed based on the sequence deposited in the EMBL/GenBank (accession number AF171085) (59). For detection of CCR7 transcripts, cDNA was subjected to PCR amplification for 28 cycles (60 s/94°C, 120 s/60°C, 90 s/72°C) using the following primers: 5-CCA GGAAAAACG TGC TGG TG3 (sense) and 5–GGC CAG GTT GAG CAG GTA GG–3 (antisense) (size of the amplified product of CCR7 cDNA was 287 bp) (10). As control, transcripts of the “housekeeping” gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified for 25 cycles (30 s/94°C, 30 s/60°C, 45 s/72°C), using the following primers: 5-CCA TGG AGA AGG CCG GGG-3 (sense) and 5-CAA AGT TGT CAT GGA TGA CC-3 (antisense) (size of the amplified product of GAPDH cDNA was 194 bp). Negative controls lacking template RNA or RT were included in each experiment. The PCR products were fractionated on a 3% NuSieve® 3:1 agarose gel (FMC BioProducts, Rockland, Mass) and visualized by ethidium bromide staining. The identity of the PCR amplified fragments was verified by size comparison with DNA standards (Promega) and by direct DNA sequencing of purified fragments (M-Medical, Roma, Italy). At least 3 mice were examined for each time point tested. In some of the EAE-affected mice used for RT-PCR studies, histopathological analysis was also performed on unfixed, frozen CNS sections to confirm the presence of immune infiltrates. The RT-PCR data shown were obtained in individual, representative mice. Immunohistochemistry. For specimen collection, mice were anesthesized with xylazine chloride hydrate and ketamine and perfused intracardiacally with PBS followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains, spinal cords and inguinal lymph nodes were removed, kept overnight in 4% paraformaldehyde at 4°C, passed in 15% and 30% sucrose, frozen in dry ice-chilled isopentane, cut in serial sections with cryostat and stored at -20°C. For immunohistochemistry, 10 m-thick sections were air dried, passed in 70%, 95% and 100% ethanol and then dried again. After rehydratation with PBS and 20minute incubation with 0.3% H2O2 in PBS (to eliminate

Lymphoid Chemokines CCL19 and CCL21 in the CNS During EAE—Columba-Cabezas et al

Figure 2. CCL19, CCL21 and CCR7 transcripts are upregulated in the CNS of SJL mice with PLP 139-151-induced EAE (A) and of Biozzi AB/H mice with whole spinal cord-induced EAE (B). RNA was extracted from different CNS areas and from control tissues (thymus and spleen), reverse transcribed and subjected to PCR amplification using CCL19, CCL21 and CCR7 specific primers, as described in Materials and Methods. RT-PCR using GAPDH specific primers is also shown, as an internal control. RNA was extracted from control and EAE-affected mice at the indicated disease stages. The PCR products were visualized by ethidium bromide staining. Results from individual mice are shown. Two relapsing SJL mice are shown, which were sacrificed at day 29 (grade 1) and 63 p.i. (grade 4), respectively. The two chronic-relapsing Biozzi AB/H mice were sacrificed at days 35 and 28 p.i. (grade 4), respectively. Lanes: 1 = cerebrum; 2 = cerebellum/brain stem; 3 = spinal cord; 4 = thymus; 5 = spleen.

endogenous peroxidase activity), sections were preincubated with 10% normal rabbit or goat serum and then incubated at 4°C overnight with the following purified primary antibodies: goat polyclonal anti-mouse CCL19 (R&D Systems, Minneapolis, Minn); rabbit polyclonal anti-mouse CCL21 (PeproTech, London, United Kingdom); goat polyclonal anti-mouse CCR7 (M18; Santa Cruz Biotechnology, Santa Cruz, Calif); rat monoclonal antibody MIDC-8, binding to a still unidentified antigen expressed by interdigitating DCs in T-cell areas of murine lymphoid organs (Serotec, Oxford, United

Kingdom) (8); rat monoclonal antibody anti-mouse CD11b/Mac-1 (5C6, Serotec); and rat monoclonal antibodies anti-mouse CD4 (RM4-5), CD45R/B220 (RA36B2), CD45RB (16A) and CD62L (MEL-14) (Pharmingen, San Diego, Calif). All antibodies were optimally diluted in PBS/1%BSA (2-5 g/ml). After extensive washings with PBS, sections were incubated with the corresponding biotinylated secondary antibodies (rabbit anti-goat IgG, goat anti-rabbit IgG and rabbit anti-rat IgG, all from Vector Laboratories, Burlingame, Calif) and avidin:biotinylated peroxidase complex

