CDlb is expressed in multiple sclerosis lesions

Journal of Neuroimmunology ELSEVIER

Journal of Neuroimmunology 67 ( 1996) l45- I5 1

Short Communication

CDlb Luca Battistini

is expressed

a3b.c3 * , Falko R. Fischer of Pothoiogy.

” Depurtmrnt

in multiple

Albert Einstein

Colie~e of Medicine.

of Neurology.

h Departmetrt

d, Cedric

sclerosis

S. Raine a.e.f, Celia F. Brosnan

1300 Morris

Hurt urd Medical

lesions

Pork Arenue,

a3e

Bron.r. NY 10461. USA

School. Boston, MA, USA

’ Eunice Kennedy Shriwr Center. Waltham. MA. USA * Department of NrLlrologx. Geor~ August C/nil er.sitJ. Giittin~en. Germane

of Neuroscience,

’ Department ’ Depurtment

qf Neurologv.

Albert Albert

Einstein Einstein

College College

ofMedicine, ofMedicine.

1300 Morris 1300 Morris

Received 16 May 1995; revised I6 March 1996; acccptcd

Pork Awnrte, Park Arenue.

Bronx. NY 10461. IJSA Bronx, NY 1046l.

USA

18March I996

Abstract

Recent observations have shown thar CDI molecules act as restriction elements in the presentation of antigens to specialized subsets of T cells. To examine the expression of CD1 molecules in multiple sclerosis (MS) lesions, frozen sections of central nervous system (CNS) tissues from nine MS and three other neurological disease (OND) patients, one patient with Wilson’s disease, and one non-neurological control were stained by immunocytochemistry. In chronic-active MS lesions, CD 1b immunoreactivity was prominent on perivascular inflammatory cells whereas macrophages within the lesion showed little reactivity. At the lesion edge. intense immunoreactivity for CD 1b was found on hypertrophic astrocytes. High level expression of CD I b in MS lesions was found to colocalize with the presence of GM-CSF in astrocytes. In chronic-silent lesions, CDlb expression was found on only a few perivascular astrocytic foot processes and the occasional perivascular macrophage. CD I b was not found in the tissues studied for control purposes. In contrast, MHC class II expression was detected on microglia in all tissues examined. The relatively low level expression of CDI b in normal-appearing tissues, chronic-silent lesions and in the OND controls supports the conclusion that the expression of CDlb in active MS lesions is significantly upregulated and could contribute to lesion development. Kewords:

Multiple sclerosis:

CDlb;

Antigen-presenting

molecules:

Inflammatory

1. Introduction The CDI family of glycoproteins are cell surface molecules, non-covalently linked to p 2-microglobulin, that are structurally related to the major histocompatibility (MHC) antigens (Calabi and Milstein, 1986). The Group I or ‘classic’ CDI family include the proteins CD1 a, b and c, which are the products of separate genes that all map to chromosome I, and have molecular masses of 49, 45 and 43 kDa, respectively. They are found on all cortical thymocytes, but not on circulating T cells or monocytes, and are differentially expressed by subpopulations of dendritic cells, Langerhans cells (CDla, CDlc), subsets of B cells (CDIc), certain T cell leukemias, and tissue macrophages in some inflammatory lesions (reviewed in Porcelli, 199.5). In the brain, CD I a has been reported on microglia by some investigators (Lowe et al., 1994) but not by others (Hauser

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cells

et al., 1983; Ulvestad et al., 1994a). CDI expression has also been reported on astrocytes (Bournsell, 1989). Further studies with human monocytes have shown that CDI a, b and c are induced by the cytokines granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-3, with IL-4 acting synergistically with GM-CSF. Other cytokines, including rIFNy, IL-I and TNF are without effect, as is IL-4 when given alone (Kasinrerk et al., 1993; Porcelli et al., 1992; Porcelli. 1995). The fact that these proteins resemble components of the MHC has led to the suggestion that cells expressing them might function as antigen-presenting cells (APC). Consistent with this hypothesis are recent observations that CDI molecules act as restriction elements in the presentation of non-peptide antigens to specialized subsets of T cells that lack expression of CD4 or CD8, co-receptor molecules for MHC class I and class II (Porcelli et al., 1989, 1992; Bendelac. 1995). These double negative (DN) T cells may express either the cup (Beckman et al., 1994; Dellabona et al., 1993) or the y6 (Faure et al., 1990: Brenner et al., 1987) T cell receptor (TCR). This finding has generated

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of Neuroinwwnology 67 llY96) 145-151

Table 1 Multiule sclerosis cases Case no.

