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(Journal of Leukocyte Biology. 2003;73:584-590.)
© 2003 by Society for Leukocyte Biology

Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS

Debora Giunti^*, Giovanna Borsellino, Roberto Benelli, Monica Marchese^*, Elisabetta Capello^*,, Maria Teresa Valle^¶, Enrico Pedemonte^*, Douglas Noonan^#, Adriana Albini, Giorgio Bernardi^**, Giovanni Luigi Mancardi^*,, Luca Battistini and Antonio Uccelli^*,

^* Neuroimmunology Unit, Department of Neurosciences, Ophthalmology and Genetics, and
Centre of Excellence for Biomedical Research, University of Genoa, Italy;
Laboratory of Neuroimmunology, IRCCS, S. Lucia, Rome, Italy;
Molecular Biology Laboratory and
^# Tumor Progression Section, National Cancer Research Institute, Genoa, Italy;
^¶ II Division of Haematology, S. Martino Hospital, Genoa, Italy; and
^** Department of Neuroscience, University of Rome "Tor Vergata," Italy

Correspondence: Antonio Uccelli, M.D., Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Via De Toni 5, 16132, Genoa, Italy. E-mail: auccelli{at}neurologia.unige.it

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The recruitment of lymphocytes across the blood brain barrier (BBB) is mediated by adhesion molecules and chemokines. The expression of activation markers and of chemokine receptors on T cells homing to the nervous system (NS) may help define their functional state. In the cerebrospinal fluid (CSF) of subjects with inflammatory neurological diseases (IND), including multiple sclerosis, we observed an increased number of T cells coexpressing CXCR3 and CCR5 as well as T cells with a CD45RO+ CCR7+ CD27+ memory phenotype. A subset of CCR7+ T cells coexpressed CXCR3 and CCR5. We also detected an increased number of interferon--producing T cells in the CSF compared with peripheral blood, mostly but not exclusively in the CD45RO+ CCR7- CD27- compartment. T helper 1 (Th1) clones, established from the CSF of individuals with IND and from a healthy subject, similarly migrated to CXCL10, CXCL12, and CCL5. CXCL10, CXCL12, and CCL19 were increased in the CSF of individuals with neuroinflammation. These findings suggest that CSF is enriched in Th1-polarized memory T cells capable of differentiating into effector cells upon antigen encounter. These cells are recruited into the CSF by inducible chemokines. Thus, CSF represents a transitional station for T cells trafficking to and from the NS.

Key Words: MS • chemokines • T lymphocytes • neuroimmunology • cell trafficking

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Immune surveillance within peripheral tissues is guaranteed by a wide array of cells from the innate and adaptive arms of the immune system. Following an inflammatory reaction, T cells of an activated/memory phenotype are recruited at the site of inflammation by the combined action of various stimuli, including the release of chemokines and the up-regulation of chemokine receptors and adhesion molecules. Chemokines are key mediators of the leukocyte recruitment process; homing chemokines, which are constitutively expressed, have been shown to display tissue specificity [¹ ]. Migration pathways correlate with specific patterns of chemokine receptor expression and are associated with different functional properties of lymphocyte subsets [² ]. For instance, effector T helper type 1 (Th1) cells up-regulate the expression of receptors for inflammatory chemokines such as CXCR3 and CCR5 [³ ]. Recent data suggest that within the memory T cell compartment, the expression of CCR7, the receptor for the lymphoid chemokines ELC and SLC (CCL19 and CCL21, respectively), may allow the distinction between CD45RO+ CCR7+ "central memory" and CD45RO+ CCR7- "effector memory" cells [⁴ ]. Thus, CCR7 expression has been used to define memory T cells during trafficking to different human tissues [⁵ ]. CD27 is a member of the tumor necrosis factor family, which upon T cell activation, is up-regulated on the cellular surface [⁶ ]. Based on CD27 surface expression, T cells can be divided in a larger CD27+ population comprising naïve and memory cells and a smaller CD27- effector subset. Moreover, CD45RO+CD27- T cells express tissue-specific homing receptors but lack receptors involved in migration to lymph nodes, supporting the hypothesis that this subset contains effector T cells [⁷ ]. Despite the involvement of inflammatory chemokines in the recruitment of leukocytes during experimental models of infectious [⁸ ] and autoimmune diseases [⁹ ] of the central nervous system (CNS), their relevance in the physiological recruitment of T cells into the cerebrospinal fluid (CSF) still needs to be fully elucidated. The role of lymphoid chemokines and their cognate receptors in the migration of T cells across the blood brain barrier (BBB) has been addressed only in experimental autoimmune encephalomyelitis (EAE) [¹⁰ , ¹¹ ], but no data are available in humans yet. Moreover, studies addressing the functional state of T cells within the CSF may provide insights into the migratory pathways of T cells from and to the nervous system (NS). In this report, we studied the expression of markers of the memory/effector state in the CSF of individuals with inflammatory diseases of the NS. Finally, we investigated the chemotactic properties of clones established from the CSF of such individuals and a healthy subject toward a panel of chemokines detected in the CSF.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects and sample preparation
For the cytofluorimetric studies, peripheral blood and CSF were obtained upon informed consent from 21 patients with multiple sclerosis (MS) and 14 subjects with other inflammatory neurological diseases (OIND) who underwent venipuncture and lumbar puncture for diagnostic purposes. MS individuals were classified according to the recommended guidelines for the diagnosis of MS [¹² ]. The clinical features and laboratory findings of the patients enrolled are given in Table 1 . Peripheral blood mononuclear cells (PBMC) were isolated on a density gradient (Ficoll-Histopaque, ICN Biomedicals, Aurora, OH), and CSF cells were collected following centrifugation at 1500 rpm for 10 min. Enzyme-linked immunosorbent assay (ELISA) was performed on CSF supernatants collected from 10 MS subjects, 10 OIND individuals, and 10 patients with noninflammatory neurological disorders (NIND), such as Alzheimer disease, stroke, epilepsy, and hydrocephalus. CSF of NIND individuals did not show oligoclonal bands nor an increased Link index.