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Lymphoid Chemokines CCL19 and CCL21 in the CNS During EAE—Columba-Cabezas et al

Figure 3. (Opposing page) Immunohistochemical detection of CCL19 in the CNS of normal and EAE-affected mice. Cryostat sections were stained for CCL19 (A, B, F-K), CD11b (C), CD4 (D) or MIDC-8 (E) and visualized with DAB, as described in Materials and methods. A-F: stainings in SJL mice. CCL19+ cells in the cerebellar meninges (A) of normal SJL mice. Serial spinal cord sections from SJL mice at EAE onset were stained for CCL19 (B), CD11b (C), CD4 (D) and MIDC-8 (E). CCL19 immunoreactivity is detected in the cytoplasm of numerous cells within a large submeningeal and perivascular infiltrate comprising mainly CD11b+ macrophages and fewer CD4+ T-cells and MIDC-8+ DCs. CCL19+ cells in perivascular infiltrates and adjacent spinal cord parenchyma during a relapse (F). G, H: CCL19 stainings in the spinal cord of Biozzi AB/H mice at EAE onset (G) and during chronic-relapsing disease (H). I-K: higher magnifications of CCL19+ cells showing typical astrocyte (arrow) and microglia (arrow head) morphologies in the spinal cord of relapsing SJL mice. Original magnifications: 500 (A-D, F-H), 1000 (E), and 1575 (I-K).

(ABC), using the ABC Vectastain Elite kit (Vector Laboratories), according to the manufacturer’s instructions. Staining reactions were performed with 3,3 diaminobenzidine (DAB; Sigma Chemical Co., St. Louis, Mo) as substrate. MIDC-8 stainings were performed using the amplification procedure described by Adams, with minor modifications (1). Following incubation with ABC, sections were incubated with 0.01% H2O2 and 28 mM biotinylated tyramine, prepared as described, for 5 minutes at room temperature. After extensive washings, sections were incubated with ABC for 20 minutes, PBS for 30 minutes, and DAB for another 2 to 5 minutes. For negative controls, the primary antibody was omitted from the diluent or replaced with preimmune serum. Sections were counterstained with hematoxylin and viewed with a Zeiss Axiophot photomicroscope. Results

Detection of CCL19, CCL21 and CCR7 mRNAs in the CNS of EAE-affected mice. To explore the expression of CCL19, CCL21, and their cognate receptor CCR7 in different phases of CNS autoimmunity, EAE was induced in 2 mouse strains, SJL and Biozzi AB/H mice, which develop a relapsing-remitting and chronicrelapsing form of paralytic disease, respectively. After immunization with the PLP peptide 139-151 in CFA, 60 to 80% of the SJL mice developed the first EAE clinical signs around days 14 to 18, followed by complete remission, and about 20% of these underwent one or more relapses (Figure 1A). In all Biozzi AB/H mice immunized with whole spinal cord homogenate in CFA, EAE clinical signs first appeared at day 13-15 after immunization and the disease progressed as a continuum of relapses and incomplete or, less frequently, complete remissions (Figure 1B). Mice immunized with PBS/CFA showed no signs of disease (not shown). Expression of transcripts specific for CCL19, CCL21 and CCR7 was investigated in control and EAE-affected mice by RT-PCR analysis. The CNS areas examined included cerebrum, cerebellum/brain