Chr. prog.

Age/Gender

Duration (yr)

Diagnosis

F/34 M/69 F/38 M/32 F/31 F/37 F/55 F/59 F/56

2 10 10 10 I1 17 20 35 40

Chr. Chr. Chr. Chr. Chr. Chr. Chr. Chr. Chr.

= chronic progressive;

Bronchopneu.

prog. prog. prog. prog. prog. prog. prog. prog. prog.

MS MS MS MS MS MS MS MS MS

= bronchopneumonia;

renewed interest in the distribution of CD1 proteins in a variety of pathological conditions. In this study, we have used immunocytochemistry to determine the distribution of CD1 proteins in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), in which cytokines such as GM-CSF have been shown to be widely disseminated (Lee et al., 1994).

2. Materials and methods Immunocytochemical studies were performed on brain tissue obtained at autopsy. Nine patients with MS, one patient with amyotrophic lateral sclerosis (49-year-old female), one stroke patient (80-year-old female), one patient with olivopontine cerebellar degeneration (3 1-year-old male), one patient with Wilson’s disease (49-year-old male), and one non-neurological disease control (metastatic cancer, 80-year-old female) were studied. For the MS tissues, frozen sections of seven chronic-active lesions, seven chronic-silent lesions and adjacent normal appearing white matter were examined. Details of the patient population are shown in Table 1. Lesions were characterized as chronic-active if they had a relatively sharp edge to the area of demyelination, along which a band of inflammatory activity occurred. The centers of such lesions were usually homogeneously demyelinated and intensely gliotic.

Lesion type

Cause of death

Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic Chronic

Cachexia Bronchopneu./adenocarcinoma Bronchopneu. UTI/septicemia Resp. failure Resp. failure Sepsis Bronchopneu. Endometrialcarcinoma

active silent active + Chronic silent silent active + Chronic silent active silent silent silent

UTI = urinary tract infection

Chronic-silent lesions comprised areas of chronically demyelinated, fibrous astrogliotic white matter. The margins of the lesions were usually distinct and slightly hypercellular due to oligodendroglial hyperplasia and mild astroglial hypertrophy. Hematogenous infiltrates were rare or absent (Cannella and Raine, 1995). Sections were air-dried and post-fixed for 10 min in ice-cold acetone. They were blocked for endogenous peroxidatic activity followed by incubation for 1 h in 2% normal goat serum or in 10% normal rabbit serum for monoclonal antibodies of the IgG,, class (Ulvestad et al., 1994a). For identification of immunoreactivity to CDl, sections were incubated with monoclonal antibodies to CDla (anti-Leu 6, IgGZb, 150, Becton Dickinson, San Jose, CA), CDlb (clone K5-lB8, IgG, , affinity-purified 150, Ancell Corporation, Bayport, MN, and IgG,, clone # 4.A7.6, Serotec, Harlan Bioproducts for Science, Indianapolis, IN), or CDlc (gift of Dr. B.R. Bloom, Albert Einstein College of Medicine, Bronx, NY) were used. An antibody to GM-CSF was purchased from Genzyme (IgG,, I : 100, Genzyme, Cambridge, MA). Astrocytes were identified using an antibody to glial fibrillary acidic protein (GFAP; 1:lOO; IgG,, DAKO, Carpinteria, CA). Microglia were identified using antibody EBM- 11 (1:25, mAb, IgG,, DAKO). Class II MHC molecules were visualized using the antibody LN3 (IgG,,, ICN Immunochemicals, Costa Mesa, CA). For negative controls, sections were incubated with the same concentration of irrele-