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Table 1. Demographic and Laboratory Features of MS Patients and Subjects with OIND

Flow cytometry
The antibodies used were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or CyChrome^TM (CyC) as indicated. Anti-CD3 (FITC), CD4 (PE or CyC), CD8 (CyC), CD19 (CyC), and CD56 (PE) antibodies were from Serotech (Oxford, UK). Antibodies against CD25 (FITC), CD62L (PE), CD70 (FITC), and CCR7 (purified) were purchased from PharMingen (San Diego, CA), and anti-CCR1 (PE), CCR2 (PE), CCR3 (FITC), CCR4 (PE), CCR5 (FITC), CCR6 (PE), CXCR3 (PE), CXCR4 (PE), and CXCR5 (PE) antibodies were from R&D Systems (Minneapolis, MN). Anti-CD27 (PE), CD45RO (FITC), CD45RA (FITC), and CD69 (PE) antibodies and the secondary antibody goat F(ab')₂ anti-mouse IgM FITC (for CCR7 detection) were from Caltag (Burlingame, CA). Control staining was performed using the following antibodies: IgM (FITC), IgG1 (FITC), IgG1 (PE), IgG1 (CyC), and IgG2 (PE; all from PharMingen). A minimum number of 10,000 CSF cells and 100,000 PBMC were stained on ice for 20 min with combinations of three of the antibodies described above as indicated. Following one wash with phosphate-buffered saline (PBS), samples were run on a FACScan, and the data were analyzed with CellQuest^TM software (BD Biosciences, Erembodegem, Belgium). In all experiments, recovered cells were labeled at the same time with anti-CD4 and anti-CD8 antibodies (both CyC). Thus, selective gating on each T cell population during analysis allowed CD4⁺ T cells (low) to be examined separately from CD8⁺ bright T lymphocytes. Intracellular staining was performed by activating T cells with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) and ionomycin (1 µg/ml) in the presence of monensin (5 nM; Sigma-Aldrich, Steinheim, Germany). These cells were then permeabilized and subsequently stained with PE-conjugated, isotype-control antibodies or FITC-labeled anti-interferon- (IFN-) and PE-conjugated interleukin IL-4 monoclonal antibodies (BD Biosciences).

Generation of T cell clones
T cell clones were established from the CSF of a healthy subject, one individual with viral meningitis (as representative of OIND), and two MS patients by single-cell sorting with the MoFlo® cytometer (Cytomation, Fort Collins, CO). Clones were maintained with repeated cycles of stimulation with phytohemagglutinin (PHA; 2 µg/ml; Sigma-Aldrich). A healthy subject was defined as an individual suffering from symptoms mimicking a neurological deficit, which was subsequently found to be unrelated to a neurological disease.

