Is there a role for NK cells in the pathogenesis of multiple sclerosis? A case study

Is There a Role for NK Cells in the Pathogenesis of Multiple Sclerosis? A Case Study Gilberto Filaci, Rosa Bacchetta, and Maurizio Zanetti ABSTRACT: We report on a patient with multiple sclerosis (MS) in which we documented an elevated percentage of activated CD561 natural killer (NK) cells in peripheral blood lymphocytes. NK cells from the patient lysed preferentially glioblastoma but not neuroblastoma cells. Killing of glial cells was not inhibited by a monoclonal antibody against a monomorphic determinant of MHC class I gene products. Lymphokine activated killer (LAK) cell function in the MS patient was comparable to that of controls. Analysis of cytokine production during resting or activated states demonstrated that this patient

ABBREVIATIONS MS multiple sclerosis NK natural killer LAK lymphokine activated killer cells MHC Major Histocompatibility Complex IFNg interferon gamma GM-CSF granulocyte-macrophage colonystimulating factor TNFa tumor necrosis factor alpha CNS central nervous system MBP myelin basic protein

INTRODUCTION Natural killer (NK) cells lyse tumor and virus-infected cells without prior sensitization or restriction by major histocompatibility complex (MHC) products. In addition to their cytotoxic activity, NK cells secrete inflammatory lymphokines such as interferon gamma (IFNg) granulocyte-macrophage colony-stimulating factor (GM-

From the Department of Internal Medicine, University of Genoa, Genoa, Italy (G.F.), TIGET, San Raffaele Hospital, Milano, Italy (R.B.), and Department of Medicine and Cancer Center, University of California at San Diego, La Jolla, California, USA (M.Z.). Address reprint requests to: Gilberto Filaci, Department of Internal Medicine, University of Genoa, Viale Benedetto XV n. 6, 16132—Genoa, Italy; Tel: 390-10-3537988; Fax: 390-10-3538994; E-Mail: Frindi @unige.it. Received November 16, 1998; accepted December 4, 1998. Human Immunology 60, 231–238 (1999) © American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc.

had a deficit in the ability to secrete T cell derived cytokines associated with increased production of TNFa, a product of NK cells. Taken together, these data indicate a possible involvement of NK cells in the pathogenesis of MS. Human Immunology 60, 231–238 (1999). © American Society for Histocompatibility and Immunogenetics, 1999. Published by Elsevier Science Inc. KEYWORDS: multiple sclerosis; natural killer cells; TNF

PLP MOG MAG TRC mAb PBL IL-2 IL-10 PHA TPA EAE

proteolipid protein myelin oligodendrocyte glycoprotein myelin-associated glycoprotein T cell receptor monoclonal antibody perpheral blood leukocytes interleukin 2 interleukin 10 phytohemagglutinin 12-O-tetradecanoylphorbol-13-acetate experimental allergic encephalitis

CSF) and tumor necrosis factor alpha (TNFa) [1]. NK cell activity has been found altered in many autoimmune diseases. A significant decrease in the number of NK cells and NK lytic activity has been described in patients with systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome, ankylosing spondylitis and progressive systemic sclerosis [reviewed in 2]. Because NK cells have suppressive effect on B cell differentiation and antibody synthesis [3] it has been proposed that an impaired NK cell number or function may contribute to polyclonal B cell activation and autoantibodies production [4]. On the other hand, a possible role for NK cells and their cytokines in the amplification of the chronic inflammatory damage sustained by autoreactive T cells or autoantibodies is unclear. 0198-8859/99/$–see front matter PII S0198-8859(98)00121-9

