Multiple sclerosis: a study of CXCL10 and CXCR3 co-localization in the inflamed central nervous system

Journal of Neuroimmunology 127 (2002) 59 – 68 www.elsevier.com/locate/jneuroim

Multiple sclerosis: a study of CXCL10 and CXCR3 co-localization in the inf lamed central nervous system Torben L. Sørensen a,*,1, Corinna Trebst b,1, Pia Kivisa¨kk b, Karen L. Klaege b, Amit Majmudar b, Rivka Ravid c, Hans Lassmann d, David B. Olsen a, Robert M. Strieter e, Richard M. Ransohoff b, Finn Sellebjerg a b

a The MS Clinic, Department of Neurology, University of Copenhagen, Glostrup Hospital, 2600 Glostrup, Denmark Department of Neurosciences, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH, USA c The Netherlands Brain Bank, Amsterdam, The Netherlands d Brain Research Institute, University of Vienna, Vienna, Austria e Division of Pulmonary Medicine, University of California, Los Angeles, Los Angeles, CA, USA

Received 7 December 2001; received in revised form 14 March 2002; accepted 15 March 2002

Abstract T-cell accumulation in the central nervous system (CNS) is considered crucial to the pathogenesis of multiple sclerosis (MS). We found that the majority of T cells within the cerebrospinal fluid (CSF) compartment expressed the CXC chemokine receptor 3 (CXCR), independent of CNS inflammation. Quantitative immunohistochemistry revealed continuous accumulation of CXCR3+ T cells during MS lesion formation. The expression of one CXCR3 ligand, interferon (IFN)-g-inducible protein of 10 kDa (IP-10)/CXC chemokine ligand (CXCL) 10 was elevated in MS CSF, spatially associated with demyelination in CNS tissue sections and correlated tightly with CXCR3 expression. These data suggest a critical role for CXCL10 and CXCR3 in the accumulation of T cells in the CNS of MS patients. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Multiple sclerosis; CXCR3; CXCL10; Central nervous system; Inflammation; Demyelination

1. Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disease of the human central nervous system (CNS) of unknown etiology (Noseworthy, 1999; Sørensen and Ransohoff, 1998). Crucial to its pathological cascade is the accumulation of T cells and phagocytic macrophages in the CNS and the formation of lesions with the typical features of inflammation, microglial activation, demyelination, glial scaring and axonal damage. Chemokines and their receptors have been implicated in migration of mononuclear cells under physiological and pathological conditions and have emerged as salient targets for investigation (Luster, 1998; Murphy et al., 2000; Trebst and Ransohoff, 2001; Zlotnik and Yoshie, 2000).

*

Corresponding author. Tel.: +45-43-23-30-55; fax: +45-43-23-39-26. E-mail address: [email protected] (T.L. Sørensen). 1 Both authors contributed equally to this work.

CXC chemokine receptor 3 (CXCR3) is expressed primarily on activated T cells, B cells, natural killer (NK) cells and proliferating endothelial cells (Qin et al., 1998; Rabin et al., 1999; Romagnani et al., 2001). Known human ligands for CXCR3 are monokine induced by interferon-g (mig)/ CXC chemokine ligand (CXCL) 9, interferon-g-induced protein of 10 kDa (IP-10)/CXCL10 and interferon-inducible T-cell a-chemoattractant (I-TAC)/CXCL11 (Cole et al., 1998; Loetscher et al., 1996). Evidence for the involvement of CXCR3 and CXCL10 in the pathogenesis of MS was first obtained in animal models of MS, including experimental autoimmune encephalomyelitis (EAE). Levels of CXCL10 were related to clinical relapses in mice with EAE (Fife et al., 2001; Glabinski et al., 1997; Godiska et al., 1995). Astrocytes were identified as the cellular source of CXCL10 production in EAE (Ransohoff et al., 1993; Tani et al., 1996). Administration of anti-CXCL10 antibodies ameliorated disease activity and reduced accumulation of pathogenic T cells in an adoptive transfer model of EAE, as well as in the chronic

0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 ( 0 2 ) 0 0 0 9 7 - 8

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demyelinating phase of mouse hepatitis virus infection of the CNS, a viral model of MS (Fife et al., 2001; Liu et al., 2001b). Descriptive evidence suggesting a role of CXCL10 and CXCR3 in MS has also emerged. Several investigators showed that MS patients exhibited significantly higher CXCL10 levels in the cerebrospinal fluid (CSF), compared to control subjects (Franciotta et al., 2001; Prat et al., 2000; Sørensen et al., 1999). Additionally, CXCL10 levels in the CSF correlated with the CSF leukocyte count, suggesting in vivo activity of CXCL10 (Sørensen et al., 1999). In a recent serial CSF study, our group found that CXCL10 CSF levels correlated significantly with other markers of intrathecal inflammation such as neopterin, matrix metalloproteinase (MMP)-9 and IgG-synthesis. Furthermore, CSF CXCL10 levels did not change after treatment with methylprednisolone, indicating a role for CXCL10 in maintaining inflammation in the CNS (Sørensen et al., 2001). Histological studies have reproducibly localized CXCL10 immunoreactivity to reactive astrocytes within active demyelinating MS lesions, suggesting astrocytes as a potential cellular source of CXCL10 in the CSF (Huang et al., 2000; Simpson et al., 2000; Sørensen et al., 1999). When CXCR3 expression on circulating and CSF T cells was compared by flow cytometry, a significant enrichment of CXCR3 bearing CD4+ and CD8+ T cells in the CSF in patients with MS was observed (Misu et al., 2001; Sørensen et al., 1999). Furthermore, CXCR3+ cells were readily detected in perivascular cuffs in active demyelinating lesions of MS autopsy material (Balashov et al., 1999; Simpson et al., 2000; Sørensen et al., 1999). Circulating CXCR3+ T cells were associated with high production of interferon (IFN)-g, arguing in favor of the functional significance of these cells (Balashov et al., 1999). The current investigation was initiated to address the relationship between CXCL10 and CXCR3 expression in the CSF and brain sections of MS patients and controls. By correlating ligand and corresponding receptor in individual patients, we were able to show a close correlation between CXCL10 expression and CXCR3+ T cells within MS brain sections, but not in the CSF of patients with demyelination or controls. Furthermore, we observed a continuous accumulation of CXCR3+ cells during MS lesion formation. These results support a specific role for the ligand/receptor pair CXCL10/CXCR3 in the accumulation of T cells in the CNS parenchyma in MS patients, but not in the accumulation of CXCR3+ T cells in the CSF compartment.