Migration assays
Chemotaxis assays were performed in quadruplicate in Boyden chambers (Neuro Probe, Cabin John, MD). Chemoattractants were placed in the lower chamber (27 µl/well) and T cell clones in the upper chamber (100,000 in 50 µl/well), both diluted in serum-free medium. These compartments were separated by a polycarbonate filter with a 5-µm pore size. The cells that migrated into the lower chamber were counted under a light microscope. Results were expressed as migration index (MI) representing the ratio for each clone between cells migrated to a given chemokine and the same cells migrated in serum-free condition The following chemokines were used as chemoattractants: CCL3 (macrophage-inflammatory protein-1 (MIP-1); 200 µg/ml), CCL5 (Rantes IP-10; 400 µg/ml), CXCL10 (IP-10; 200 µg/ml), and CXCL12 (stromal cell-derived factor-1 (SDF-1); 500 µg/ml); all purchased from Pepro-Tech EC Ltd., London, UK). For chemotaxis inhibition experiments, T cell clones responsive to CCL5 were incubated for 30 min at 4°C with antibodies against CCR1 (7 µg/ml), CCR3 (25 µg/ml), and CCR5 (7 µg/ml), the three known CCL5 receptors (all from R&D Systems). Anti-CCL5-, -CXCL12-, and -CXCL10-blocking antibodies (from R&D Systems) were also used at different concentrations (1, 3, 6, and 9 µg/ml) to block the bioactivity of each chemokine, incubating the responsive clone for 30 min at room temperature (RT).

ELISA
Levels of IFN- and IL-4 were measured in triplicate on the supernatants of CSF T cell clones 48 h following PHA stimulation using an ELISA method (Quantikine, R&D Systems). Similarly, levels of CXCL10, CXCL12, CCL5, CCL19, and CCL21 were analyzed on CSF supernatants by commercial ELISA kits (Quantikine for CXCL10, CXCL12, and CCL21; DuoSet for CCL5 and CCL19; all from R&D Systems).

Statistical analysis
To calculate significant differences between CSF and PBMC and between two groups of patients, a two-tailed Mann-Whitney U-test was used. ELISA results were compared by a nonparametric ANOVA test (GraphPad Prism 3.0, GraphPad Software, San Diego, CA). Significance was assigned at P< 0.05.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CSF is enriched in CCR7+ CD27+ T cells
To characterize the functional phenotype of T cells detected in the CSF of individuals with inflammatory diseases, we conducted a detailed analysis of CD4 and CD8 T cells by flow cytometry. Increased numbers of CD4⁺ and CD8⁺ T cells coexpressing CXCR3 and CCR5 were detected in the CSF as compared with the peripheral blood (Table 2 ). A high proportion of CD45RO memory T cells in both subsets was positive for CD27 and CCR7, suggesting a memory phenotype. In contrast, low expression of these molecules was observed in peripheral blood T cells (Table 2) . No differences were detected for these subpopulations when comparing T cells within the same compartment, CSF or peripheral blood, of individuals with MS and subjects with OIND (Table 2) . Despite a significant number of CSF T cells coexpressing CCR5 and CXCR3 (Table 2) , only less than 20% of CCR7+ T cells coexpressed CCR5. A similar proportion of CD27+ T cells was CCR5-positive. In contrast, CCR7+ were largely CXCR3-positive. Hence, CCR5 and CXCR3 were coexpressed on a minority of CCR7+ CSF T cells (Fig. 1 ). It is interesting that up to 90% of CCR7+ T cells in CD4⁺ and CD8⁺ subsets coexpressed CD62L (data not shown). Thus, memory/activated T cells, expressing the receptors required to home to secondary lymphoid organs, enter the CNS possibly to sustain a chronic inflammation.

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Table 2. Expression of Markers Suggesting a Th1 Memory Phenotye on CD4+ and CD8+ T Cells from Peripheral Blood Mononuclear Cells (PBMC) and Cerebrospinal Fluid (CSF) of Subjects with Multiple Sclerosis (MS) and Other Inflammatory Neurological Diseases (OIND)

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Figure 1. Coexpression of CCR7 and CXCR3 (a), CCR7 and CCR5 (b), and CD27 and CCR5 (c) on CSF T cells. (d) Coexpression of CCR5 and CXCR3 gating on CCR7+ T cells.