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Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by a prominent lymphocyte and macrophage infiltration into the white matter, and by demyelination. This pathology is associated with neurological dysfunctions like paralysis, sensory deficit and visual problems. T cells reactive with several myelin antigens, such as myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG) have been found in MS patients [5–9] suggesting that autoreactive T cells may be involved in the pathogenesis of the disease. However, oligoclonality in the T cell response to MBP is restricted to single patients and no restriction in T ceel receptor (TCR) Va or Vb chains usage has been detected among different patients [7, 10 –12]. Moreover, MBP-reactive T cells exist in patients with other neurological diseases and even in healthy individuals [13–16]. On the basis of these considerations, the pathogenic role of T cells reactive with MBP is still controversial. Here, we report on a patient with MS displaying a high number of activated NK cells in the peripheral blood and a preferential NK-mediated cytotoxic activity against glial cells. The possible role of NK cells in sustaining and/or amplifying the inflammatory damage of the CNS in relapsing MS is discussed. SUBJECTS, MATERIALS AND METHODS Subjects Patient CM is a 46-year-old male Caucasian with intermittent paresthesias and sight impairment since June 1989. A diagnosis of MS was made in December 1990. Between June 1991 and May 1992 he had relapses at monthly frequency. Our study is based on the analysis performed between February and May 1992. During this period, the patient experienced a progressive deterioration of sensitive, motile and visceral functions with loss of vibratory sensitivity and ataxia, migrating pain, weakness, stiffness, hyperreflexia and both bladder and bowel incontinency. An HLA typing indicated that patient CM is HLA-A 1,3; HLA-B 7,8; HLA-C w7, w8; HLA-DR 3, 14, w52; HLA-DQ w2, w6. A normal individual of the same age and sex of patient CM was selected as control after that his phenotypic and functional lymphocyte parameters were compared with and found representative of those of the healthy population. Antibodies The murine monoclonal antibody (mAb) to CD56 antigen was supplied by Becton & Dickinson (Mountain View, California). Goat antibody to mouse Ig conjugated with fluorescein-isothiocyanate (FITC) was purchased

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from Biomeda (Foster City, CA). Murine mAb W6/32 to human MHC class I antigens was a gift of Dr. F. Indiveri (University of Genoa, Italy). g1(RGD)3 antigenized mAb was produced as described [17]. Isolation of Nonadherent Peripheral Blood Leukocytes (PBL) PBL were isolated from heparinized peripheral blood by Ficoll-Hypaque gradient centrifugation. Cells were washed with isotonic saline solution, resuspended at a concentration of 2 3 106 cells/ml in RPMI 1640 culture medium supplemented with 4 mM glutamine, 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin, and incubated in culture flasks for 2 hours at 37°C in a 5% CO2 atmosphere to deplete adherent monocytes. The medium containing nonadherent PBL was removed and cells were collected by centrifugation. Cell Lines Human erytroleukemia cells K562, neuroblastoma cells SK-N-SH and glioblastoma cells U87 were obtained from the American Tissue Cell Collection (ATCC). Human glioblastoma cells LNZ 308 were obtained from Dr. W. Cavenee (Ludwig Institute, University of California, San Diego). Human medulloblastoma Daoy 324 was obtained from Dr. A. L. Yu (University of California, San Diego). Cells were grown in DMEM supplemented with 4 mM glutamine, 0.1 mM non essential aminoacids, 1 mM sodium pyruvate, 10 mM Hepes, 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin, and were harvested by centrifugation. Fluorescence-Activated Cell Sorter Analysis Non adherent PBL were suspended (106 in 200 ml) in DMEM supplemented with 10 mM Hepes, 0.02% sodium azide and 1% bovin serum albumin, and incubated for 1 h at 4°C with the phycoeritrin conjugated murine mAb to human CD56 (1 mg/ml). The cells were then washed, resuspended in 200 ml of 1% paraformaldehyde in phosphate-buffered solution and analysed with an Ortho Cytofluorograph Iis. A gate was established in the forward vs. side light scatter in order to exclude monocytes and granulocytes, dead cells, debris and doublets for studies on PBL. In some experiments cells were incubated with the unconjugated mAb to human CD56 or with the unconjugated mAb to human CD3, washed and incubated for an additional period of 1 hour at 4°C with a FITC-conjugate goat antibody to mouse Ig (1: 100). NK and LAK Cell Activity NK cell lytic activity of nonadherent PBL was tested in a 4 h 51Cr release assay. Tumor cells K562, SK-N-SH, LNZ-308, Daoy and U87 were labelled with 150 mCi of

