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From the Departments of Neurosciences* and Cell Biology, The Lerner Research Institute, and the Department of Neurology, the Mellen Center for Multiple Sclerosis Treatment and Research, the Cleveland Clinic Foundation, Cleveland, Ohio; Berlex Biosciences, Richmond, California; the Department of Pathology, University of California, San Francisco, California; the Department of Neurology,¶ The Johns Hopkins University, Baltimore, Maryland; the Department of Neurology, || Mayo Clinic, Rochester, Minnesota; and the Brain Research Institute,** University of Vienna, Vienna, Austria
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Abstract |
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This diverse pattern of expression in vitro left unclear what parts the CCR8/CCL1 system might play in any specific immune or inflammatory process. The role of CCR8 and CCL1 in the pathogenesis of CNS diseases has recently been clarified significantly through studies in experimental autoimmune encephalomyelitis (EAE), a model of CNS inflammatory demyelination. Initial experiments a decade ago showed that production of the murine CCL1 homologue was tightly associated with the capacity of proteolipid protein-specific T-cell clones to mediate EAE.15,16
Analysis of eae7, a polymorphic locus that controls susceptibility to EAE in mice revealed amino acid substitutions in CCL1, and two other ß-chemokines.16
Both CCL1 and CCR8 mRNA were highly expressed in the CNS of mice with EAE and this up-regulation was entirely contingent on secretion of tumor necrosis factor-
by infiltrating hematogenous leukocytes17,18
(personal communication, J. Sedgwick, DNAX Research, Palo Alto, CA). Microglia were the predominant cellular source of CCL1 in the brains of mice with EAE, suggesting the possibility of an autocrine signaling to these cells through CCR8 (personal communication, J. Sedgwick). CCR8-/- mice exhibited a significant delay in the onset of EAE as compared with controls. The severity of EAE was significantly attenuated in CCR8-/- mice, with knockouts never achieving the mean neurological disease scores observed in wild-type animals. Leukocyte infiltration into the parenchyma of CCR8-/- mice with EAE was not impaired, suggesting that reduced EAE severity in CCR8 knockouts was because of defective activation of microglia and macrophages (personal communication, J. Sedgwick). A previous analysis of CCR8-/- mice described a specific defect in the generation of type 2 inflammatory responses to protozoal pathogens or airway-allergen challenge, while type 1 reactions were unaffected.19
Therefore, it was plausible that the afferent limb of the type 1 immune response to myelin antigens was not impaired in CCR8-/- mice with EAE. Furthermore, these studies demonstrated that the effects of deleting CCR8 on macrophage activation in the CNS were dominant over the type 2 cytokine defect, as revealed in the EAE model.
The current study addressed the expression of CCR8 in multiple sclerosis (MS). We found that CCR8 was not expressed on T cells but was associated with phagocytic macrophages and activated microglia in both pattern II and pattern III lesions. To establish possible factors that may govern CCR8 expression in vivo, the study was extended to analysis of tissue sections from other CNS pathologies. CCR8 was consistently expressed on phagocytic macrophages and activated microglia in stroke and progressive multifocal leukoencephalopathy (PML). In other disorders that lacked phagocytic macrophages, such as Rasmussen’s encephalitis, neurosarcoidosis, or herpes simplex viral encephalitis, microglia failed to express CCR8. In two cases (one each of cerebral lymphoma and cerebral toxoplasmosis) in which concentrations of intense CCR8 immunoreactivity were detected, focal cerebral infarcts were also identified. Further, CCR8 was not expressed on activated microglia in cases with senile changes of the Alzheimer’s type, raising the possibility that such microglial activation differs from that observed in MS, PML, and stroke. CCR8 was not expressed by T cells or monocytes in peripheral blood, but its expression at protein and message levels was positively modulated by macrophage differentiation and activating stimuli in vitro. Our data demonstrated that CNS CCR8 expression was associated with phagocytic macrophages and activated microglial cells, but not with T cells, in these human CNS diseases. These findings suggest that CCR8 expression may be a marker for a selective program of mononuclear phagocyte gene expression, present in some but not all CNS pathologies that induce microglial activation and monocyte infiltration.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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CCR8 expression was analyzed in paraffin-embedded archival autopsy material of eight patients with MS. Five patients had lesions consistent with pattern II and three patients with pattern III.20
A total of 16 tissue sections with 38 active lesions were available for this study (Table 1)
. The material was collected and characterized at the Brain Research Institute, University of Vienna, Vienna, Austria.