CCR7- and CCR7+ CSF cells produce IFN-
As the expression on T cell surfaces of CXCR3 and CCR5, which are receptors for the inflammatory chemokines CXCL10 and CCL5, respectively, has been associated with a Th1 phenotype, we measured production of IFN- by CSF T cells by intracellular staining. We detected an over twofold increase of the percentage of T cells producing IFN- in the CSF compared with the peripheral blood. We subsequently determined whether CD4⁺ and CD8⁺ CD45RO+ cells, which have lost CD27 and CCR7, indicating their differentiation to effector cells, are present in the CSF during an inflammatory response. Only a small fraction of the lymphocytes in the CFS had the phenotype of effector cells in individuals with MS and in subjects with OIND (Table 3 ). CD45RO+ CD27- and CD45RO+ CCR7- lymphocytes from the CSF of both groups were always slightly higher in the CD4 as compared with the CD8 subset; in any case, the median was never above 7.3% (Table 3) . A higher percentage of cells with such phenotypes was detected in the peripheral blood of both groups. It is interesting to note that a modest increase of CD4⁺ CD45RO+ CD27- and CD4⁺ CD45RO+ CCR7- was observed in the peripheral blood of subjects with OIND as compared with MS individuals (Table 3) . The increased number of CD45RO+ CCR7- effector cells in the peripheral blood resided exclusively in two individuals with on-going, systemic, bacterial infections (neuroborreliosis) with subsequent involvement of the NS, where a direct role for systemic effector CD4⁺T cells has been established [¹³ ]. IFN- produced within the CSF was mostly detected into CCR7- CSF T cells, confirming their effector nature (Fig. 2a ). Although most of CCR7+ CSF T cells did not produce IFN-, a minority of these cells were shown capable of producing it (Fig. 2b) . Only a few T cells coexpressed markers of early activation, such as CD25 and CD69 (median values for MS: 7.9; for OIND: 6.3). These markers were expressed exclusively on the CCR7- subset. Thus, a limited IFN--producing CCR7- T cell population resides in the CSF of subjects with neuroinflammation. Of interest, within the larger CCR7+ subset, a small, unrestricted population can also produce IFN-, therefore suggesting some effector potential.

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Table 3. Effector Phenotye on CD4+ and CD8+ T Cells from PBMC and CSF of Subjects with MS and OIND

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Figure 2. Production of IFN- by CCR7-negative and CCR7-positive CSF T cells. (a) The majority of IFN--producing T cells is CCR7-. (b) Almost 100% of CSF T cells are CCR7+, and 23% of those produce IFN-.

T cell clones established from the CSF of individuals with OIND and a healthy donor have a similar phenotype
As a result of the limited number of T cells within the CSF, chemotaxis could not be performed on T cells sorted ex vivo. Thus, we decided to characterize the migratory properties of CSF-derived cells by means of T cell clones established by repeated cycles of stimulation with PHA following single T cell sorting. T cell clones were obtained from two individuals with MS, one patient with viral meningitis, and one healthy subject. All clones obtained expressed a CD4⁺ CD45RO+ memory phenotype. Twenty-four out of 28 (85%) were CXCR3-, CCR5-, CCR7-, CCR3-, CCR4-, CD70-, and CD62L-positive. A minority of clones were CD27-, CXCR4-, and/or CCR1-positive. CCR2, CCR6, and CXCR5 were absent on these clones (data not shown). The expression of these markers was confirmed at different time points, suggesting a relative stability with time in vitro.

T cell clones migrate to a panel of inflammatory chemokines
To analyze the chemotactic properties of the T cell clones, their migratory response was tested in a series of experiments using a panel of chemokines including CCL5, CXCL10, CCL3, and CXCL12. A significant response was detected in CXCL12, CCL5, and CXCL10. No difference in migration capabilities was detected among clones from the three groups (Fig. 3 ). We then verified whether migration to these chemoattractants was mediated by a specific ligand/receptor interaction. Indeed, migration to CCL5 was inhibited by an anti-CCL5 antibody in a dose-dependent manner. Moreover, a partial inhibition of migration to CCL5 was obtained by the addition of a blocking antibody for each of the known CCL5 receptors (CCR1, CCR3, and CCR5). Almost complete inhibition was observed when all of the blocking antibodies were used at the same time in the chemotaxis assay (data not shown). A similar pattern of inhibition of migration was observed by blocking the chemokine/chemokine receptor interaction for CXCL10 and CXCL12 (data not shown). To characterize the functional phenotype of the clones, the production of IFN- and IL-4 was measured by ELISA. All clones, regardless of their origin or phenotype, produced high concentrations of IFN- (mean level: 524 pg/ml) and little or no IL-4 (mean level: 24.67 pg/ml), suggesting a Th1 profile. These results were in agreement with the expression of high levels of CXCR3 and CCR5.