stem and spinal cord, whereas thymus and spleen were used as positive control tissues for CCR7 and for CCL19 and CCL21, respectively. Very low levels of CCL19 mRNA, but no CCL21 and CCR7 mRNAs, were detected in the CNS, mainly cerebrum and cerebellum/brain stem, of control SJL (Figure 2A) and Biozzi AB/H (Figure 2B) mice (untreated and injected with PBS/CFA). In the CNS of preclinical SJL mice (day 10 to 13 post-immunization), levels of CCL19 and CCL21 mRNAs were either unchanged or only slightly increased, whereas CCR7 mRNA was weakly expressed. During the first peak of disease, expression of CCL19 and CCR7 mRNAs was increased in all CNS areas from SJL mice, whereas levels of CCL21 transcripts remained low. All transcripts analyzed returned to baseline levels during the early remission phase (24 h after disappearance of clinical signs). Following EAE relapses, CCL19 and CCR7 mRNAs were detected in the CNS of all SJL mice examined, with CCR7 being expressed predominantly in the spinal cord and at higher levels compared to disease onset. CCL21 mRNA was weakly induced only in some relapsing SJL mice. In the CNS of Biozzi AB/H mice, CCL19 mRNA was slightly induced before (day 10 p.i.) and immediately after the appearance of clinical signs but its levels markedly increased during the chronic-relapsing phase, the highest expression being detected in the cerebellum/brain stem and spinal cord (Figure 2B). Similarly, CCR7 mRNA levels were low at disease onset and increased as disease progressed, particularly in the spinal cord. CCL19 and CCR7 mRNAs were detected in the CNS also during partial remissions (Figure 2B). While CCL21 mRNA was not detected in the CNS of Biozzi AB/H mice before and immediately after EAE onset, low levels of CCL21 mRNA were induced in the cerebellum/brain stem and spinal cord of Biozzi AB/H mice undergoing chronic-relapsing disease (Figure 2B). Immunohistochemical detection of CCL19 and CCL21 in the CNS of EAE-affected mice. The expression of CCL19 and CCL21 was then evaluated by

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Figure 4. Immunohistochemical detection of CCL21 in peripheral lymph node and CNS of normal and EAE-affected mice. A-C: stainings in SJL mice. Lymph node section showing positive reaction for CCL21 on HEV (A). Absence of CCL21 immunoreactivity in the normal spinal cord (B). CCL21 immunoreactivity on an intrameningeal blood vessel in the spinal cord of a SJL mouse developing chronic EAE (C); the meninges and intrameningeal leukocytes are negative. D-F: stainings in the spinal cord of Biozzi AB/H mice during chronic-relapsing EAE. CCL21 is expressed in cells lining the lumen of inflamed blood vessels. In a severely affected Biozzi AB/H mouse (day 49 p.i., EAE grade 4) (F), CCL21 immunoreactivity is also detected in some process-bearing cells scattered in the adjacent white matter. Original magnifications: 500 (B, D) and 1000 (A, C, E, F).

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Lymphoid Chemokines CCL19 and CCL21 in the CNS During EAE—Columba-Cabezas et al

immunohistochemistry on cryostat sections of normal and EAE-affected CNS using polyclonal anti-mouse CCL19 and CCL21 antibodies. Sections of inguinal lymph nodes were also stained to check the specificity of the two anti-chemokine antibodies. Consistent with the distribution of CCL19 and CCL21 in lymphoid tissue (15, 24, 38, 43), anti-CCL19 antibody labeled dendritiform cells, likely DCs and stromal cells, in T-cell zones (not shown), whereas anti-CCL21 antibodies labeled HEV endothelia and stromal cells in T-cell zones of normal mouse lymph nodes (Figure 4A). In control SJL and Biozzi AB/H mice, anti-CCL19 antibody labeled few, distinct cells in the cerebral, cerebellar and spinal cord meninges (Figure 3A). No staining of normal CNS tissue was observed with rabbit anti-CCL21 antibody (Figure 4B). After the appearance of the first clinical signs (EAE grade 3-4), CCL19 immunoreactivity was found associated with numerous immune cells accumulating in the meninges, and in the perivascular and submeningeal lesions of the brain stem and spinal cord of both mouse strains (Figure 3B, G). Rare CCL19+ cells were also present in the parenchyma adjacent to the inflammatory infiltrates. Compared to Biozzi AB/H mice, SJL mice exhibited more prominent submeningeal cell infiltration, but fewer perivascular lesions. At these sites, the cell infiltrates comprise a major population of CD11b+ macrophages, and less numerous CD4+ T-cells and MIDC-8+ DCs (Fig. 3 C-E; only stainings performed in SJL mice are shown). Conversely, very few CCL19+ cells were detected in the perivascular inflammatory cuffs of the cerebellum, which contain mainly T-cells and very few macrophages and DCs (not shown). These observations, together with previous findings that CCL19 is produced by macrophages and DCs, but not Tcells (15), suggest that macrophages, and to a lesser extent DCs, are the major source of CCL19 in EAE lesions. Numerous CCL19+ cells were also detected in EAE lesions of relapsing SJL mice (EAE grade 2) and chronically affected Biozzi AB/H mice (EAE grade 3-4) (Figure 3F, H). Staining was particularly abundant in the CNS of Biozzi AB/H mice which exhibited the most invasive and widespread lesions (Figure 3H). As disease progressed, CCL19 immunoreactivity was also detected in some astrocyte-like and microglia-like cells in the CNS parenchyma of both mouse strains (Figure 3I-K). At EAE onset, no CCL21+ cells were detected in CNS lesions of either SJL or Biozzi AB/H mice (not shown). Despite weak induction of CCL21 mRNA (see Figure 2A), CCL21 immunoreactivity remained unde-

