Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein, and myelin basic protein in multiple sclerosis

Journal of Neuroimmunology, 13 (1986) 99-108

99

Elsevier JNI 00419

Cell-Mediated Immunity to Myelin-Associated Glycoprotein, Proteolipid Protein, and Myelin Basic Protein in Multiple Sclerosis David Johnson 1 David A. Hafler 1, Robert J. Fallis 1 Marjorie B. Lees 2 Roscoe O. Brady 3 Richard H. Quarles 3 and Howard L. Weiner 1 i Multiple Sclerosis Unit, Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 2 Biochemistry Department, E.K. Shriver Center, Waltham, MA, and ~ Section on Myelin and Brain Development, NINCDS, NIH, Bethesda, MD (U.S.A.)

(Received 24 February, 1986) (Revised, received 5 May, 1986) (Accepted 5 May, 1986)

Summary Peripheral blood lymphocytes (PBL) from active and stable multiple sclerosis (MS) patients, patients with other neurologic diseases (OND), and control subjects were tested for sensitization to two myelin antigens not previously examined in multiple sclerosis, using a [3H]thymidine incorporation assay. The antigens investigated were myelin-associated glycoprotein (MAG) and proteolipid protein (PLP). In addition, sensitization to myelin basic protein (MBP) was also tested. Lymphocyte stimulation indices in active MS patients that were greater than 2 standard deviations above controls were as follows: 9 / 3 0 for MAG, 0 / 1 7 for PLP, and 8/81 for MBP. No control subjects responded to M A G or PLP, and only 1 / 2 9 control subjects responded to MBP. Three of the patients that responded to M A G also responded to MBP. Although the mean proliferative response to M A G and to MBP was greater in the population of active MS patients than in stable MS, ONDs, or controls, the difference was not statistically significant. The O N D group was the only population which proliferated to PLP (6/16). The only statistically significant differences among the groups for all myelin antigens tested were the proportion of individuals with active MS vs. controls that responded to M A G ( P < 0.05), and O N D vs. controls and active MS that responded to PLP ( P < 0.025). The greatest

Address reprint requests to Dr. Johnson, Center for Neurologic Diseases, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, U.S.A. 0165-5728/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

100 individual responses to the three antigens tested were to MBP in active MS patients. Elimination of the T8 (cytotoxic/suppressor) subset amplified the responses to myelin antigens in some patients and ONDs studied. These studies have demonstrated reactivity to MAG but not PLP in some patients with active MS, and reactivity to PLP in some patients with other neurologic diseases. Key words: Immunity, cell-mediated Myelin-associated glycoprotein protein - Myelin basic' protein - Multiple sclerosis

Proteolipid

Introduction

Although the etiology and pathogenesis of multiple sclerosis (MS) is unknown, the presence of a marked inflammatory response associated with demyelination has been viewed as evidence that MS may be a cell-mediated autoimmune disease (McFarlin and McFarland 1982; Waksman and Reynolds 1984). In addition to the presence of lymphocytes and macrophages in areas of demyelination (Prineas 1985), there have been reports of a variety of immune abnormalities in MS patients. Current theories propose that these immune abnormalities are somehow related to an immune attack on the myelin sheath or its supporting cell, the oligodendrocyte (McFarlin and McFarland 1982; Waksman and Reynolds 1984). The marked localization of lesions in central nervous system white matter has been viewed as strong evidence that the target of a putative autoimmune attack is a myelin-related antigen. Myelin basic protein (MBP) has been regarded as a prime candidate, primarily due to its highly antigenic nature and its ability to cause experimental allergic encephalomyelitis (EAE) in animals (Alvord et al. 1984). There have been several descriptions of humoral and cellular immunity to MBP in MS patients (Knight 1977; Eisak et al. 1984). This report describes the investigation of cell-mediated immunity in MS patients to two other well-defined white matter antigens, proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) that has not been previously reported. PLP is specific to central nervous system myelin, where it constitutes the major protein, comprising about 50% of total myelin protein (Lees and Brostoff 1984). MAG is a quantitatively minor constituent of both central nervous system and peripheral nervous system myelin (Quarles 1980) and is thought to be concentrated in the periaxonal region of the myelin sheath (Trapp and Quarles 1984).

Methods

Patients 103 patients with clinically definite multiple sclerosis (Rose et al. 1976) were studied from the out-patient Multiple Sclerosis Clinical and Research Unit at the Brigham and Women's Hospital. Patients were classified as being clinically active or

101 inactive. Active patients (n = 81) were either in an actively progressive stage, in which they had declined at least one grade on the Kurtzke disability scale or ambulation index (Hauser et al. 1983) in the past 9 months (n = 75), or in the midst of an acute exacerbation (n = 6). The mean duration of chronic progressive disease before blood sampling was 3.0 years, and the range on the Kurtzke disability status scale was between 3 and 7 with a mean of 5.6. Patients with inactive (stable) disease (n = 22, 14 females, 7 males, mean age 34) had been clinically stable for at least 9 months prior to sampling and had an identical range of Kurtzke disability scores (mean 5.1). The other neurologic disease patients (n = 20, 10 females, 10 males, mean age 40) included patients with seizures, meningitis, cerebrovascular accidents, transient global ischemia, brain tumors, dementia, and lower back pain. The control population consisted of healthy volunteers (n = 29, 14 females, 15 males, mean age 42).