2. Materials and methods 2.1. Patients Twenty-one patients with clinical relapses of MS, 19 patients with monosymptomatic optic neuritis (ON), 18 pa-

Table 1 Flow cytometry study: patient demographics Diagnosis Gender

Age (years) CSF CSF Lesions pleocytosis oligoclonal on MRI bands

ON (19) MS (21) CON (18) HC (24)

35 41 57 30

11 17 13 18

F/8 M F/4 M F/5 M F/6 M

(21 – 50) (24 – 59) (34 – 79) (20 – 44)

9 (47%) 10 (48%) 0 (0%) NA

11(58%) 21(100%) 0 (0%) NA

12 (63%) 21 (100%) NA NA

ON, optic neuritis; MS, multiple sclerosis; CON, control subjects without neurological conditions; HC, healthy controls; F, female; M, male; CSF, cerebrospinal fluid; NA, not available; MRI, magnetic resonance imaging; pleocytosis > 4 cells/Al.

tients with noninflammatory neurological diseases (CON) and 24 healthy individuals (HC) were included in our studies (Table 1). ON patients had no history of previous neurological symptoms and were diagnosed using established clinical criteria (Francis, 1991). MS diagnosis was based on established criteria for clinical research (McDonald et al., 2001). None of the patients was receiving any immunomodulatory drugs at the time of simultaneous phlebotomy and lumbar puncture. Magnetic resonance imaging (MRI) was performed on MS and ON patients and the results were graded according to established criteria (Paty et al., 1988). The presence of oligoclonal bands in the CSF was assayed as previously described (Sellebjerg and Christiansen, 1996). The local scientific ethics committee approved the study, and informed written consent was obtained from all participants. 2.2. Analysis of CXCR3 expression by flow cytometry Expression of CXCR3 on CD4+ and CD8+ T cells was examined by flow cytometry in peripheral blood and CSF. CSF was obtained directly on ice and cells were counted in a Jessen chamber. Within 10 min of lumbar puncture, CSF cells were collected by centrifugation at 250  g for 10 min at 4 jC. The CSF supernatant was removed by pipetting and immediately stored at 80 jC for subsequent analysis of CXCL10 by ELISA (see below). CSF cells were washed once in ice-cold phosphate-buffered saline (PBS) with 1% human serum albumin and 0.1% sodium azide (FACS buffer). Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway), washed three times at 4 jC in Hank’s PBS with 1% human serum albumin and resuspended in ice-cold FACS buffer. Whole blood staining was also performed on random samples with an intraassay variability of less than 5% (not shown). One hundred microliters of CSF cells (minimum, 4000 mononuclear cells) or 100 Al of PBMC (100,000 mononuclear cells) were incubated at 4 jC with antibodies for 30 min and washed twice in FACS buffer and fixed with 1% paraformaldehyde (PFA) before data acquisition on a fourcolor FACSCalibur flow cytometer (Becton Dickinson

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Immunocytometry Systems, San Jose, CA). List-mode data were analyzed with CellQuest software (Becton Dickinson). Cells were gated according to forward- and side-light scattering properties and positively or negatively selected for CD4 and CD8 or CD14 expression, respectively. 2.3. Antibodies For these studies, PE-conjugated anti-CXCR3 (Clone 1C6/CXCR3, Pharmingen, San Diego, CA), FITC-conjugated anti-CD4 (Clone SK3, Becton Dickinson), PerCPconjugated anti-CD8 (Clone SK1, Becton Dickinson), APC-conjugated anti-CD3 (Clone SK7, Becton Dickinson) and APC-conjugated anti-CD14 (Clone MNP9, Becton Dickinson) were used. Isotype control antibody was PEconjugated mouse IgG1 (Clone MOPC-21, Becton Dickinson). 2.4. Analysis of CXCL10 CSF levels Frozen CSF samples from 17/21 MS patients, 17/19 patients with ON and 15/18 CON patients were available for the determination of CXCL10 levels. Enzyme-linked immunosorbant assay (ELISA) was performed as previously described. Chemokine concentrations were measured twice in separate, coded ELISA runs. Intraassay variability has previously been determined to be less than 10%, and lower detection limit was 0.01 ng/ml (Sørensen et al., 1999). 2.5. MS autopsy material for immunohistochemistry studies of CXCR3 and CD3 expression CXCR3 and CD3 expression was analyzed in paraffinembedded archival autopsy material of five patients with MS—diagnosed based on the presence of characteristic MS pathology. A total of nine tissue sections with 19 active lesions were available for this study (Table 2). The material was collected and characterized at the Brain Research Institute, University of Vienna, Austria. All tissue sections showed pattern II demyelination (Lucchinetti et al., 2000), with prominent deposition of immunoglobulins and complement (as indicated by presence of C9 neoantigen) at sites of active myelin destruction.