tectable also in the CNS of relapsing SJL mice. This suggests that in a EAE model characterized by acute disease attacks followed by complete remissions, the self-limiting inflammatory process is not sufficient to trigger CCL21 protein synthesis, at least at levels detectable by immunohistochemical techniques. Interestingly, only in one SJL mouse developing an atypical form of chronic stable EAE (grade 3 up to day 30 p.i.), CCL21 immunoreactivity localized to the endothelial cells of an intrameningeal blood vessel in the spinal cord (Figure 4C). In Biozzi AB/H mice undergoing disease progression, anti-CCL21 antibody reacted with endothelial cells of intrameningeal vessels (not shown) and of some inflamed vessels in the spinal cord and brain stem white matter (Figure 4D, E). Although the intensity of CCL21 staining was variable, we calculated that about 40% of the inflamed vessels examined expressed CCL21. Only in one severely diseased Biozzi AB/H mouse, some CCL21+ cells displaying an elongated or irregular cell body and thin cytoplasmic processes were found in a discrete area of the spinal cord white matter containing scattered inflammatory infiltrates (Figure 4F). Immunohistochemical detection of CCR7-expressing cells and potential cellular targets for lymphoid chemokines in the EAE-affected CNS. Immunohistochemical analysis of CCR7 expression was performed using a polyclonal anti-mouse CCR7 antibody. While strong CCR7 immunoreactivity was detected on lymphocytes in T- and B-cell zones of control lymph nodes, no CCR7 staining was detected in normal CNS (not shown). A few CCR7+ cells were present in CNS lesions of SJL and Biozzi AB/H mice at EAE onset (Figure 5A, B). In agreement with the RT-PCR data, more numerous CCR7+ cells were found in the CNS of relapsing SJL and chronically-affected Biozzi AB/H mice, mainly within perivascular and intrameningeal cell infiltrates (Figure 5C, D). Because CCR7 has been reported to be expressed on mature DCs, B-cells, naive and central memory T-cells (7, 50, 59, 61), we next examined the expression of markers specific for these cell types in spinal cord lesions. As previously shown (51), cells labeled by MIDC-8 antibody, a marker for mature interdigitating DCs, enter the CNS at disease onset (see also Figure 3E) and markedly increase in number during EAE progression in both mouse strains (Figure 5E; only staining in Biozzi AB/H mice is shown). CD45R/B220+ B-cells were detected only in the spinal cord of relapsing SJL and chronic-relapsing Biozzi AB/H mice, mainly in intrameningeal and perivascular locations (Figure 5F). We also stained for L-selectin

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Figure 5. (Opposing page) Presence of CCR7+ cells and potential cellular targets for lymphoid chemokine action in EAE lesions. At disease onset, CCR7 immunoreactivity is detected on the surface of some round cells in the spinal cord lesions of SJL (A) and Biozzi AB/H (B) mice. In relapsing SJL (C) and chronic-relapsing Biozzi AB/H (D) mice, numerous CCR7+ cells are present in the meningeal and perivascular infiltrates. E-H: Sections from the spinal cord of a chronic-relapsing Biozzi AB/H mouse were stained for MIDC-8 (E), a marker for mature DC, B220/CD45R (F), a marker for B-cells, CD62L (G) and CD45RB (H), two molecules highly expressed in naive T-cells. Sections were cut from a 200-m thick tissue block; F and G show adjacent sections. Original magnifications: 500 (E) and 1000 (A-D, F-H).