Isolation of peripheral blood lymphocvtes Peripheral blood lymphocytes were isolated from heparinized venous blood by means of Ficoll-Hypaque gradient density centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ). Lymphocytes were cultured with or without antigen (5-10 fig/well) at a concentration of 2 x 106 cells/ml with RPMI 1640 media supplemented with 10% human AB serum (Pel Freez Biologicals, Rogers, AK) in a 96-well round-bottom microtiter plate (Linbro, Flow Laboratories, McLean, VA) in a humidified atmosphere with 5% CO 2. After 5 days, 1 ffCi of [3H]thymidine (New England Nuclear, specific activity 2 C i / m M ) was added to each well and cells harvested after 2 days of further incubation. Thymidine incorporation was measured by standard techniques (Bradley 1980). Each incubation was performed in quadruplicate and proliferation expressed as stimulation index (SI), which was defined as the radioactivity incorporated by lymphocytes in the presence of antigen divided by the radioactivity incorporated by lymphocytes in the absence of antigen. A positive response for an individual was defined as an SI greater than 2 standard deviations above the mean SI of lymphocytes of control subjects incubated with the same antigen. Such individuals were termed 'responders'. As shown in Table 1 (legend), background counts were comparable among groups studied. In terms of absolute counts, increases in stimulation indices in responders were due to an increase in the numerator and in some instances both an increase in the numerator and a decrease in the denominator. Lymphocytes were incubated with 25 /,tg of pokeweed mitogen (Sigma Chemical Co.) as a positive control for cell proliferation.

Subset lysis The T8 subset of lymphocytes was lysed by incubation of whole peripheral blood lymphocyte preparations with saturating concentrations (1:250) of anti-T8 monoclonal antibody for 15 rain at room temperature, followed by rabbit complement (Pel Freeze Biologicals, Rogers, AR) for 1 h at 37°C. These conditions had previously been determined to be optimal for T8 lysis by titration of antibody concentrations and there were less than 5% T8-positive cells in the T8-1ysed population, as measured by analysis on a fluorescence-activated cell sorter (Rein-

102 TABLE 1 P R O L I F E R A T I O N OF P E R I P H E R A L BLOOD L Y M P H O C Y T E S TO MY ELIN A N T I G E N S

Results are expressed as stimulation indices: cpm in presence of white matter antigen/cpm in media alone. Background counts (in the presence of media alone) were as follows: controls = 767 + 465 (SD), O N D = 707 ± 601, multiple sclerosis active 891 -+ 112, MS stable 6"/7 ± 316. Using Student's t-test, there were no statistically significant differences between the groups for the three antigens tested. n

Mean±SD

81 22 20 29

2.22_+ 2.99 1.30 _+0.62 1.41 _+ 1.2 1.28 ± 1.11

30 10 10 10

1.66 -+ 1.85 1.18-+0.50 1.31_+ 1.05 0.85 -+ 0.42

17 17 16 12

0.7 +0.27 0.85-+0.39 2.26-+3.9 0.65 ± 0.29

Basic protein Active MS Stable MS OND Control

Myelin-associated glycoprotein Active MS Stable MS OND Control

Proteolipid protein Active MS Stable MS OND Control

herz et al. 1979). Cells remaining after lysis were washed extensively and cultured with antigen as described above.

Preparation of antigens Basic protein was prepared from et al. (1972). Proteolipid apoprotein method of Lees and Sigura (1978). from human myelin as described by

human white matter by the method of Diebler was prepared from bovine white matter by the Myelin-associated glycoprotein was prepared Quarles et al. (1983).

Results The proliferative response of peripheral blood lymphocytes to myelin antigens is shown in Figs. 1 - 3 and is summarized in Table 1. The distribution of the proliferative response is shown in Figs. 1-3. It is evident from the figures that the majority of MS patients, both active and stable, had a similar proliferative response to controls, in that there was little or no response to any of the three myelin antigens tested. However, when subjects were considered individually, it is also clear that some patients were definite responders to these myelin antigens. For myelin basic protein, peripheral blood lymphocytes from eight of 81 active MS patients had a response greater than 2 standard deviations from controls compared to zero of 22 stable MS, two of 16 O N D s (stroke and oligodendroglioma), and one of 29 controls.