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Within active MS lesions, individual regions of different demyelinating activity were classified according to previously published criteria (Bru¨ck et al., 1994, 1995; Lucchinetti et al., 2000). Early active (EA) demyelinating regions were diffusively infiltrated by macrophages immunoreactive for all myelin proteins including myelin oligodendrocyte glycoprotein (MOG). Late active (LA) demyelinating regions were more advanced with respect to myelin degradation, and were immunoreactive for the major myelin proteins myelin basic protein (MBP) and proteolipid protein (PLP), but not for MOG. Inactive (IA) demyelinated regions were completely demyelinated with no signs of active demyelination. 2.6. Control autopsy material for CXCR3 and CD3 expression Additionally, 13 paraffin-embedded archival brain tissue sections from three individuals without known neurological, inflammatory or metastatic disorder were collected at the Cleveland Clinic Foundation and served as controls. All three individuals (mean age: 69 years; two females, one male) died from sudden cardiac attack. Similar brain sections, as in the MS cases, were chosen for comparison. 2.7. Autopsy material for CXCR3 and CXCL10 co-localization The analysis of CXCL10 expression by immunohistochemistry was not possible in paraffin-embedded archival autopsy material (Sørensen et al., 1999). Frozen autopsy material, collected and characterized at the Netherlands Brain Bank, Amsterdam, The Netherlands, was therefore used for our co-localization studies of CXCL10 and CXCR3 expression. Six frozen tissue sections of three individuals with MS (mean age: 39 years; all female) containing a total of 10 demyelinated MS lesions were available for this study. Additionally, two sections of individuals (mean age: 51 years, both female) without neurological diseases served as controls. Due to a rapid autopsy protocol employed at the Netherlands Brain Bank, the mean postmortem delay was 7:15 h (5:15 – 10:55 h).

Table 2 MS autopsy material for CXCR3 and CD3 quantitation: patient characteristics Case number

Gender

Age (years)

Disease course

Disease duration (months)

Number of tissue sections

Number of lesions

Early-active regions

Late-active regions

Inactive regions

Periplaque WM regions

1 2 3 4 5 5 cases

F F F M F 4 F/1 M

47 46 28 52 34 41 (28 – 52)

Acute Acute SP Acute SP 3 acute/2 SP

3.5 0.4 12 1.5 144 32.3 (0.4 – 144)

2 3 2 1 1 9

3 4 7 3 2 19

3 4 6 3 2 18

2 4 4 2 2 14

2 4 3 0 2 11

2 3 2 1 1 9

F, female; M, male; SP, secondary progressive; WM, white matter.

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2.8. Immunohistochemistry Immunohistochemistry was performed as previously described (Sørensen et al., 1999; Trebst et al., 2001). In brief, 5 Am sections were placed on Superfrost slides. Paraffin-embedded tissue sections were deparaffinized with xylenes and rehydrated in ethanol. Frozen tissue sections were fixed with 4% PFA prior to staining. After antigenretrieval, slides were incubated overnight with primary antibody at 4 jC, washed in PBS, incubated with secondary antibody at room temperature for 40 min, washed and incubated with avidin – biotin – horseradish peroxidase complex (Vectastain Elite, Vector Laboratories, Burlingame, CA). After development with 3,3-diaminobenzidine (DAB) substrate (Sigma), slides were dehydrated with ethanol and mounted in Permount (Fisher Scientific, Pittsburgh, PA). The following antibodies were used: murine monoclonal anti-CXCR3 (clone 1C6.2), kindly provided by Leukosite, rat monoclonal anti-CD3 (clone CD3-12, Serotec, Raleigh, NC), murine monoclonal anti-CXCL10 (kindly provided by Leukosite) and rabbit-polyclonal anti-GFAP (DAKO, Carpinteria, CA). For analysis of co-localizations of CXCR3 with CXCL10, and CXCL10 with glial fibrillary acid protein (GFAP), sections were simultaneously labeled with primary antibodies and then incubated with Texas Red- and fluorescein isothiocyanate-conjugated secondary antibodies (Southern Biotechnology Associates, Birmingham, AL). In controls, primary antibodies were omitted, and tests for cross-reactivity by secondary antibodies were performed. 2.9. Quantitation The number of immunoreactive cells was determined in at least four standardized fields (146,200 mm2, defined by a morphometric grid) from each of the distinct lesion areas. Immunostained sections were photographed on a Leica DMR microscope (Leica Wetzlar, Heidelberg, Germany) and an Optronix Magnafire digital camera system and analyzed using Image ProR Plus (Media Cybernetics, Silver Spring, MD). 2.9.1. Statistics Nonparametric tests (Mann – Whitney test and Wilcoxon signed rank test) were applied because the data were not normally distributed (Kolmogorov –Smirnov test). A p-value < 0.05 was considered statistically significant.

3. Results 3.1. The majority of CD4+ and CD8+ T cells in the CSF express CXCR3 CXCR3 expression on CD4+ and CD8+ T cells in peripheral blood and CSF was compared in patients with

Table 3 Expression of CXCR3 on CD4+ and CD8+ CSF T-cells

MS ON CON HC

Percentages of CXCR3 expression on CD4

Percentages of CXCR3 expression on CD8

Blood

CSF

Blood

CSF

32 35 30 31

82* (51 – 97) 84* (72 – 95) 85* (73 – 96) NA

67 69 66 66

92* (63 – 100) 94* (79 – 100) 92* (71 – 100) NA

(16 – 70) (18 – 51) (17 – 54) (20 – 51)

(22 – 99) (47 – 87) (48 – 82) (48 – 84)

MS, multiple sclerosis; ON, optic neuritis; CON, control subjects without neurological conditions; HC, healthy controls; CSF, cerebrospinal fluid; values are given as median and range; NA, not available. * Expression of CXCR3 was significantly higher in the CSF compared to peripheral blood ( p < 0.0001).