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), there were 172 eosinophils/mm2, 2838 granulocytes/mm2, 1161 macrophages/mm2, and 301 T cells/mm2.
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Normal appearing white matter was defined as an area that showed no evidence of demyelination by macroscopic inspection and histology within the area and the surrounding tissue. Periplaque white matter (PW) represented a strip of tissue of 5 mm adjacent to the border of active (PW active) or inactive (PW inactive) plaques.
Control Autopsy Material
Thirteen 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 noninflammatory controls. All three individuals (mean age, 69 years; two females and one male) died from sudden cardiac attack.
Autopsy and Biopsy Material from Other CNS Pathologies
Archival autopsy and biopsy material from a variety of other cases with neurological disorders were collected at the Cleveland Clinic Foundation, the University of California at San Francisco, and the Johns Hopkins University. All cases underwent routine gross and histopathological evaluation at the sites of collection. Diagnoses were based on established criteria. A total of 43 tissue sections of 31 individual cases were included in our analysis of CCR8 expression (Table 2)
. Diagnoses were as follows: Rasmussen’s encephalitis (one case), lymphoma (two cases), chronic encephalitis (two cases), cerebral toxoplasmosis (two cases), Herpes simplex virus encephalitis (three cases), neurosarcoidosis (two cases), PML (one case), rabies encephalitis (two cases), CNS malaria (one case), senile changes of the Alzheimer’s type (eight cases), and ischemic stroke (seven cases).
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Immunohistochemistry
Immunohistochemistry was performed as previously described.22,23 In brief, 5-µm sections were placed on Superfrost (Fisher Scientific, Pittsburgh, PA) slides. Paraffin-embedded tissue sections were deparaffinized with xylenes and rehydrated in ethanol. After antigen retrieval by steaming in citrate buffer, slides were incubated overnight with primary antibody at 4°C, washed in phosphate-buffered saline (PBS), incubated with biotinylated secondary antibody at room temperature for 40 minutes, washed, and incubated with avidin-biotin-horseradish peroxidase complex (Vectastain Elite; Vector Laboratories, Burlingame, CA). After development with 3,3-diaminobenzidine substrate (Sigma Chemical Co., St. Louis, MO), slides were dehydrated and mounted in Permount (Fisher Scientific, Pittsburgh, PA). For analysis of co-localization of CCR8 with CD68 and CCR8 with CD3, sections were simultaneously labeled with primary antibodies, followed by incubation with species-specific Texas Red- and fluorescein isothiocyanate-conjugated secondary antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL). In controls, primary antibodies were omitted, and tests for cross-reactivity by secondary antibodies were performed. A rabbit polyclonal anti-CCR8 antibody was prepared at Berlex Biosciences (Richmond, CA).24 Confirmatory stainings were done with a goat polyclonal antibody preparation from Alexis Biochemicals (catalog no. 210-764-R100). This reagent has been shown to stain T-regulatory cells generated in vitro.12 Murine monoclonal anti-CD68 (clone KP1, mouse IgG1) was obtained from DAKO Corp., Carpinteria, CA; a goat polyclonal antibody anti-CCR3 from Santa Cruz Biotechnology, Santa Cruz, CA; and a rat monoclonal anti-CD3 (clone CD3–12) from Serotec Inc., Raleigh, NC.
Identification and Quantitation of CCR8-Positive Cells
CD68+ cells were classified as either monocytes, phagocytic macrophages, or activated microglial cells based on morphological criteria. Within CD68+ cells, the presence of vesicular structures and foamy cytoplasmic inclusions were considered as histological criteria for phagocytic macrophages. Small round CD68+ cells in the perivascular space and in the parenchyma, with no indicators of phagocytosis, were classified as monocytes. Activated microglial cells were defined as CD68+ cells with either elongated or round cell bodies and thickened asymmetric processes.
The number of immunostained cells was determined in at least four standardized fields (146,200 µm2, defined by a morphometric grid) from each lesional area. Immunostained sections were photographed using a Leica DMR microscope (Leica Wetzlar, Heidelberg, Germany) microscope and an Optronix Magnafire digital camera system and analyzed using Image Pro Plus (Media Cybernetics, Silver Spring, MD).