103 MYELIN

BASIC PROTEIN

'5I 14

13

12

10~

Control

Active

I

i

Slable

Multiple ScJerosIs

Other Neurological Diseases

Fig. 1. The proliferative response of peripheral blood lymphocytes to myelin basic protein. Each point represents a single individual. The shaded area represents 2 standard deviations above the mean value of all the control subjects tested. The number of individuals with active MS responding to MBP was not significantly different than controls (X 2 1.5, P > 0.1).

Using the same criterion, nine of 30 active MS patients, two of ten stable MS, one of seven a N D s (stroke), and zero of ten controls showed proliferative response to myelin-associated glycoprotein; zero of 17 active MS, two of 17 chronic MS, zero of

MYELIN-ASSOCIATED

Control

L

Active

GLYCOPROTEIN

Stable

Olher I Neurological Multiple Sclerosis Diseases

Fig. 2. The proliferative response of peripheral blood lymphocytes to myelin-associated glycoprotein. Each point represents a single individual. The shaded area represents 2 standard deviations above the mean value of all the control subjects tested. The number of individuals with active MS responding to MAG was greater than controls (X 2 3.87, P < 0.05).

104 PROTEOLIPID PROTEIN !

!

,2L

!

6

J

I

I Control

L

Achve

Stable

Multiple Sderosis

Olher j Neurological Diseoses

Fig. 3. The proliferative response of peripheral blood lymphocytes to proteolipid protein. Each point represents a single individual. The shaded area represents 2 standard deviations above the mean value of all the control subjects tested. The number of O N D individuals responding to PLP was greater than controls (X 2 5.73, P < 0.025), and greater than active MS (X 2 7.9, P < 0.01).

12 controls, and six of 16 O N D s (oligodendroglioma, syringomyelia, stroke, Parkinson's disease, amyotrophic lateral sclerosis) responded to proteolipid protein. Of the nine MS patients who had stimulation indices to M A G greater than 2 standard deviations from control values, three also responded to myelin basic protein; five of the 81 patients who responded to basic protein did not respond to MAG. The MS patient who had a stimulation index of 10 to M A G also had one of 8.3 to MBP. Although the mean proliferative response to both MBP and M A G by peripheral blood lymphocytes from patients with active MS was greater than the other three populations studied (stable MS, ONDs, and controls), the differences were not statistically significant when the population was treated as a whole (Student's t-test). However, when the number of individuals that were responders (as defined by a proliferative response greater than 2 standard deviations from controls) in a given group were compared to nonresponders, more active MS patients responded to M A G than controls (chi-square analysis, P < 0.05, Fig. 2). Similarly, although the mean proliferative response to PLP of O N D patients was greater than controls or MS patients, the group differences were not statistically different. However, when the reactivity of individual members of each population was compared, there were more responders in the O N D group as compared to control ( P < 0.025, chi-square) or active MS ( P < 0.01, chi-square) (Fig. 3). No statistically significant differences were found in the number of responders among groups in response to MBP (Fig. 1). In an attempt to enhance or uncover responses to white matter antigens, proliferative responses were studied following lysis of T8 + lymphocytes in an attempt to remove suppressive influences (Table 2). Of 16 MS patients who did not respond to MBP, five responded following T8 lysis. Of 19 MS patients who did not respond to

105 TABLE 2 RESPONSE OF PERIPHERAL BLOOD LYMPHOCYTES TO MYELIN ANTIGENS FOLLOWING LYSIS OF THE T8 SUBSET Controls consisted of five OND patients and four normal controls. Proliferation of peripheral blood lymphocytes to myelin antigens. Unseparated mononuclear cells

Following lysis of T8 cells

0/16 '~ 0/9

5/16 0/9

0/19 0/9

4/19 0/9

Basic protein Multiple sclerosis Controls

Proteolipid protein Multiple sclerosis Controls

a Number of patients whose response was greater than 2 standard deviations from controls/number of patients tested.

PLP, four showed a proliferative response following T8 lysis. In the control and other neurologic diseases population investigated in this series of experiments (n = 9), no proliferation to proteolipid protein or basic protein was observed after T8 lysis.