ON, MS, control subjects (CON) and healthy individuals (blood only). Approximately 80% of all CD4+ and approximately 90% of all CD8+ T cells in the CSF expressed CXCR3, significantly higher than found in peripheral blood ( p < 0.001, Table 3, Fig. 1). In this regard, there were no differences between patients with MS, ON or controls. CXCR3 expression on circulating T cells did not differ among the three patient groups or healthy individuals (Table 3). We concluded that CD4+ and CD8+ T cells in the intrathecal compartment were enriched for expression of CXCR3, without regard to the presence of CNS inflammation. Because patients with ON and MS exhibited CSF pleocytosis (Table 1), absolute numbers of CSF CXCR3+ T cells were higher in these patients. Percentages of CXCR3+ T cells in the blood and in the CSF did not correlate with: time from onset of symptoms to lumbar puncture, the presence of oligoclonal bands, CSF leukocyte counts, or MRI abnormality scores. 3.2. CXCL10 levels in CSF are higher in MS and ON, compared to CON, and do not correlate with CXCR3 expression on CSF T cells To address the relation between CSF concentrations of CXCL10 and expression of CXCR3 on CSF T cells, analysis of CXCL10 in the CSF of patients with ON, MS and other neurological diseases (CON) was performed by ELISA and correlated with CXCR3 expression on CSF T cells. Patients with MS and with ON exhibited a significantly higher level of CXCL10 in the CSF, compared to CON patients (MS: 1.64 ng/ml [0.054 – 6.2], ON: 3.53 ng/ ml [0.036 –28], CON: 0.59 ng/ml [0.024 – 3.4], p < 0.05). There were no differences between the CXCL10 levels of MS patients and patients with ON ( p = 0.8). CXCL10 levels showed a positive correlation with CSF leukocyte counts in all patient groups (r = 0.449, p < 0.01). There was no correlation between CXCL10 levels and CXCR3 expression on CSF T cells (normalized to the individual absolute number of CSF cells) of patients with MS, ON or in the controls.

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Fig. 1. CXCR3 expression on CD4+ and CD8+ T cells in peripheral blood and CSF. CXCR3 expression was analyzed by flow cytometry. Cells were gated according to forward- and side-light scattering properties and positively selected for CD4 (A) or CD8 (B) expression, respectively. Immunofluorescence levels above the isotype control expressed by 1% of the cells were considered positive. Shown are representative histograms of a single patient. A significant increase in percent expression of CXCR3 on both, CD4+ and CD8+ T cells was observed in the CSF, compared to peripheral blood.

3.3. T cells in actively demyelinating MS lesions are CXCR3+ and accumulate during lesion development To follow the fate of CXCR3+ T cells during MS lesion development, CD3 and CXCR3 expression was studied in 19 actively demyelinating lesions in nine tissue sections from five MS autopsy brains. Periplaque white matter was studied in each of the nine tissue sections as an internal control. CD3+ cells were almost exclusively found within actively demyelinating lesions compared to their infrequent detection in the periplaque white matter. Within actively demyelinating lesions, the majority of CD3+ cells were localized to the perivascular space with some dispersion into the parenchyma (Fig. 2A). The distribution patterns of CXCR3 and CD3 immunoreactivity on serial sections were markedly similar (Fig. 2B). Quantitative immunohistochemistry was performed to define the relationship between CXCR3+ T-cell accumulation and demyelinating activity. Within the 19 actively demyelinating lesions, 43 distinct areas of different demye-

linating activity were available: 18 early active areas, 14 late active areas and 11 inactive areas (Table 2) as well as 9 areas outside the lesions (periplaque white matter). There were significantly more cells expressing CXCR3 and CD3 within lesions at all stages, as compared to the periplaque white matter ( p < 0.001, Fig. 2C and D). Interestingly, we observed a significant increase in the number of CXCR3+ cells (early active vs. inactive, p < 0.05, Fig. 2D) during lesion development, suggesting a continuous accumulation of CXCR3+ cells. Spearman Rank correlations of individual fields on serial sections documented a robust relationship between numbers of CXCR3+ and CD3+ cells (r = 0.845, p < 0.0001). Tight correlations were observed in all different lesional stages (early active: r = 0.838, p < 0.0001; late active: r = 0.626, p < 0.05; inactive: r = 0.957, p < 0.0001). Quantitative analysis within the 13 tissue sections of three control brains revealed similar numbers for CD3+ and CXCR3+ cells (CD3: 7.27 F 2.07 cells/mm2; CXCR3: 4.71 F 1.41 cells/mm2) as in the periplaque white matter of MS cases.

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Fig. 2. CD3 and CXCR3 expression in autopsy material from patients with MS. CD3 (A) immunoreactivity within actively demyelinating MS lesions was predominately observed around vessels (A: arrows) with some dispersion into the parenchyma (A: arrowhead). CXCR3 (B) immunoreactivity resembled CD3 staining pattern with accumulation of CXCR3+ cells around vessels (B: arrows) and only occasional CXCR3+ cells within the parenchyma (B: arrowhead). VL, vessel lumen; bars indicate 100 Am. Quantitation (C and D) was employed to characterize the relationship between CD3 (C) and CXCR3 (D) expression and different demyelinating activity. Eighteen areas of early active (EA) demyelination, 14 areas of late active (LA) demyelination and 11 inactive (IA) demyelinated areas were available for these studies. Periplaque white matter (WM) served as an internal control in all nine tissue sections. Values are displayed on a logarithmic scale. Values are given as cells/mm2 and represent means F S.E.M.