Fig. 1. Chronic-active MS lesions immunoreacted for CD1 b (mAb IgG,, red) and counterstained with hematoxylin. (A) Low-power field across the edge of the lesion showing a blood vessel towards the lesion center (top) surrounded by perivascular infiltrating cells some of which are CDlb-positive. At the edge of the lesion (lower left), CDlb-positive hypertrophic astrocytes are present (arrows). X 110. (B) A section of a chronic-active MS lesion from another case is taken across the lesion edge with the lesion center to the upper left. Note the intense CDlb reactivity around the blood vessel (asterisk) and on the numerous hypertrophic astrocytes (arrows) along the lesion edge, X 110. (C) Detail of another inflamed blood vessel from the same case as in (A) to show the intense CDlb reactivity (red) on the perivascular macrophages, X480. (D) Detail of the blood vessel (in B, asterisk) depicting the CDlb immunoreactive perivascular macrophages, X 750. (E) Higher magnification of CDlb-positive hypertrophic astrocytes (red, arrows) from the same case as illustrated in (A). Note the attenuated astrocyte processes and the membrane staining for CDlb, X 480. (F) Isotype-matched IgG,-negative control to show lack of immunoreactivity around the same vessel as shown in (B), X480. (G) Isotype-matched IgG,-positive control with an antibody against MBP. The center of this chronic-active MS lesion, same as in (A), shows MBP-positive macrophages (red) around the blood vessel and throughout the demyelinated parenchyma, X 110. (H) Detail of MBP-positive lipid-laden macrophages from (G), X 750.

L. Battistini et al. /Journal

of Neuroimmunolog~

vant isotype-matched mouse immunoglobulins. For positive controls, an antibody to myelin basic protein (MBP) was used for mouse monoclonal antibodies of the IgG, subclass (Boehringer Mannheim, Indianapolis, IN) and to

67 (1996) 145-151

147

smooth muscle actin for mAb of the IgG2, subclass (DAKO). All primary antibodies were incubated overnight at +4”C. After washing, isotype-specific secondary antibodies coupled to either horseradish peroxidase or alkaline

148

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phatase were used as secondary reagents and develphosl (Sigma, St. Louis, MO), oped with 3,3’-diaminobenzidine nicke :I-enhanced DAB (Pierce, Rockford, IL) or hexazo-

67 (1996) 345-151

tized triaminotrimethyltriphenylmethane (HistoMark R 4 Kirkegaard and Perry, Gaithersburg, MD) as chromage :ns. For double-staining 3,3’-diaminobenzidine and nitrobluc :te-

L. Battistini et al. /Journal

trazolium gens.

(Boehringer-Mannheim)

of Nruroimmunology

were used as chroma-

3. Results and discussion In chronic-active MS lesions, immunoreactivity for CDlb was prominent on perivascular inflammatory cells within the lesion center and around vessels in adjacent white matter (Fig. lA-D). At the lesion edge, hypertrophic astrocytes also showed prominent immunoreactivity on both the cell body and cell processes (Fig. 1A, B and El. In contrast, immunoreactivity for CDlb was negligible to non-existent in adjacent normal appearing white matter. In parallel sections reacted with isotype-matched irrelevant control antibodies, no immunoreactivity was observed at these same sites (Fig. 1F). Staining of the same chronic-active MS lesions with an isotype-matched antibody to MBP gave intense immunoreactivity with debris-laden foamy macrophages within the lesion center (Fig. 1G and H). With the IgG,, mAb to CDlb, immunoreactivity was initially observed on microglia. However, as previously reported (Van de Winkle and Anderson, 199 1; Ulvestad et al., 1994a,b), monoclonal antibodies of this subclass have a propensity to bind to human Fc receptors, a binding pattern that can be abrogated by preincubation of the sections with 10% normal rabbit serum (Ulvestad et al., 1994a). Following this blocking procedure, all of the immunoreactivity on microglia was lost. Thus, when the results with the two antibodies tested were compared, immunoreactivity for CD 1b was found only on perivascular inflammatory cells and hypertrophic astrocytes. In chronic silent lesions, staining for CD1 b was much reduced and only an occasional astrocytic foot process and perivascular macrophage showed positive reactivity (Fig. 2A and B). No staining of the astrocyte cell body, or of associated endothelial cells, was detected. Three chronic-active MS lesions and three chronic-silent MS lesions were also tested for the expression of CDla and CD lc. Immunoreactivity for both of these molecules was rarely detected and only an occasional perivascular macrophage showed positive staining (Fig. 2C and D). Since GM-CSF has been found to be the principal cytokine involved in upregulation of CDlb in cells of the monocyte/macrophage series, the sections were stained for GM-CSF. As reported previously (Lee et al., 1994), positive staining for GM-CSF was detected in reactive