NK Cell Activity and Multiple Sclerosis

Na51CrO4 per 1 3 106 cells for 1 h at 37°C in a 5% CO2 atmosphere, then washed, resuspended in culture medium and plated in a 96-well flat bottomed microtiter plate at a concentration of 5 3 103 cells/well in 100 ml volume. 100 ml of culture medium containing nonadherent PBL were then plated at an effector:target (E:T) ratio of 50:1. The plate was incubated for 4 h at 37°C in 5% CO2, then centrifuged at 1000 g for 5 min. 100 ml of supernatants were removed and the radioactivity counted in a gamma counter. Spontaneous and maximum 51Cr release were determined by incubating target cells in medium alone or in the presence of 1% Tryton X-100, respectively. The cytotoxic activity was calculated from triplicate wells as follows: percent cytotoxicity 5 (experimental release 2 spontaneous release) / (maximum release 2 spontaneous release) 3 100. In some experiments the cytotoxic assay was performed in the presence of W6/32 mAb at a final concentration of 10 mg/ml or of gl(RGD)3 (100 mg/ml)l. Lymphokineactivated killer (LAK) cells were generated by incubating nonadherent PBL (2 3 106/ml) with 400 U/ml of recombinant interleukin 2 (IL-2) for 4 days at 37°C. Their cytotoxic activity was tested as specified for NK cell activity. In Vitro Cytokine Production and Quantitation PBL (1 3 106 cells/ml) were cultured in 24-well plates in Yssel’s medium [18] with or without phytohemagglutinin (PHA) (1 mg/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA) (1 ng/ml) plus a mAb to CD3 antigen (0.1 mg/ml). Supernatants were harvested after 48 h and stored at 220°C until tested for their cytokine content. IL-2, IFNg, IL-10, GM-CSF and TNFa were quantified by ELISA, as described [19]. The sensitivity of the ELISA assays was 50 pg/ml for TNFa and IL-10, 40 pg/ml for IFNg and 20 pg/ml for IL-2 and GM-CSF.

RESULTS Elevated Expression of CD56 Antigen in PBL from Patient CM The number of CD561 cells and the level of surface expression of the CD56 molecule were determined by cytofluorimetric analysis on PBL from patient CM and the healthy control. The results shown in Table 1 refer to analyses performed over a period of 3 consecutive months. The percentage of CD561 circulating lymphocytes and the linear mean fluorescence were higher in the patient’s PBL at each determination. Double staining analysis indicated that the great majority of CD561 lymphocytes did not express CD3 molecule (97%) and most of them were CD161 (80%), confirming the NK nature of the CD561 lymphocytes.

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TABLE 1 Augmented expression of CD56 on PBL of patient CM CD56 Expression Cell donor

% of positive cellsa

LMFb

3.2.92

Patient Control

28 16

39 13

3.13.92

Patient Control

29 16

36 8

4.30.92

Patient Control

20 7

31 7

5.21.92

Patient Control

45 11

33 18

Date

a

A gate was established in the forward vs. side light scatter in order to exclude non-lymphocyte cells (namely monocytes and granulocytes), dead cells, debris and doublets. b Linear mean fluorescence.

NK Cell Cytotoxicity and Preferential Lysis of Glial Cells Nonadherent PBL from patient CM were tested for their ability to lyse a number of human target cells including erytroleukemia cells K562, neuroblastoma cells SK-NSH, glioblastoma cells LNZ-308 and U87, and medulloblastoma cells Daoy 324. The assays were performed during relapses as well as remissions of the disease without significant differences in the pattern of cytotoxicity. The results of three representative experiments (performed in a follow up period of 5 months) are shown in Table 2 and indicate that lysis of K562 cells, a conventional target, was similar for patient’s and control’s cells whereas lysis of glioblastoma and medulloblastoma cells were markedly and consistently higher using patient’s cells. The fact that the lysis of glioblastoma cells LNZ308 by control PBL was marginal together with the fact that both patient’s and control’s cells lysed K562 cells similarly, prompted a study on LAK cells. When LAK cells were generated from the patient’s and control’s PBL, significant lysis of glioblastoma cells and increased lysis of K562 cells were obtained in both instances (Fig. 1). Lysis of Glial Cells is Not Restricted by MHC Class I Molecules and is Inhibited by gl(RGD)3 Because the glioblastoma and medulloblastoma cells used in these experiments expressed elevated levels of MHC class I antigens (not shown), it was important to exclude lysis by CD81 T cells. To this end, the cytotoxic assay was repeated in the presence of mAb W6/32 which

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TABLE 2 Preferential lysis of glial cells by patient CM

inhibition) by patient PBL. These results confirm, therefore, that lysis of glioblastoma cells is not MHC-restricted and is due to NK cells.