(CD62L) and CD45RB, since these molecules are known to be highly expressed on murine naive and central memory T-cells that preferentially home to lymphoid tissues (54, 59). Numerous CD62L+ and CD45RB+ cells were detected in the meninges and some inflamed blood vessels of relapsing SJL and chronic-relapsing Biozzi AB/H mice (Figure 5G, H). Although anti-CD62L and anti-CD45RB antibodies may also stain B-cells, we would exclude labeling of other cell types (eg, neutrophils, monocytes/macrophages) based on the observation that cells expressing CD62L or CD45RB were absent or only occasionally seen in CNS lesions at EAE onset (not shown). CNS tissues from control mice failed to stain with all the above antibodies. Taken together, these findings indicate that, during EAE progression, the inflamed CNS provides a suitable environment for recruiment and positioning of immune cell types that are potential targets for CCL19 and CCL21. Discussion

Major unsolved issues regarding the immunopathology of MS concern the nature of the immunologic drive responsible for continuous T-cell accumulation/activation in the CNS and the site for priming and expansion of CNS-targeted T-cells. Because DCs are thought to play an essential role in the pathogenesis of autoimmunity (5, 18, 37), signals regulating DC-T-cell homing and interactions in the diseased CNS may be the key to understanding how the inflammatory self-recognition process is sustained. The presence of DCs in the CSF of MS patients (47) and CNS lesions of EAE-affected rodents (39, 51, 53) suggests that these potent antigen presenting cells may internalize CNS antigen(s) and either transport it to secondary lymphoid organs or present it to T-cells locally to maintain autoimmune responses. The first scenario is supported by studies showing that progression to chronicity in EAE and MS is associated with the appearance of circulating T-cells with new CNS-antigenic specificities, a phenomenon referred to as epitope spreading (34, 57), and by the presence of macrophages/DCs containing myelin components in the cervical lymph nodes of EAE-affected monkeys (16). The second scenario is favored by the demonstra-

tion of T-cell clonal expansions in the CSF and active lesions of MS patients, most of which are not identifiable in blood (3). Furthermore, Targoni et al (56) have recently shown that in SJL mice with PLP 139-151 peptide-induced EAE a second wave of effector T-cells recognizing PLP 178-191 peptide is detected first in the CNS and only at later time points in the spleen, suggesting that priming of new autoreactive T-cells could occur in the target organ itself. The characteristic intrathecal synthesis of oligoclonal immunoglobulins (2) and the accumulation of clonally related B-cells in the CSF and brain lesions of MS patients (6, 13) are also suggestive of antigen-driven, intrathecal B-cell clonal expansion which, according to the requirements for B-cell stimulation, should be sustained by local B-, T-cell and DC interactions (32, 41). This study supports the concept that compartmental leukocyte homing to the CNS may contribute to autoimmunity by showing that EAE development in SJL and Biozzi AB/H mice is associated with the intracerebral expression of 2 chemokines, CCL19 and CCL21, that regulate trafficking of lymphocytes and DCs in lymphoid organs (15, 41, 50). While CCL19 mRNA and immunoreactivity are detected in the CNS already at EAE onset, CCL21 expression is much lower and confined to some CNS blood vessels during chronic disease. The demonstration that immune cells expressing CCR7, the common receptor for CCL19 and CCL21, accumulate in the CNS during EAE progression further implies a role for these chemokines in maintaining neuroinflammation. CCL19 and CCL21 have been shown to stimulate
2 integrin-mediated adhesion of lymphocytes to intercellular adhesion molecules (ICAM)-1 and cause rapid arrest of circulating lymphocytes to the vascular endothelium (9, 14). CCL21 also induces 4
7 integrinmediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule-1 (MadCAM-1) expressed in mucosal HEV (46).The unique combination of traffic molecules found on HEV, including ICAM-1, MadCAM-1, peripheral node addressin (PNAd) or CD62L ligand, and CCL21, is thought to be essential for efficient lymphocyte homing into secondary lymphoid organs (22, 50). In EAE, blood-brain barrier endothelia