3.4. CXCL10 is abundantly expressed on astrocytes within demyelinated MS lesions Immunohistochemistry for CXCL10 was performed in frozen autopsy material. A total of 10 inactive demyelinated lesions in six sections of three individuals with MS were available. Periplaque white matter in all six sections served as internal controls. Two frozen sections of two individuals without neurological disease were analyzed as controls. Immunoreactivity for CXCL10 was predominantly found within areas of demyelination with the majority of staining localized around blood vessels (Fig. 3A). In some MS lesions, CXCL10 staining associated with astrocytic processes was observed throughout the lesion. Only occasionally was CXCL10 immunoreactivity associated with an astrocyte cell body (Fig. 3D). In periplaque white matter, CXCL10 staining was occasionally observed around

blood vessels. The staining pattern within the two control sections resembled the staining in the periplaque white matter. Astrocytes were identified as the cellular sources of CXCL10 immunoreactivity within the examined brain sections by double-label experiments of CXCL10 with GFAP, showing an almost complete overlap between the two staining patterns (Fig. 3D – F). For quantitation, CXCL10 expression was defined as immunostained cellular elements per square millimeter, as most of the expression was observed on astrocytic processes, rather than on somata. Quantitative evaluation of CXCL10 immunoreactivity revealed a clear association with demyelinated MS lesions: an average of 1056.8 cellular elements/mm2 were immunoreactive for CXCL10 within lesions, whereas only 59.9 CXCL10+ cellular elements/ mm2 could be identified in the periplaque white matter

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Fig. 3. Co-localization of CXCL10 with CXCR3 and GFAP expression in frozen autopsy material from patients with MS. CXCL10 (A and D), CXCR3 (B) and glial fibrillary acid protein (GFAP, E) expression was analyzed in frozen autopsy material of patients with multiple sclerosis (MS). CXCL10 expression was predominantly found around blood vessels (A) and only occasionally is associated with an astrocyte cell body (D: arrow). CXCL10 and CXCR3 expression colocalized in the majority of perivascular cuffs (C). GFAP+ astrocytes were identified as cellular source of CXCL10 in MS lesions (F). BV, blood vessel; bars indicate 100 Am.

patients to evaluate the relationship between the two expression patterns. Double-labeling experiments for CXCR3 and CXCL10 on selected tissue sections complimented this analysis. The majority of perivascular cuffs were immunoreactive for both CXCL10 and CXCR3 (Fig. 3A –C). When correlating the number of perivascular accumulation of CXCL10+ cellular elements and the number of CXCR3+ perivascular cuffs within entire tissue sections of MS patients, we observed a tight relationship (Spearman Rank correlation r = 0.89, p < 0.01). These data underline the potential biological significance of CXCL10 in the accumulation of CXCR3+ cells in the perivascular space.

(Table 4). Expression levels of CXCL10 in the control brains were comparable to periplaque white matter levels (65 CXCL10+ cellular elements/mm2). None of the available MS lesions contained areas of early active or late active demyelination. Therefore, a correlation of CXCL10 immunoreactivity with lesion formation was not possible. 3.5. CXCR3 and CXCL10 expression are closely related in MS tissue sections Immunohistochemistry for CXCL10 and CXCR3 were performed on serial sections of all six frozen sections of MS Table 4 Expression of CXCL10 in frozen autopsy material from patients with MS Case number

Age (years)

Postmortem time (h)

1

35

5:45

2

45

10:55

3

38

6:55

Tissue section 1A 1B 2A 2B 3A

3B

CXCL10+ (cellular elements/mm2) Lesion Lesion Lesion Lesion Lesion Lesion Lesion Lesion Lesion Lesion

1 2 3 4 5 6 7 8 9 10

759 544 551 1589 325 2130 953 1262 954 1503 1056.8 F 177.6

CXCL10+ (cellular elements/mm2) WM

19

WM WM WM WM

0 111 115 10

WM

104 59.9 F 22.6

WM, periplaque white matter. Expression of CXCL10 was significantly higher within lesions, as compared to periplaque white matter and controls ( p < 0.001).

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4. Discussion 4.1. What are the relations between CXCR3 expression on CD4+ and CD8+ T cells and CXCL10 levels in the CSF? MS patients exhibit higher percentages of CXCR3 expressing T cells in the CSF, compared to peripheral blood (Sørensen et al., 1999). Here, we compared CXCR3 expression on peripheral and CSF CD4+ and CD8+ T cells of patients with MS, ON and individuals with noninflammatory CNS conditions, finding expression of CXCR3 on the majority of CD4+ and CD8+ CSF T cells in all patient groups. In vitro activation of T cells is associated with a dramatic increase in expression of CXCR3 (Rabin et al., 1999). Furthermore, it has been shown in animal models that activated T cells are capable of entering and leaving the CSF regardless of the presence of CNS inflammation (Hickey et al., 1991; Wekerle et al., 1991). Expression of CXCR3 on T cells along with activation-dependent integrins such as VLA4 might enable a subset of peripheral T cells to enter the CSF compartment for physiological immune surveillance, independent of CNS pathology. However, it has recently been shown that encephalitogenic T lymphocytes change their chemokine receptor expression just before they enter the CNS from the circulation in a mouse model. Interestingly, in contrast to other chemokine receptors such as CCR1, CCR2, CCR3, CCR5 and CCR7, CXCR3 is not up-regulated under these conditions in comparison to in vitro activated T cells, which only have a limited capacity to home into established inflammatory CNS lesions (Flu¨gel et al., 2001). Thus, as will be discussed below, CXCR3 expression on T cells may be less important in their recruitment through the blood – brain barrier than in their further migration into the CNS parenchyma. CXCR3 has been described on lymphocytes within a variety of inflamed tissues including rheumatoid arthritis, inflammation of the skin, chronic vaginitis, ulcerative colitis, alveolitis and pulmonary sarcoidosis (Agostini et al., 1998, 2000; Flier et al., 2001; Qin et al., 1998), indicating that the accumulation of CXCR3+ T cells at sites of inflammation is not a CNS-specific phenomenon. CXCL10 has previously been shown to be elevated in CSF of patients with MS, compared to control subjects (Franciotta et al., 2001; Prat et al., 2000; Sørensen et al., 1999). In this report, we confirmed previous findings of elevated CXCL10 levels in MS CSF. CXCL10 CSF levels and CXCR3 expression on CSF T cells were evaluated in individual subjects, allowing correlation between ligand and receptor-expressing CSF cells. Interestingly, no correlation between CXCL10 CSF levels and CXCR3+ T cells was observed, either in direct correlation or when corrected for number of CSF cells in individuals. These results have to be interpreted with some caution, as the analysis is performed in the lumbar CSF. Even though the lumbar CSF is in equilibrium with the extracellular space of the CNS white matter, in this particular analysis it has to be taken into