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Figure 3. Chemotaxis of T cell clones established from the CSF of homeostatic chemokines (open bars), OIND (shaded bars), and MS individuals (solid bars). MI for each clone represents the ratio between cells migrated to a given chemokine and the same cells migrated in serum-free condition. Each bar indicates the mean migration index to a given chemokine of clones established from each group. Nonstatistically significant differences among groups were detected.

Inducible chemokines are detected in the CSF
To evaluate whether the ligands of CCR7 and the chemokines used for the migration assays are present in the CSF, we analyzed the CSF from 10 MS subjects, 10 OIND individuals, and 10 patients with NIND. CXCL10 levels were increased in the CSF of subjects with OIND and to a lesser extent, in MS individuals compared with NIND. Increased CXCL12 levels were also detected in OIND compared with NIND and MS subjects. CCL19 levels were slightly augmented only in subjects with OIND compared with MS and NIND (Table 4 ). Comparable, low concentrations of CCL5 were observed in a few samples from all the groups, and CCL21 was never detected in any CSF sample. Thus, inflammatory and homeostatic chemokines can be detected in the CSF of individuals with IND, being higher in subjects with overt inflammation.

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Table 4. Levels (pg/ml) of CXCL10, CXCL12, and CCL19 Measured in the CSF of MS, OIND, and NIND Individuals

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Most inflammatory chemokines are up-regulated in nonlymphoid tissues under inflammatory conditions and recruit T cells at the site of inflammation [¹⁴ ]. Recent reports on MS and EAE strongly support a pathogenic role for inflammatory chemokines expressed by heamatogenous and neural cells within CNS lesions and their receptors on infiltrating leukocytes [⁹ , ¹⁵ , ¹⁶ ]. Similarly, these chemokines are involved in the immune response during CNS infections [⁸ , ¹⁷ , ¹⁸ ]. Recently CCL19 and CCL21 have been shown to be expressed at the BBB and to chemoattract CCR7+ encephalitogenic T cells in mouse EAE [¹⁰ ]. In addition CCR7+ T cells have been detected in inflammatory lesions during EAE progression [¹¹ ]. We report a striking expansion of the CD4⁺ and CD8⁺ subsets with a memory phenotype in the CSF of subjects with inflammatory diseases of the NS as compared with the peripheral compartment. This was especially true for cells expressing memory markers such as CD45RO, CCR7, and CD27. CSF T cells also coexpressed CCR5 and CXCR3, confirming a preferential recruitment of Th1 T cells during inflammatory reactions in the NS. Similar findings have been reported in the CSF of MS subjects [¹⁹ , ²⁰ ]. A preferential compartmentalization of Th1 T cells in the CSF was confirmed by an increased percentage of IFN--producing cells compared with the peripheral blood in patients with an ongoing inflammatory reaction of the NS. It is interesting that IFN--producing T cells were observed mainly but not exclusively in the CD45RO+ CCR7- CD27- compartment. The presence of IFN--producing T cells also in the CCR7+ CD27+ subset is in agreement with the hypothesis that CCR7+ T cells may still retain an effector function [²¹ ]. The coexpression of CCR7, CXCR3, and to a lesser extent, CCR5 extends the results obtained in the EAE model [¹⁰ , ¹¹ ] to humans, suggesting that polarized, memory T cells enter the CSF upon recruitment by inflammatory chemokines such as CXCL10 and CCL5 as well as homeostatic chemokines such as CCL19 and possibly CXCL12. Further differentiation to effector cells leads to the down-regulation of CCR7 and up-regulation of CCR5 [²² ]. Taken together, these results favor the hypothesis that memory T cells patrolling the NS evolve to effector cells through distinct differentiation steps: CD45RO+ CD27+ CCR7+ CCR5- CXCR3- no IFN- producers (true central memory) CD45RO+ CD27+ CCR7+ CCR5+/- CXCR3+ low IFN- producers (memory/effectors) CD45RO+ CD27- CCR7- CCR5+ CXCR3+ strong IFN- producers (true effector cells). This type of differentiation lineage has been proposed recently for antigen-specific CD8⁺ T cells [²³ ].