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acquire some properties of HEV, like expression of ICAM-1 and MadCAM-1 (11, 45). Although the lack of PNAd in inflamed CNS blood vessels (11) implies that these do not sustain lymphocyte emigration as efficiently as HEV, it is conceivable that the combined expression of CCL21, ICAM-1 and MadCAM-1 is sufficient to enable the intracerebral migration of circulating lymphocytes that normally home to lymphoid tissues (15, 41, 50). This idea is consistent with the accumulation of cells expressing B-cell (CD45R/B220), naive and central memory T-cell (CD45RB, CD62L) markers in CNS lesions of mice undergoing EAE progression. Interestingly, in a severely diseased Biozzi AB/H mouse, CCL21 was found expressed by some intraparenchymal process-bearing cells whose identity remains to be established. A possible explanation for this finding is that CNS inflammation facilitates the recruitment and/or induces the local differentiation of cells with properties of stromal cells, another major source of CCL21 in lymphoid T-cell zones (15, 38). Lymphotoxin- and tumor necrosis factor, which have a pivotal role in regulating the synthesis of CCL19 and CCL21 in lymphoid tissues (26, 42), are both produced in the EAE-affected CNS (27, 29) and could induce production of lymphoid chemokines during inflammation. In secondary lymphoid organs, CCL19 is produced by T zone stromal cells and interdigitating DCs and is thought to facilitate interaction of naive T-cells with DCs (15, 38). Interestingly, perivascular CCL19 can be transcytosed to the luminal surfaces of HEV and may also participate in CCR7-mediated triggering of leukocyte arrest (4). The observation that during EAE CCL19 is expressed by CNS-infiltrating immune cells, likely macrophages and DCs, as well as by resident microglia and astrocytes, suggests that this chemokine may help to retain CCR7-expressing cells in the lesion site and favor encounter of mature DCs with pathogenic T-cells. Notably, DCs present in EAE lesions are stained by mAb MIDC-8, which recognizes interdigitating DCs in T-cell zones of lymphoid organs, where T-cell responses are initiated (8, 51). Thus, a number of similarities in the cellular and chemokine environment of lymphoid Tcell zones and chronic EAE lesions suggest that DCs maturing in the inflamed CNS, where myelin destruction leads to continuous exposure of self-antigens, could support the local generation of autoreactive T-cells. The findings that in the normal CNS CCL19 mRNA is expressed at low levels and that some cells in the meninges stain positively for CCL19 raise the question of whether CCL19 regulates the constitutive trafficking of specific leukocyte subsets to the CNS. Because

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CCL19 has been reported to attract hematopoietic progenitor cells, particularly macrophage progenitors (31), we propose that it could contribute to the normal turnover of macrophages residing in the meningeal compartment. Recent studies have shown that ectopic expression of CCL21 in the pancreas is sufficient to trigger lymphoid neogenesis (19), whereas transgenic mice expressing CCL21 in oligodendrocytes develop CNS inflammation and neurologic abnormalities, but show neither lymphocyte recruitment nor lymphoid neogenesis in the CNS (12). Although these findings highlight the importance of tissue-specific environments in regulating chemokine effects, they do not exclude that lymphoid chemokines might play a role in CNS immunopathologies. So far, only limited morphological evidence has been provided that lymphoid-like structures develop in the CNS during autoimmune disease (48, 49). The simultaneous presence of CCR7+ cells, mature DCs, Bcells and cells expressing naive/central memory T-cell markers in the inflamed meninges and CNS perivascular spaces during EAE progression suggests that these may represent preferential sites for the establishment of functional interactions mimicking those occurring in lymphoid organs. In conclusion, this study describes the expression of lymphoid chemokines in EAE lesions and suggests their possible involvement in promoting cellular interactions that could be important for priming and expansion of autoreactive lymphocytes within the inflamed CNS. It remains to be seen if blockade of the intracerebral production or activity of lymphoid chemokines might represent an effective therapeutic approach for inhibiting EAE progression, and ultimately for the treatment of MS patients. Acknowledgments

We thank Luciano Adorini for critical reading of the manuscript. This work was supported by Project “Dendritic cells in central nervous system autoimmunity” of the Istituto Superiore di Sanità, and Projects “Multiple Sclerosis” and “Inflammatory, oxidative and autoimmune mechanisms in disabling CNS diseases” of the Italian Ministry of Health. Note Added in Proof

After the submission of this manuscript, Alt et al reported that CCL19 and CCL21 are expressed in venules surrounded by inflammatory cells in the CNS of EAE-affected SJL mice (Eur J Immunol 32:21332144.)

Lymphoid Chemokines CCL19 and CCL21 in the CNS During EAE—Columba-Cabezas et al

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