account that CXCL10 may be immobilized on astrocytic processes and the extracellular matrix as evidenced by the observation of CXCL10 immunoreactivity mainly on the surface of astrocyte processes and therefore, may not be evenly distributed in the CSF (Huang et al., 2000). Receptor internalization after binding the cognate ligand should probably also be taken into account. The fact that we do not find a correlation between CXCL10 levels and receptorexpressing cells in the CSF stresses on the other hand the hypothesis that trafficking of lymphocytes from the bloodstream into the CSF compartment is not only an inflammation-independent process, but likely is not governed by the presence of CXCL10 in the CSF. Interestingly in this regard are findings by Kieseier et al. (2002), who described elevated levels of CXCL10 in CSF of patients with Guillain – Barre´ syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) despite normal cell counts in the CSF. The latter observation furthermore stresses that the presence of CXCL10 in the CSF has not necessarily to be reflected in the cellular composition of the CSF. This does not imply (as seen below) that the accumulation of CXCR3+ T cells in the CNS parenchyma is independent of the presence of CXCL10. In interpreting our results, a potential role of the other known human ligands for CXCR3, CXCL9 and CXCL11 in accumulating CXCR3-bearing cells in the CSF has to be considered. Preliminary data suggest increased levels of CXCL9 in the CSF of MS patients as compared to controls (de Groot and Woodroofe, 2001; Sørensen et al., 1999). A distinct role for CXCL9 in promoting a protective Th1 response against viral infection of the CNS has been demonstrated in mouse hepatitis virus infection of the CNS (Liu et al., 2001a). CXCL11 has not been investigated to date. A comprehensive understanding of the roles of CXCR3 and all its ligands in accumulating cells in the CNS in different pathological conditions await further studies. 4.2. What is the relationship between accumulation of CXCR3+ cells and expression of CXCL10 within MS lesions? CXCR3 immunoreactivity has been abundantly detected on perivascular lymphocytes within MS lesions (Balashov et al., 1999; Simpson et al., 2000; Sørensen et al., 1999). We performed quantitative correlations between the number of CXCR3+ T cells and demyelinating activity in active MS lesions to elucidate the role of CXCR3 in lesional T-cell accumulation in MS. We found a significant increase of CXCR3+ cells when comparing early and late stages of MS lesions, suggesting a continuous accumulation of CXCR3+ T cells in active MS lesions. This has particular impact on the design of protocols for therapeutic intervention as it implies that the continuous migration of CXCR3+ T cells into the CNS parenchyma may be targeted not only at early stages of lesion development. Furthermore, numbers of CXCR3+ cells and CD3+ cells correlated in a field to field

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analysis of serial sections in lesion areas at all different stages, indicating that most CD3+ cells within MS lesions expressed CXCR3 regardless of the stage of activity. This finding corresponded with our flow cytometry studies, which showed that the majority of T cells in the CSF compartment expressed CXCR3. CXCR3 expression on T cells was only occasionally found in control autopsy brain tissues. This contrasts to our observation of CXCR3+ T cells within the CSF compartment in the control group. We therefore propose that trafficking from the peripheral blood into the CSF compartment can occur independently of CNS pathology, whereas retention within the perivascular space and invasion of the CNS parenchyma depends on a local inflammatory microenvironment. CXCL10 immunoreactivity was abundantly detected on astrocytic processes within demyelinated MS lesions, but only occasionally in periplaque white matter, indicating a close relationship between inflammatory demyelination and CXCL10 expression as previously reported (Balashov et al., 1999; Simpson et al., 2000; Sørensen et al., 1999). Quantitative co-localization of CXCL10 and CXCR3 within the same tissue sections was performed and revealed a strong correlation between CXCL10 and CXCR3 immunoreactivity, underlining the proposed biological significance of CXCL10– CXCR3 interactions in vivo. CXCL10 immunoreactivity was only occasionally observed in the control brain sections. This observation agrees with our findings of low CSF CXCL10 levels in controls and demonstrates specificity of CXCL10 expression during CNS inflammation. In summary, this report provides new insights into the role of the chemokine/chemokine receptor pair CXCL10/ CXCR3 in accumulating T cells into the CSF compartment, the CNS perivascular space and the CNS parenchyma during MS lesion formation. In particular, we showed that the majority of T cells within the CSF compartment express CXCR3, independently of CNS inflammation. Furthermore, quantitative immunohistochemistry revealed the continuous accumulation of CXCR3+ T cells during MS lesion development. CXCL10 expression in the CNS parenchyma was associated with inflammatory demyelination and closely correlated with the accumulation of CXCR3+ T cells. CXCL10 and CXCR3 expression was only occasionally found in control brain sections. Based on these observations, we propose the following of CXCL10 and CXCR3 in the accumulation of T cells to the CNS under physiological and pathological conditions: CXCR3+ T cells are capable of entering the CSF compartment under any conditions. In noninflammatory circumstances, CXCR3+ cells fail to encounter CXCR3 ligands (such as CXCL10) and are not retained within the CSF. In inflammatory settings, such as MS, CXCL10 is abundantly expressed within the CNS. Under these conditions, CXCR3+ T cells encounter CXCL10 in the CSF compartment and receive signals that activate adhesion molecules, resulting in retention in the perivascular space, a location favoring access to the CNS parenchyma.