67 (lY961 145-151

149

astrocytes, particularly hypertrophic astrocytes in the lesion center and edge of actively demyelinating MS lesions (Fig. 2E). Normal appearing areas of CNS tissues from MS brains, and normal white matter from non-neurological cases, showed no immunoreactivity for GM-CSF (data not shown). Double-staining with antibodies to CDlb and GM-CSF was occasionally detected at the lesion edge in hypertrophic astrocytes, a staining pattern that was confirmed with double-staining for CD1 b and GFAP (data not shown). The same tissues were also stained with antibodies to class II MHC. In actively demyelinating MS lesions, reactivity for HLA-DR was abundant on microglia (Fig. 2F), perivascular macrophages and phagocytic macrophages in agreement with previous reports (Hauser et al., 1983; Hayes et al., 1987; Lee et al., 1990; Bo et al., 1994). No staining was noted on astrocytes or endothelial cells. In addition, all of the human tissues studied showed a low level reactivity for HLA-DR on microglia, a finding also consistent with previous reports of constitutive expression of these molecules on microglia, particularly in white matter (Hayes et al., 1987; Mattiace et al., 1990). The identification of astrocytes and microglia was confirmed by positive reactivity to anti-GFAP and EBM-11 antibodies, respectively (Fig. 2G or H). The results of this study show, therefore, that positive immunoreactivity for CD1 b is present in actively demyelinating MS lesions whereas immunoreactivity for this protein is essentially absent in normal autopsy brain tissue. CDlb on perivascular cells associated with the lesions could, therefore, function as antigen-presenting molecules to specialized subsets of T cells lacking the coreceptors CD4 or CD8 (Porcelli, 1995). Since these T cells have been implicated in the response to non-peptide antigens, the results further suggest that the spectrum of antigens to which T cells may respond in MS lesions could be greater than previously thought. Alternatively, induced expression of CD1 b on inflammatory cells could function as part of an immunoregulatory process in the lesions since these molecules have been shown to function as restriction elements in the activation of T cells expressing NKl.1 (McDonald, 1995). Interestingly, CDlb could also be identified on a subset of astrocytes at the lesion edge whereas astrocytes outside of the lesion were generally non-reactive. These data suggest that products of inflammatory cells are pivotal for the induction of CDlb on astrocytes. In studies with blood monocytes and tissue macrophages, it has now been well

Fig. 2. (A) and (B) Chronic-silent MS lesions immunoreacted for CDlb. Note the reaction product on astrocytic endfeet around the blood vessels, X 240. (C) A blood vessel located in the white matter adjacent to a chronic-active MS lesion shows a few perivascular macrophages that are positive for CD1 a, X 110. (D) Same case as in (C). This blood vessel outside a chronic-active MS lesion displays two CDlc-positive macrophages within the Virchow-Robin space. The lesion itself was non-reactive, X 110. (E) A chronic-active MS lesion shows GM-CSF reactivity on hypertrophic astrocytes within the lesion center, X 480. (F) An area adjacent to a chronic-active MS lesion shows I-LA-DR reactivity on microglia, X 480. (G) Astrocytes in the same lesion as in (E) are stained for GFAP, X 480. (H) Microglia from the same lesion as in (F) are reacted with EBM-11 and display intense immunoreactivity, X 480.