Isolation and Culture of Human Monocytes
Monocytes were obtained from freshly donated human peripheral blood from three healthy donors (two females and one male; mean age, 28 years (range, 22 to 33 years). The blood was immediately diluted 1:1 with PBS and underlayered with Ficoll-Paque (Pharmacia, Piscataway, NJ) for separation of mononuclear cells by density centrifugation. Monocytes were separated by adherence to serum-coated flasks according to the method of Kumagai and colleagues26 and subsequently detached using 0.5 mmol/L of ethylenediaminetetraacetic acid. Isolated monocytes were cultured in Dulbecco’s modified Eagle medium (Mediatech Cellgro Inc., Herndon, VA) supplemented with L-glutamine, 4.5 mg/L glucose, and 10% bovine calf serum (Hyclone, Logan, UT) at 37°C and 10% CO2. Under these conditions monocytes differentiate into macrophages after 7 days.26,27 Granulocyte-macrophage colony-stimulating factor was omitted from the cell culture medium because this might alter chemokine surface expression levels.27 Where indicated, monocytes were stimulated with opsonized zymosan (ZOP) for 24 hours. Zymosan was obtained from ICN Biochemicals (Cleveland, OH) and opsonized and used at a concentration of 2 mg/ml to activate human monocytes.28
CCR8 Expression on Monocytes in Vitro
CCR8 expression on cultured monocytes was examined by quantitative immunohistochemistry as previously described.29 Live cells were rinsed with PBS and then fixed in situ with 4% paraformaldehyde (pH 7.3). After 1 hour the fixative was removed and the cells carefully washed with PBS and stored in PBS at 4°C until the time of staining. Before staining, cells were blocked overnight at 4°C with 0.2 mg/ml of normal goat IgG (Caltag Laboratories, Burlingame, CA). The blocking solution was removed, and the primary antibody solution was added without a rinse step. After incubation for 2 hours at 37°C, cells were rinsed and incubated with a biotinylated goat anti-rabbit secondary antibody for 1 hour. Finally, a fluorescein isothiocyanate-conjugated avidin (Southern Biotechnology Associates, Inc.) was added for 1 hour. After a final rinse step, cells were mounted with Vectashield mounting media containing 4'-6-diamidine-2'-phenylindole (DAPI) for nuclear counterstain. Analysis was performed on a Leica DMR microscope. CCR8-immunostained cells were counted in at least five entire microscopic fields at high (x40) magnification (diameter, 620 µm). Immunostained cells were expressed as percentages of absolute number of cells per field as assessed by DAPI nuclei counterstain.
RNA Extraction
RNA was extracted from monocyte cultures and nonadherent cells using TRIzol (Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s instructions. Briefly, cells were washed once with PBS and mixed with 250 µl of TRIzol. RNA was precipitated with isopropanol and washed with 75% ice-cold ethanol. The supernatant was removed with a Pasteur pipette after centrifugation at 7500 x g for 5 minutes at 4°C. RNA was resuspended in sterile MilliQ-water and the concentration determined by spectrophotometry. One µg of RNA was DNase treated (Life Technologies, Inc.) according to the manufacturer’s instructions.
Reverse Transcription-Coupled Polymerase Chain Reaction (PCR)
First-strand cDNA was synthesized using 1 µg of DNase treated RNA, oligo dT primers, and Super-Script II (Life Technologies, Inc.) according to the manufacturer’s instructions. The product of this reaction was amplified by PCR using primer pairs and TaqDNA polymerase (Boehringer Mannheim Corp., Indianapolis, IN). Primer pair sequences were as follows: 5'-TTT ACC AAG TGG CCT CTG AA; 3'-CCA AGA TGT GCA TAC TGT GCA A.30 PCR conditions were set as follows: first denaturation, 94°C, 3 minutes; annealing, 60°C, 1 minute; extension, 72°C, 1 minute; denaturation, 94°C, 1 minute. Optimal concentration of Mg2+ was empirically determined to be 1.5 mmol/L. The product of 40 cycles of PCR was a unique band of expected length (297 bp) as revealed by electrophoretic analysis on ethidium bromide-stained agarose gels. Cloning and subsequent sequencing of the product verified amplification of CCR8 mRNA.