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Therefore, targeting the CXCR3 receptor via small molecular antagonists could be a reasonable approach to intervene with T-cell trafficking in the CNS in MS (Ransohoff and Bacon, 2000). Acknowledgements The Danish Multiple Sclerosis Society, United States National Institutes of Health (PO1 NS38667 to RMR), Deutsche Forschungsgemeinschaft (TR 463/1-1 to CT), Bundesministerium fu¨r Bildung, Wissenschaft und Kultur, Austria (GZ 70.056/2-Pr/4/99), Christian and Otilia Brorsons Legat, The Beckett Foundation, Desiree and Niels Yde’s Foundation, Dagmar Marshall’s Foundation, Grosserer Madsen’s Foundation and the Danish Toyota Foundation supported this work. We thank Anne Petersen, Marie Burdick and Barbara Tucky for technical help and the Nancy Davis Center Without Walls for providing a morphometric image analysis station. References Agostini, C., Cassatella, M., Zambello, R., Trentin, L., Gasperini, S., Perin, A., Piazza, F., Siviero, M., Facco, M., Dziejman, M., Chilosi, M., Qin, S., Luster, A.D., Semenzato, G., 1998. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. J. Immunol. 161, 6413 – 6420. Agostini, C., Facco, M., Siviero, M., Carollo, D., Galvan, S., Cattelan, A.M., Zambello, R., Trentin, L., Semenzato, G., 2000. CXC chemokines IP-10 and mig expression and direct migration of pulmonary CD8+/CXCR3+ T cells in the lungs of patients with HIV infection and T-cell alveolitis. Am. J. Respir. Crit. Care Med. 162, 1466 – 1473. Balashov, K.E., Rottman, J.B., Weiner, H.L., Hancock, W.W., 1999. CCR5(+) and CXCR3(+) T cells are increased in multiple sclerosis and their ligands MIP-1a and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. U. S. A. 96, 6873 – 6878. Bru¨ck, W., Schmied, M., Suchanek, G., Bru¨ck, Y., Breitschopf, H., Poser, S., Piddlesden, S., Lassmann, H., 1994. Oligodendrocytes in the early course of multiple sclerosis. Ann. Neurol. 35, 65 – 73. Bru¨ck, W., Porada, P., Poser, S., Rieckmann, P., Hanefeld, F., Kretzschmar, H.A., Lassmann, H., 1995. Monocyte/macrophage differentiation in early multiple sclerosis lesions. Ann. Neurol. 38, 788 – 796. Cole, K.E., Strick, C.A., Paradis, T.J., Ogborne, K.T., Loetscher, M., Gladue, R.P., Lin, W., Boyd, J.G., Moser, B., Wood, D.E., Sahagan, B.G., Neote, K., 1998. Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187, 2009 – 2021. de Groot, C.J., Woodroofe, M.N., 2001. The role of chemokines and chemokine receptors in CNS inflammation. Prog. Brain Res. 132, 533 – 544. Fife, B.T., Kennedy, K.J., Paniagua, M.C., Lukacs, N.W., Kunkel, S.L., Luster, A.D., Karpus, W.J., 2001. CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis. J. Immunol. 166, 7617 – 7624. Flier, J., Boorsma, D.M., van Beek, P.J., Nieboer, C., Stoof, T.J., Willemze, R., Tensen, C.P., 2001. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J. Pathol. 194, 398 – 405. Flu¨gel, A., Berkowicz, T., Ritter, T., Labeur, M., Jenne, D.E., Li, Z., Ellwart, J.W., Willem, M., Lassmann, H., Wekerle, H., 2001. Migratory activity and functional changes of green fluorescent effector cells before

68

T.L. Sørensen et al. / Journal of Neuroimmunology 127 (2002) 59–68

and during experimental autoimmune encephalomyelitis. Immunity 14, 547 – 560. Franciotta, D., Martino, G., Zardini, E., Furlan, R., Bergamaschi, R., Andreoni, L., Cosi, V., 2001. Serum and CSF levels of MCP-1 and IP-10 in multiple sclerosis patients with acute and stable disease and undergoing immunomodulatory therapies. J. Neuroimmunol. 115, 192 – 198. Francis, D.A., 1991. Demyelinating optic neuritis: clinical features and differential diagnosis. Br. J. Hosp. Med. 45, 376 – 379. Glabinski, A.R., Tani, M., Strieter, R.M., Tuohy, V.K., Ransohoff, R.M., 1997. Synchronous synthesis of alpha- and beta-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am. J. Pathol. 150, 617 – 630. Godiska, R., Chantry, D., Dietsch, G.N., Gray, P.W., 1995. Chemokine expression in murine experimental allergic encephalomyelitis. J. Neuroimmunol. 58, 167 – 176. Hickey, W.F., Hsu, B.L., Kimura, H., 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28, 254 – 260. Huang, D., Han, Y., Rani, M.R., Glabinski, A., Trebst, C., Sørensen, T., Tani, M., Wang, J., Chien, P., O’Bryan, S., Bielecki, B., Zhou, Z.L., Majumder, S., Ransohoff, R.M., 2000. Chemokines and chemokine receptors in inflammation of the nervous system: manifold roles and exquisite regulation. Immunol. Rev. 177, 52 – 67. Kieseier, B.C., Tani, M., Mahad, D., Oka, N., Ho, T., Woodroofe, N., Griffin, J.W., Toyka, K.V., Ransohoff, R.M., Hartung, H.P., 2002. Chemokines and chemokine receptors in inflammatory demyelinating neuropathies—a central role for IP-10. Brain, 125, 823 – 834. Liu, M.T., Armstrong, D., Hamilton, T.A., Lane, T.E., 2001a. Expression of Mig (monokine induced by interferon-gamma) is important in T lymphocyte recruitment and host defense following viral infection of the central nervous system. J. Immunol. 166, 1790 – 1795. Liu, M.T., Keirstead, H.S., Lane, T.E., 2001b. Neutralization of the chemokine cxcl10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis. J. Immunol. 167, 4091 – 4097. Loetscher, M., Gerber, B., Loetscher, P., Jones, S.A., Piali, L., Clark-Lewis, I., Baggiolini, M., Moser, B., 1996. Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 184, 963 – 969. Lucchinetti, C., Bru¨ck, W., Parisi, J., Scheithauer, B., Rodriguez, M., Lassmann, H., 2000. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann. Neurol. 47, 707 – 717. Luster, A.D., 1998. Chemokines—chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338, 436 – 445. McDonald, W.I., Compston, A., Edan, G., Goodkin, D., Hartung, H.P., Lublin, F.D., McFarland, H.F., Paty, D.W., Polman, C.H., Reingold, S.C., Sandberg-Wollheim, M., Sibley, W., Thompson, A., van den, N.S., Weinshenker, B.Y., Wolinsky, J.S., 2001. Recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis. Ann. Neurol. 50, 121 – 127. Misu, T., Onodera, H., Fujihara, K., Matsushima, K., Yoshie, O., Okita, N., Takase, S., Itoyama, Y., 2001. Chemokine receptor expression on T cells in blood and cerebrospinal fluid at relapse and remission of multiple sclerosis: imbalance of Th1/Th2-associated chemokine signaling. J. Neuroimmunol. 114, 207 – 212. Murphy, P.M., Baggiolini, M., Charo, I.F., Hebert, C.A., Horuk, R., Matsushima, K., Miller, L.H., Oppenheim, J.J., Power, C.A., 2000. International union of pharmacology: XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52, 145 – 176. Noseworthy, J.H., 1999. Progress in determining the causes and treatment of multiple sclerosis. Nature 399, A40 – A47. Paty, D.W., Oger, J.J., Kastrukoff, L.F., Hashimoto, S.A., Hooge, J.P., Eisen, A.A., Eisen, K.A., Purves, S.J., Low, M.D., Brandejs, V.,