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established that these cells normally express low to nondetectable levels of CD1 molecules but high expression follows exposure to cytokines such as GM-CSF and IL-3 (Porcelli et al., 1992; Kasinrerk et al., 1993 and Porcelli, 1995). Other cytokines that are active in inducing class I and class II MHC molecules, such as IFNy, do not induce CD1 molecules and may suppress GM-CSF-induced expression, suggesting that these two groups of molecules are reciprocally regulated (Porcelli, 1995). In this regard, in the MS lesions, CD1 b and MHC class II were found on distinct populations of glial cells. Upregulation of CD1 b has been noted in a number of inflammatory and infectious conditions including expression on dendritic cells in the kidney in association with the development of glomerulonephritis (Cuzic et al., 19921, in skin lesions of patients with the tuberculoid form of leprosy (Modlin et al., 19831, and in various tumors of probable dendritic cell etiology (Cattoretti et al., 1987). Our observations in MS, therefore, add to the growing list of conditions in which a specific member of this group, CD1 b, is upregulated in the target tissue. Sequence analysis has shown that CD1 proteins are non-polymorphic with little or no allelic diversity and are likely to bind hydrophobic ligands. These observations have led to the hypothesis that CD1 molecules might function to present antigen to DN T cells whose TCRs express conserved or ‘canonical’ sequences. In studies that have examined T cell clones or lines derived following antigen selection of PBMC, both CD1 b- and CDlc-restricted DN T cells reactive to mycobacterial sonicates of either M. tuberculosis or M. leprae have now been demonstrated. Interestingly, for one of the CD 1b-restricted T cell lines, the antigen recognized was the mycobacterial lipid, mycolic acid (Beckman et al., 1994), and for two other DN op TCR+ T cell lines restricted by CDlb or CDlc, the antigen was the glycolipid lipoarabinomanaam from M. leprae (Sieling et al., 1995). These studies add to the accumulating evidence that T cells respond to nonpeptide ligands (Tanaka et al., 1994; Constant et al., 1994) and suggest that the CD1 family of molecules may have evolved to function as antigen-presenting molecules for these ligands to specific subsets of T cells (Porcelli, 1995; Bendelac, 1995). These lipid antigens are found only in certain bacteria, thus it is not clear what relevance these observations may have to the pathogenesis of MS at the present time. Nevertheless, it is intriguing to note that lipids, particularly glycolipids, are a major component of the myelin sheath, the target of immunological destruction in the MS lesion. Staining for the closely-related molecules CDla and CDlc was barely detectable in the same tissues. cDNA probes have shown that the CDla, b,and c proteins are encoded by distinct genes (Seed and Aruffo, 19871, and following transfection of COS cells, a hierarchy of exclusion has been observed in which CDlc excludes the expression of both CDla and b, and CDlb excludes the

67 119961 145-151

expression of CDla (Aruffo and Seed, 1989). The absence of Cdl a expression on CNS glial cells is in agreement with the results of Hauser et al. (1983) and Ulvestad et al. (1994a). Thus of this family of glycoproteins, only CD1 b appears to be significantly upregulated in MS lesions.

Acknowledgements The authors would like to thank Dr. Jack Ante1 of the Montreal Neurological Institute for functioning as the handling editor for this manuscript. Our thanks are also extended to E. Swanson for the preparation of frozen sections. Supported in part by USPHS Grants NS 11920 and NS 08952, and RG 1001-H-8 and FA 1095 from the National Multiple Sclerosis Society, the New York Community Trust and the Gladstein Foundation; and by grants from the Italian Multiple Sclerosis Society (AISM). Falko Fischer was a participant in the Biomedical Sciences Exchange Program and was partially supported by a grant from the German Academic Exchange Service.

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