Target cells Exp # Cell donor K562 SK-N-SH LNZ 308 U87 DAOY 324 1

Patient Control

38a 35

2

Patient Control

60 45

39 13

3

Patient Control

29 25

22 6

1 0

20 6

18 1

17 3

a

The values are expressed as percent cytotoxicity. The experiments were conducted at an effector:target ratio of 50:1. Maximum (Max) and spontaneous (Min) 51Cr release from each target during experiment #1 were as follows: K562 SK-N-SH LNZ 308 U87 DAOY 324 (cpm6SD) (cpm6SD) (cpm6SD) (cpm6SD) (cpm6SD) Max 66026405

3074663

31856622

70926175

52916255

Min 520636 425660 37866147 886624 705644 Cpm and Max/Min ratios in experiments #2 and #3 were comparable.

is directed against a monomorphic determinant of human MHC class I molecules. Results presented in Fig. 2 indicate that the addition of anti-MHC class I antibody did not affect lysis. Accordingly, gl(RGD)3 mAb, an antigenized antibody which inhibits specifically NK cell activity [17], inhibited lysis of glial targets (57% of

FIGURE 1 LAK cell activity against erytroleukemia K562 and glioblastoma LNZ 308 cells in patient CM and control. The experiment was conducted at an effector: target ratio of 50:1. Maximum and spontaneous 51 Cr releases were 13105 6 163 and 3780 6 144 for K562 cells and 17782 6 1014 and 4917 6 411 for LNZ 308 cells, respectively.

Cytokine Production by Nonadherent PBL To verify whether the observed abnormality in NK cell number and function in patient CM was associated with abnormalities in cytokine secretion, studies were undertaken to measure the concentration of IL2, IL-10, IFNg, GM-CSF and TNFa in the supernatant of nonadherent PBL unstimulated or after activation with PHA or TPA plus an anti-CD3 mAb. Two normal donors served as controls. The results shown in Table 3 demonstrate a decreased secretion of IL-2, IL-10 and IFNg associated with a normal or increased release of GM-CSF and TNFa from patient’s cells. Unstimulated PBL from the patient produced two to threefold higher levels of TNFa compared to control PBL. Following activation, NK cellderived cytokines increased comparably to controls. Secretion of T cell-derived cytokines was consistently reduced whether cells were resting or activated. Taken together these data indicate that patient’s PBL have an augmented NK secretory function combined with an impaired secretion of T cell-derived cytokines. DISCUSSION MS is an acquired demyelinating disease of the CNS of unknown etio-pathogenesis. The involvement of the im-

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235

FIGURE NK cell activity of CM patient and control against erythroleukemia K562 and glioblastoma LNZ 308 cells in the presence of the W6/32 mAb specific for HLA class I antigens. The experiment was conducted at an effector:target ratio of 50:1. Maximum and spontaneous 51 Cr releases were 10319 6 327 and 2337 6 38 for K562 cells and 9337 6 362 and 1789 6 171 for LNZ 308 cells, respectively.

mune system is suggested by the fact that lymphocyte infiltrates are always present in the proximity of the areas of demyelination [20]. Both T and B lymphocyte subpopulations have been identified in the infiltrates [21] while the presence of NK cells has not been investigated. We observed an elevated percentage of circulating NK cells in a patient with MS. These cells showed markedly high density of CD56 antigen, a marker of NK cells, whose expression raises upon cell activation [22]. When functional analysis was performed against K562 cells, no differences were detected between the lytic activity of patient’s and control’s PBL. In contrast, glioblastoma and medulloblastoma cells were lysed markedly more by patient’s NK cells than control. The finding was consistent during the four months observation period. No meaningful variations were observed between remission and relapsing phases of the disease. Recently, the expression of CD56 antigen by a subpopulation of CD41 T cells has been related to their capacity to lyse in MHC unrestricted manner glial tumor cells [23]. On this basis, it is tempting to speculate that the presence in the patient CM of an elevated number of NK cells expressing CD56 at high density could be cytotoxic against autologous glial cells. In agreement with this hypothesis NK cells from patient CM, but not those of healthy controls, were able to lyse in vitro glial tumor cells. To corroborate our hypothesis is the recent observation on the existence of a positive correlation between