Flow Cytometry
Freshly isolated peripheral blood mononuclear cells, 24-hour monocytes after adherence, and 7-day macrophages after culture were isolated and diluted to 106 cells/ml in cold fluorescence-activated cell sorting (FACS) buffer (PBS containing 1% fetal calf serum and 0.1% sodium azide). One hundred µl of the cell suspension was blocked with 0.2 mg/ml normal goat IgG (Caltag Laboratories) for 15 minutes at room temperature. Cells were incubated with anti-CCR8 (Berlex Biosciences) for 15 minutes at room temperature and washed twice with FACS buffer. In a second staining step, cells were incubated with anti-CD3 PE (clone SK7; BD Biosciences, San Jose, CA) or anti-CD14 PE (SK3, BD Biosciences) and goat anti-rabbit fluorescein isothiocyanate (Southern Biotechnology Associates) for 15 minutes at room temperature, followed by two washes with FACS buffer and subsequent fixation with 1% paraformaldehyde. Cells were acquired on a FACScan flow cytometer (BD Biosciences) and analysis performed using WinList software (Verity Software House Inc., Topsham, ME). Cells were gated according to forward- and side light-scattering properties, and were positively selected for CD3 or CD14.
Statistics
Nonparametric tests (Mann-Whitney test and Wilcoxon signed rank test) were applied because the data were not normally distributed (Kolmogorov-Smirnov test). Reported P values are two-tailed and considered statistically significant at a P value <0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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CCR8 expression was analyzed by immunohistochemistry in 10 tissue sections of five patients with pattern II MS lesions and in six sections of three patients with pattern III lesions. Twenty-one active lesions exhibiting pattern II and 17 exhibiting pattern III were identified within the available sections. In both pattern types, CCR8 immunoreactivity was predominantly found within MS lesions, as compared to the periplaque and normal appearing white matter (Figure 1)
. CCR8 immunoreactivity was detected on cells with the morphology of phagocytic macrophages (Figure 1, c and g)
at the expanding lesion edge and in areas of active ongoing demyelination. The distributions of CD68 and CCR8 immunoreactivity were similar (Figure 1; a, b, e, and f)
. Within the periplaque and normal appearing white matter, CCR8 expression was observed on cells morphologically consistent with activated microglia (Figure 1, d and h)
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. CCR3 immunoreactivity was also expressed on T cells in cases of Devic’s neuromyelitis optica, associated with other indicators of humoral immunity.33 CCR8 Expression on Phagocytic Macrophages Is Associated with Active Demyelination in MS Lesions
Quantitative immunohistochemistry was applied to establish the relationship between CCR8 expression and demyelinating activity. Within the 38 actively demyelinating lesions, 86 distinct areas of different demyelinating activity were identified: 36 EA areas, 25 LA areas, and 25 inactive (IA) areas (Table 1)
. There were significantly more cells expressing CCR8 in areas of ongoing demyelination (EA and LA areas) than in normal appearing white matter in both pattern II and pattern III lesions (data not shown).
Because CCR8 was co-localized with CD68+ on cells in these tissues (Figure 2)
, numbers of CCR8+ cells were normalized to the total CD68+ macrophage-lineage cells. In pattern II lesions, approximately half of all CD68+ cells in EA and LA areas expressed CCR8; EA, 52% (SEM = 6.7); LA, 47% (SEM = 9.4) with no significant differences between areas (Figure 4a)
. Only 23% (SEM = 8.2) of CD68+ cells in IA areas of pattern II lesions expressed CCR8, which was significantly less (P = 0.01) than in EA areas (Figure 4a)
. In pattern III lesions, CCR8 expression normalized for CD68+ cells, was less than in pattern II; EA, 31% (SEM = 4.7); LA, 37% (SEM = 7.8); IA, 20% (SEM = 3.0) [EA(II) versus EA(III); P = 0.001, Figure 4b
]. The total numbers of CD68+ cells in EA, IA, and LA zones of individual pattern II and III lesions were not significantly different (data not shown).23
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To characterize CCR8 expression in other neurological disorders, tissue sections from CNS inflammatory conditions such as Rasmussen’s encephalitis and neurosarcoidosis; from viral infections such as PML, herpes simplex virus encephalitis, and rabies encephalitis; from nonviral infectious conditions such as cerebral toxoplasmosis and CNS malaria; and from patients with ischemic stroke, senile changes of the Alzheimer’s type, chronic encephalitis of undetermined etiology, and cerebral lymphoma were analyzed (Table 2)
.
The most prominent CCR8 expression was observed in cases of ischemic stroke (Figure 5, a and b)
and in one case of PML (Figure 5, c and d)
. In both instances, CCR8 was expressed on the majority of macrophages within lesions (PML, 62% of CD68+ cells expressed CCR8; an average of 67% in ischemic stroke cases). CCR8 expression on activated microglia was also detected in nondemyelinated areas in PML.