Discussion

The majority of previous studies on white matter reactivity in MS have focused on myelin basic protein. There have been many reports of cellular reactivity to MBP using a wide range of assays, including production of migration inhibition factor, active E-rosetting cytotoxicity, and lymphocyte proliferation. Although results have been variable, the body of investigations to date would suggest that peripheral blood lymphocyte sensitization to MBP is detectable in some MS patients, that there has been a variable relationship to disease activity, and that the sensitization is also detectable in other neurologic disorders (Knight 1977; McFarlin and McFarland 1982; Lisak et al. 1984; Waksman and Reynolds 1984). Although the mean proliferative response of lymphocytes from active MS patients to MBP in the present study was not significantly different from those of control patients, 10% of patients were responders to MBP, a number greater than that observed with stable MS patients, suggesting that there might be a connection between MBP sensitization and disease activity in these patients. In agreement with previous studies (Lisak et al. 1984), this relationship was not absolute, and sensitization to MBP was not specific to MS. Nonetheless, the greatest individual responses to the three antigens tested were to MBP in active MS patients. Because of the relative ease of preparation of purified MBP and its marked encephalitogenic properties, little attention has been paid to other well-defined myelin components and sensitization to MAG and PLP has not been previously reported in MS patients. MAG is a quantitatively minor component of both CNS

106 and PNS myelin, being less than 1% of myelin protein. It has recently generated much interest as the putative autoantigen in peripheral neuropathies associated with IgM paraproteinemia (Braun et al. 1982), although it now appears that the initial target antigen may in fact be a glycosphingolipid of peripheral nerve (Chou et al. 1985) and that the detectable humoral sensitization to M A G may represent a cross-reaction with the carbohydrate epitope shared with M A G (Ilyas et al. 1984). The pathogenetic role of the IgM is still not clear (Mendell et al, 1985). Earlier studies from our group have shown that M A G loss is seen early in the development of some MS plaques, suggesting that it may be an early target for autoimmune attack (Itoyama et al. 1980; Johnson et al. 1986). A recent study reported low levels of anti-MAG antibodies in CSF of MS patients (Wajgt and Gorney 1983: Moller, Johnson, and Quarles, unpublished data), However. cellular responses to M A G in MS patients have not been reported. The results described here show M A G sensitization does occur in some active MS patients, although it is at a low level. Although proliferative responses to M A G were not statistically significant from controls when the whole population of active MS patients is grouped together, it is clear that some individuals with active disease are sensitized to M A G and that these may represent a greater proportion than are sensitized to MBP. It is not clear, however, whether the statistical difference in M A G responsiveness compared to that to MBP is significant. The standard deviation of the MBP control group is higher due to the single high responder and this may give the range encompassed by 2 standard deviations in this group an artificially high value. In addition, the lower number of M A G controls reduced the chances of detecting a rare M A G responder. A larger number of controls and MS patients will need to be studied to obtain the true significance of this observation. There does appear to be some connection with disease activity, as there was a greater incidence of M A G reactivity in active MS than in stable MS patients. It appears that sensitization to M A G is not unique to MS, since one stroke victim also responded to MAG. Studies to examine sensitization to M A G in other inflammatory conditions of the CNS, such as acute disseminated encephalomyelitis or encephalitis are currently in progress. One-third of the MS patients that responded to M A G also responded to MBP, raising the possibility that these responses might have represented a generalized response to myelin breakdown. Of note, however, is that no active MS patients responded to PEP and only two of 17 stable MS patients responded to PLP, whereas a relatively high number of O N D subjects proliferated to PEP. Since PLP is specific to CNS myelin and represents a major component of CNS myelin, one would have expected proliferative responses to PLP as well as to MBP and M A G in MS patients were the responses to MBP and M A G simply a secondary result of myelin breakdown. Proteolipid protein has been reported to cause an EAE-like disease in experimental animals (Madrid et al. 1982; Cambi et al. 1983). In an attempt to amplify or uncover responses to the myelin antigens tested, the T8 (cytotoxic/suppressor) lymphocyte subset was deleted using monoclonal antibodies (Reinherz et al. 1980). Following T8 lysis, some of the peripheral blood lymphocytes from MS patients which had not previously shown a response to basic

107 protein or proteolipid protein did proliferate to these antigens. This was not seen in any of the O N D or control cases examined and raises the possibility that there may be suppression to sensitization to some myelin antigens in MS patients. This is currently being investigated further in our laboratory. In summary, our results have demonstrated some reactivity to myelin-associated glycoprotein in approximately one-third of the active MS patients studied; while sensitization of PLP was only seen after deletion of the T8 subset, and then in only a few patients. The significance of these results in terms of the pathogenesis of the disease is unknown. If there is cellular autoimmunity to a single white matter protein in MS, either the antigen has yet to be identified or the soluble, isolated form of the protein does not resemble sufficiently that encountered by the immune system in vivo. In addition, the responsible cells may not be present in sufficient number or appear too transiently in the peripheral blood to be consistently detected by current techniques. In this regard, sensitivity to MBP has been demonstrated in the cerebrospinal fluid of some MS patients (Lisak and Zweiman 1977), and it may be productive to examine this compartment for M A G and PLP reactivity.

Acknowledgements Supported by N I H grant NS17812. Anti-T-cell monoclonal antibodies were the gift of Dr. Ellis L. Reinherz. We are grateful to Christine Lucas for valuable technical assistance and to Pat Lamy for expert assistance in preparing the manuscript.

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