the degree of NK cell function and the number of active lesions detected by magnetic resonance imaging in patients with relapsing/remitting MS [24]. The finding of elevated NK but not LAK cell activity cell activity against relevant neurological targets in the PBL from the MS patient suggests that in the patient CM NK cells were present in an activation status. The reasons for activation of NK cells in the patient CM remain unclear. These could involve genetic background and/or environmental stimulators such as cytokines or recognition of activating signals on target cells. The latter possibility is of particular interest since in the last years both activating and inhibitory receptors have been identified at the surface of NK cells [25]. Furthermore, the existence of different NK cell repertoires among individuals has been demonstrated studying patterns of alloantigen recognition by different NK cell clones [26, 27]. It is possible that in patient CM NK cells were activated and killed preferentially selected (glial) targets perhaps due to the recognition of a modified phenotypic pattern on their membrane modified by an external agent such as a virus. Activation of NK cells also induces the release of cytokines. Accordingly, PBL of patient CM showed increased spontaneous production of TNFa, a cytokine considered to play an important role in the induction of pathology in the CNS of MS patients. Several arguments

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TABLE 3 Preferential lysis of glial cells by patient CM Cytokines Cell donor

Cell treatment

IL-2 a

IFNg

IL-10

GM-CSF

TNFa

Patient

None PHA TPA 1 anti-CD3

35 52 108

0 2,820 0

507 1,175 0

29 494 675

450 2,500 700

Control 1

None PHA TPA 1 anti-CD3

39 559 3,546

0 11,073 1,251

515 2,174 51

0 606 514

180 2,200 700

Control 2

None PHA TPA 1 anti-CD3

56 719 6,730

0 20,000 3,776

415 6,044 113

0 402 492

150 1,600 800

a

The values are expressed as pg/ml.

suggest that TNFa could be implicated in the pathogenesis of MS: 1. TNFa causes gliosis of oligodendrocytes in areas of demyelination [28 –30]; 2. elevated serum concentrations of TNFa [31] and TNFa-receptor [32] precede relapses of MS and correlate with blood-brain damage in patients with active disease [33, 34]; 3. TNFa has been identified in active acute and chronic lesions but not in silent chronic lesions [35]; 4. T cell clones derived from the CSF of MS patients produce significantly higher amounts of TNFa than control clones [36]; 5. inhibitors of TNFa, like phosphatidylserine [37] or antibodies to TNFa [38], prevent autoimmune demyelination. The demyelinating lesions of MS resemble the lesions that occur in experimental allergic encephalitis (EAE), the rodent model of the human disease. In EAE, T lymphocytes recognizing MBP (or other myelin antigens) are responsible for the onset of the disease [39, 40]. Characteristically, these cells show a highly restricted pattern of the T cell receptor (TCR) Va and Vb gene products [41– 43]. T lymphocytes from PBL and cerebral spinal fluid (CSF) reactive with several myelin antigens have been identified in MS patients [5–9]. However, studies on the TCR Va and Vb gene rearrangement failed to demonstrate the existence of restricted gene usage in these autoreactive T lymphocytes when different patients were considered. Oligoclonality in TCR V gene utilization was only demonstrated at the single patient level [7, 10 –12]. Accordingly, the treatment of MS patients by oral MBP administration to induce tolerance has met little if any success [44, 45]. Interestingly, it has

been shown that CSF cells from MS patients transferred by intracisternal injection into SCID mice led to paralysis and ataxia in 6 weeks with pathological lesions (demyelination and gliosis) clearly resembling those of MS 46. No lymphocytes but only macrophages and granulocytes were detected in the areas of lesions. These observations suggest that the cerebral pathological changes in MS may be triggered and/or sustained by pro-inflammatory cells and release of factors without restriction to a particular cell type (i.e., lymphocytes). Collectively, our data support the view that the pathogenesis of CNS lesions in MS is different and more complex than EAE. Although a role for autoreactive T cells can not be ruled out, other cells, including NK cells, may be involved in the pathogenesis due to their ability to induce glial damage. It is possible that the variability of clinical behaviour and progression in different MS patients could reflect the multiplicity of pathogenic mechanisms and agents able to determine the pathological lesions typical of the disease. In this sense, MS might be regarded as a syndrome, rather than a single disease, in which demyelination is the common clinical pathologic manifestation and various cell types including NK may play a pathogenic role.

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