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CCR8 Is Expressed by Activated Microglia in Some but Not All CNS Pathological Processes
In the remaining cases, CCR8 expression was inconsistently present, despite the presence of activated microglia. CCR8 expression was readily identified in the microglial nodules of cerebral toxoplasmosis, where 47% of microglia within nodules expressed CCR8 (Figure 5, e and f)
. The dissociation between microglial activation and CCR8 expression was most striking in change of the Alzheimer’s type, in which diffuse, prominent microglial activation was observed, with clusters of such cells around senile plaques, but CCR8 expression was not detected (Figure 5, g and h)
. Further, CCR8 expression was not detected in either Rasmussen’s encephalitis and neurosarcoidosis, despite a report of abundant activated CD68+ microglial cells in the former pathology.34
In viral encephalitides, CCR8 expression by CD68-positive microglial cells was variable (Table 2)
. CCR8 expression was not detected in three control brains.
In Vitro, CCR8 Expression Is Up-Regulated by Macrophage Differentiation and Activating Stimuli
Using flow cytometry, the expression of CCR8 was analyzed on monocytes cultured for 24 hours and 7 days. The expression of CCR8 was detected on macrophages derived from monocytes after 24 hours of culture and the expression of CCR8 up-regulated after 7 days of culture (Figure 6a)
. CD3+ T cells and CD14+ monocytes in preparations of freshly isolated peripheral blood mononuclear cells showed negligible levels of CCR8 expression (data not shown). The absence of CCR8 immunoreactivity in freshly isolated T cells was confirmed by the lack of CCR8 mRNA using reverse transcriptase (RT)-PCR, whereas CCR8 transcripts were detected in monocytes obtained after 24 hours and 7 days of culture (Figure 6b)
.
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. Monocytes cultured for 24 hours in the absence of any stimuli served as controls for the stimulation experiments. In other cultures, monocytes were allowed to differentiate into macrophages by maintenance for 7 days.
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. Without stimulation, CCR8 expression decreased to an average of 63% after 24 hours in culture (Figure 7, b and e)
. In contrast, monocytes stimulated with ZOP for 24 hours (Figure 7c)
maintained the expression of CCR8 (mean 90% positive cells; Figure 7e
). ZOP-stimulated monocytes displayed evidence of cell maturation with changed morphology, characterized by increased number of vesicles and formation of cell clusters (Figure 7c)
. Mature macrophages derived from monocytes by 7 days of culture (without further stimulation) had become enlarged or spindle shaped with extended processes (Figure 7d)
and 90% expressed CCR8 (Figure 7e)
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In MS tissues, CCR8 expression by CD68+ macrophages was primarily associated with ongoing active demyelination. CCR8 expression decreased significantly during lesion evolution in pattern II and III. In IA areas of both pattern II and pattern III lesions, where myelin phagocytosis was completed, only a minority of CD68+ macrophages expressed CCR8 (Figure 4)
. We concluded that macrophages expressed CCR8 only transiently during active myelin phagocytosis in MS lesions. In this regard, it is important to note that all lesions analyzed here were highly active as required for classification according to patterns of demyelination. Furthermore, in the majority of these cases, MS was fatal within months. Therefore the expression of CCR8 in the chronic lesions of MS remains to be defined.
CCR8 was also observed on microglial cells within MS lesions and in the periplaque and normal appearing white matter. Furthermore, significantly more cells expressed CCR8 in the periplaque white matter (PW) at the borders of active demyelinating lesions (PW active), when compared to PW IA and normal appearing white matter (data not shown). CCR8 expression was not observed on microglial cells in normal control brain sections. These data suggested that CCR8 expression on microglial cells in MS lesions is a reflection of specific microglial activation rather than an intrinsic property of microglial cells.
There are several lines of evidence that microglial cells in normal appearing and PW in MS brains are activated.35-37 Immune-mediated inflammation is thought to play a critical role in the pathogenesis of pattern II lesions, whereas primary injury to the oligodendrocyte is proposed to initiate the pattern III lesion.20 We found similar profiles of CCR8 expression in pattern II and pattern III lesions, and also found extensive CCR8 expression in cerebral ischemia and PML. These results indicate that varied pathways to destruction of neural tissue, and myelin in particular, can induce CCR8 expression on the resident and infiltrating mononuclear phagocytes.