1988. MRI in the diagnosis of MS: a prospective study with comparison of clinical evaluation, evoked potentials, oligoclonal banding, and CT. Neurology 38, 180 – 185. Prat, E., Baron, P., Meda, L., Galimberti, D., Livraghi, S., Ardolino, G., Clerici, R., Scarpini, E., Scarlato, G., 2000. Interferon-g-inducible protein-10 is increased in the cerebrospinal fluid of patients with multiple sclerosis. Mult. Scler. 5, 83 (abstract). Qin, S., Rottman, J.B., Myers, P., Kassam, N., Weinblatt, M., Loetscher, M., Koch, A.E., Moser, B., Mackay, C.R., 1998. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101, 746 – 754. Rabin, R.L., Park, M.K., Liao, F., Swofford, R., Stephany, D., Farber, J.M., 1999. Chemokine receptor responses on T cells are achieved through regulation of both receptor expression and signaling. J. Immunol. 162, 3840 – 3850. Ransohoff, R.M., Bacon, K.B., 2000. Chemokine receptor antagonism as a new therapy for multiple sclerosis. Expert Opin. Investig. Drugs 9, 1079 – 1097. Ransohoff, R.M., Hamilton, T.A., Tani, M., Stoler, M.H., Shick, H.E., Major, J.A., Estes, M.L., Thomas, D.M., Tuohy, V.K., 1993. Astrocyte expression of mRNA encoding cytokines IP-10 and JE/MCP-1 in experimental autoimmune encephalomyelitis. FASEB J. 7, 592 – 600. Romagnani, P., Annunziato, F., Lasagni, L., Lazzeri, E., Beltrame, C., Francalanci, M., Uguccioni, M., Galli, G., Cosmi, L., Maurenzig, L., Baggiolini, M., Maggi, E., Romagnani, S., Serio, M., 2001. Cell cycledependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J. Clin. Invest. 107, 53 – 63. Sellebjerg, F., Christiansen, M., 1996. Qualitative assessment of intrathecal IgG synthesis by isoelectric focusing and immunodetection: interlaboratory reproducibility and interobserver agreement. Scand. J. Clin. Lab. Invest. 56, 135 – 143. Simpson, J.E., Newcombe, J., Cuzner, M.L., Woodroofe, M.N., 2000. Expression of the interferon-g-inducible chemokines IP-10 and Mig and their receptor, CXCR3, in multiple sclerosis lesions. Neuropathol. Appl. Neurobiol. 26, 133 – 142. Sørensen, T.L., Ransohoff, R.M., 1998. Etiology and pathogenesis of multiple sclerosis. Semin. Neurol. 18, 287 – 294. Sørensen, T.L., Tani, M., Jensen, J., Pierce, V., Lucchinetti, C., Folcik, V.A., Qin, S., Rottman, J., Sellebjerg, F., Strieter, R.M., Frederiksen, J.L., Ransohoff, R.M., 1999. Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J. Clin. Invest. 103, 807 – 815. Sørensen, T.L., Sellebjerg, F., Jensen, C.V., Strieter, R.M., Ransohoff, R.M., 2001. Chemokines, CXCL10 and CCL2: differential involvement in intrathecal inflammation in multiple sclerosis. Eur. J. Neurol. 8, 665 – 672. Tani, M., Glabinski, A.R., Tuohy, V.K., Stoler, M.H., Estes, M.L., Ransohoff, R.M., 1996. In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis. Am. J. Pathol. 148, 889 – 896. Trebst, C., Ransohoff, R.M., 2001. Investigating chemokines and chemokine receptors in patients with multiple sclerosis: opportunities and challenges. Arch. Neurol. 58, 1975 – 1980. Trebst, C., Sørensen, T.L., Kivisa¨kk, P., Cathcart, M.K., Hesselgesser, J., Horuk, R., Sellebjerg, F., Lassmann, H., Ransohoff, R.M., 2001. CCR1+/CCR5+ mononuclear phagocytes accumulate in the central nervous system of patients with multiple sclerosis. Am. J. Pathol. 159, 1701 – 1710. Wekerle, H., Engelhardt, B., Risau, W., Meyermann, R., 1991. Interaction of T lymphocytes with cerebral endothelial cells in vitro. Brain Pathol. 1, 107 – 114. Zlotnik, A., Yoshie, O., 2000. Chemokines: a new classification system and their role in immunity. Immunity 12, 121 – 127.