As a result of the technical restrictions imposed by the low number of cells, it is virtually impossible to perform chemotaxis assays with CSF cell populations obtained from healthy subjects, as the detection of an increased number of cells in the CSF is suggestive of inflammation. To overcome this problem, we established T cells clones from a healthy individual and from subjects with MS and OIND. Despite the possibility that in vitro expansion of CSF T cells may have influenced the overall phenotype of clones, they remarkably mirrored the phenotype expressed in vivo by CSF cells. These clones showed a memory phenotype associated with high expression of CXCR3, CCR5, CD62L, and CCR7. Moreover, they produced high levels of IFN- and little IL-4, suggesting a Th1 profile, in agreement with their pattern of chemokine receptors. Clones from individuals with inflammatory diseases of the NS and from the healthy subject migrated equally well to CXCL10 and to a lesser extent to CCL5 and CXCL12. As the levels of these chemokines appear to increase within the CSF upon the presence of overt inflammation, it is likely that they may mediate the recruitment of T cells into the NS under physiological and pathological conditions and that quantitative more than qualitative stimuli may tune-up their migration across the BBB. Indeed, we detected increased levels of CXCL10 and CXCL12 in subjects with an ongoing CNS inflammatory response. Accordingly, CXCL10 has been previously reported to be elevated in subjects with MS and other inflammatory disorders of the NS [²⁴ , ²⁵ ]. It is likely that the relatively small difference between MS individuals and subjects with NIND, which we observed and which does not reach statistical significance, is probably a result of the limited size of our cohorts. CXCL12 was recently shown to play a pivotal role for the recruitment of dendritic cells to the CSF during bacterial infections of the NS [¹⁸ ]. In the three groups, we also identified significant levels of CCL19, being higher in the OIND group. CCL19 may be involved in trafficking memory and activated T cells to secondary lymphoid organs [²⁶ ], being expressed on inflamed CNS venules of EAE mice [¹⁰ ] and neural cells such as astrocytes and microglia [¹¹ ]. In contrast, CCL21, which is also present on inflamed mice endothelium [¹⁰ ] and on the endothelium of the high endothelial venules (HEV), was not detectable in any sample.

The presence of CCR7 together with CCR5 and CXCR3 on the surface of T cells within the CSF suggests that memory/effector T cells are recruited into the CSF using chemotactic pathways that are mediated by inflammatory and at least in part, by homeostatic chemokines. This hypothesis is in agreement with the results obtained in the mouse model of MS, where it has been shown that lymphoid chemokines expressed on the surface of CNS-inflamed venules and on some neural cells may contribute to the migration of encephalitogenic T cells across the BBB [¹⁰ , ¹¹ ]. Depending on the nature of the recruitment trigger (e.g., autoimmune vs. infectious), an increased number of T cells with a memory/effector phenotype may migrate into the CSF depending on the levels of inducible chemokines. Subsequently, upon antigen recognition and under the influence of inflammatory chemokines [⁹ ] and possibly other yet unknown chemotactic stimuli, memory cells may acquire a fully effector phenotype, down-regulating CCR7 and further up-regulating CCR5 to enter the NS [²² ]. Conversely, the expression of CCR7 and CD62L on CSF T cells indicates that these cells may still migrate to a secondary lymphoid organ and possibly contribute to the formation of ectopic lymphoid structures sustained by homeostatic chemokines [¹¹ , ²⁷ ]. Whether T cells recirculate to a lymphoid-like structure possibly located into the CNS, as suggested by the detection of a compartmentalized accumulation of B cell clones in the CSF [²⁸ ], or to draining lymph nodes remains to be established. We were not able to identify any pattern of chemokine receptor expression that defined any tissue or disease specificity, as indicated by the similar expression pattern of surface molecules on CSF cells from different inflammatory conditions. Altogether, the CSF may represent an intermediate station during trafficking of memory T cells in their function of immune surveillance along the NS.

 
This work was supported by the Federazione Italiana per la Sclerosi Multipla (FISM), the Ministero dellÍstruzione, dellÚniversitá e della Ricerca (MIUR COFIN and COFINLAB), the Ministero della Sanitá (Progetto Finalizzato), and the Progetto Strategico Neuroscienze (National Research Council). Dompè Biotec funded part of this work. We thank Drs. Vito Pistoia and Marco Gattorno for critically reviewing the manuscript.

Received December 9, 2002; revised January 19, 2003; accepted January 22, 2003.

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