In the current study, CCR8 expression was strikingly associated with phagocytic macrophages. CCR8+ macrophages were observed in all MS cases, in PML, and in all cases of stroke. Furthermore, we unexpectedly found CCR8-positive macrophages co-localized with foci of incidental ischemic pathology in two additional cases (one case each with primary CNS lymphoma and toxoplasmosis). Despite the common endpoint of CCR8 expression, molecular mechanisms that lead to generation of phagocytic macrophages in these cases are likely to differ. In MS, macrophages actively strip myelin from axons, although the factors that target the process to myelin internodes remain uncertain. PML is a primary viral infection of oligodendrocytes, which leads to oligodendrocyte cell death and demyelination. In PML, accumulation of phagocytes to remove myelin debris is presumed to be a secondary process.38,39 Most leukocytes found in lesions of focal cerebral ischemia are macrophages, derived either from monocytes or microglia.40 Despite different activation mechanisms and origins (ie, resident microglia versus hematogenous monocytes), most phagocytic macrophages in MS, PML, and stroke expressed CCR8.
Expression of chemokine receptors on the cell surface is a result of a complex interplay of regulatory mechanisms including cytokine-regulated transcription and ligand-induced internalization, occurring in the context of differentiation of receptor-bearing cells. We previously reported that other chemokine receptors, CCR1 and CCR5, are differentially regulated during monocyte maturation in vitro.23 Here, we report that the expression of CCR8 on monocytes in vitro is dependent on the activation state of the cells. After 2 hours of adherence to serum-coated flasks, 83% of cultured monocytes expressed CCR8. Without further stimulation CCR8 expression decreased during initial culture (63% after 24 hours), whereas stimulation with ZOP or during 24 hours of culture maintained CCR8 expression. The presence of increased CCR8 immunoreactivity was confirmed by the detection of increased mRNA.
Various types of injury to the CNS, such as infection, trauma, autoimmune inflammation, and neurodegeneration are known to elicit microglial activation.41 Regardless of the nature of damage inflicted on the CNS, microglial activation is generally associated with a change in morphology into an amoeboid appearance with shortened cytoplasmic processes and a rounded cell body accompanied by increased expression of genes involved in immune reactions. It has become clear that microglia may display different activity states and have different functional properties under different pathological conditions.42 For example, microglial cells play an important role in the defense against toxoplasma infection. Because cytokines secreted by human microglial prevent the intracellular entry of Toxoplasma gondii.43 Our detection of CCR8 expression in cerebral toxoplasmosis is consistent with this function and supports the importance of CCR8 in defense against protozoal pathogens, recently demonstrated in CCR8-/-.19
A different, detrimental role of microglia has been suggested in degenerative CNS diseases such as Alzheimer’s dementia. It is hypothesized that ß-amyloid precursor protein can directly activate microglia cells and enhance their neurotoxic capabilities.41 Unexpectedly, we did not detect CCR8 in brain sections with changes of the Alzheimer’s type. This observation is provocative, in view of the recent proposal that microglial activation may be deficient, rather than excessive, in Alzheimer’s dementia.44
In summary, we report that CCR8 expression in the human CNS is limited to cells of the macrophage lineage and is found in actively demyelinating MS lesions, in PML, and in cerebral ischemia. CCR8 expression was furthermore observed on activated microglial cells in some, but not all cases with diffuse microglial activation. We conclude that CCR8 expression is strongly associated with phagocytic properties of monocytes and activated microglia cells and that CCR8 identifies a subset of specifically activated microglia cells in different CNS pathologies. These interpretations are limited, however, by insufficient knowledge concerning distribution of CCL1 in human CNS disorders, because of lack of suitable reagents for its detection. CCR8 was not found on T cells, making it unlikely that CNS entry by polarized type 2 lymphocytes or CD4+/CD25+ T cells is mediated by CCR8. Taken in the context of reduced EAE severity in CCR8-/- mice, these findings suggest that CCR8 may be considered a target for therapeutic intervention in CNS diseases in which phagocytic macrophages are deemed pathogenic.
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Acknowledgements |
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Note Added in Proof: Sedgewick J: Interactions between hemotopoietically-derived TNF and central nervous system resident glial chemokines underlies initiation of autoimmune inflammation in the brain. J Immunol, in press.
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Footnotes |
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Supported by National Institutes of Health (PO1NS38667 to R. M. R.) and Bundesministerium für Bildung, Wissenschaft, und Kultur, Austria (GZ 70.056/2-Pr/4/99 to H. L.).
Current address of K. D. A.: MD Anderson Cancer Center, University of Texas, Houston, TX.
Accepted for publication October 14, 2002.
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