Defective T lymphocyte function in nonthymectomized patients with myasthenia gravis

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

60,

93-105 (191)

Defective T Lymphocyte Function in Nonthymectomized Patients with Myasthenia Gravis RICHARD E. AHLBERG,* RITVA PIRSKANEN,~ AND ANN KARI L,EFVERT* *Department

of Medicine,

Karolinska

Hospital, Stockholm,

and ?Department Sweden

of Neurology,

South

Hospital,

In vitro functional properties of peripheral blood mononuclear cells were evaluated in 29 patients with myasthenia gravis and in 11 healthy controls. Spontaneous cell proliferation was higher in patients than in controls. The production of interleukin-2 and interferon-y and the proliferative response to different mitogens were reduced in the patients. A positive correlation was found between the production of interleukin-2 and interferon-y. These defects in T cell function were the most pronounced in nonthymectomized patients. Patients with severe disease had a higher percentage of cells bearing the interleukin-2 receptor and a higher spontaneous production of tumor necrosis factor a in cell culture than in patients with mild disease. There was no difference between patients and controls in the level of soluble interleukin-2 receptor in cell culture supernatants or in sera. The results indicate a partially suppressed T cell function in myasthenia gravis. This defect was less pronounced in patients studied after thymectomy. in 1991 Academic Press, Inc.

INTRODUCTION

Myasthenia gravis (MG) is caused by an autoimmune attack on the nicotinic acetylcholine receptors on the muscle endplate. The production of specific autoantibodies against the acetylcholine receptor is a T cell-dependent phenomenon (I), and receptor-specific T cells are present in the blood and in the thymus gland (2, 3). One site for the T cell autosensitization in MG might be the thymus since certain thymocytes bear acetylcholine receptors (4). Thymic hyperplasia with a germinal center formation is found in over 70% of the myasthenic patients and thymomas in 15% (5, 6). Thymectomy generally improves the clinical symptoms in patients with thymic hyperplasia (7). Despite these indications the role of the thymus in the pathogenesis of the disease is still not clear. The search for a disturbance of immunoregulatory mechanisms as revealed by the intrinsic cytokine network is a new approach in the study of autoimmune diseases. The cytokines interleukin-1 (IL-I) and tumor necrosis factor (TNF) are released by activated macrophages. TNF interacts in a synergistic fashion with IL-l and other cytokines and regulates the functional activities of several types of cells (8, 9). IL-l functions as an essential activating signal in all T cell-dependent antigen-specific immune responses (10). It induces IL-2 production as well as the expression of a membrane receptor for IL-2 (IL-2R). Following T cell activation and IL-2R expression, a soluble form of IL-2R (sIL-2R) is released under both in vivo and in vitro conditions (11). IL-2 is synthesized by T cells in response to antigen or mitogen stimulation. It acts on activated T cells, B cells, NK cells, and thymocytes; is required for the proliferation of activated T cells; and induces the 93 0090-1229/91 $1.50 Copyrkht 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

94

AHLBERG,

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AND

LEFVERT

release of interferon-y (IFN-7) (12) and other lymphokines. IFN-y induces the proliferation of macrophages and increases their expression of MHC class II molecules. It is also involved in the regulation of T and B cell proliferation. An impaired capacity of peripheral blood mononuclear cells (PBMC) to produce IL-I, IL-2, and IFN-y in response to mitogens has been described in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome, and type 1 diabetes mellitus (13-18). There are also reports of normal in vitro production of IL-l (17, 18), IL-2, and IFN-y (19). The level of sIL-2R in serum in systemic lupus erythematosus (20), rheumatoid arthritis (21), and type I diabetes mellitus (22) was elevated, while the in vitro production was reduced in type I diabetes (23) and was normal in rheumatoid arthritis (21). Increased amounts of cells expressing the receptor for IL-2 (CD25) were found in several autoimmune diseases in the active phase such as multiple sclerosis and systemic lupus erythematosus (24). No difference in the percentage of CD25 positive cells between patients and controls was found in a recent study of MG (25). In another study of MG, PBMC from four out of nine patients failed to produce IFN-y after stimulation with phytohemagglutinin (PHA) or suboptimal amounts of concanavalin A (Con A) (26). Other abnormalities reported in MG are a depressed lymphoproliferation in response to mitogens (27), hyperreactivity of PBMC in response to rIL-2 in nonthymectomized patients (25), diminished suppressor cell activity (28.29), increased spontaneous production of total immunoglobulins. and reduced production of IgG and IgM in response to mitogens (30). In the present investigation the cytokine production and the stage of mononuclear cell activation were studied. The results indicate defects in T cell function which was the most pronounced in nonthymectomized patients. MATERIALS

AND METHODS

Patients

Twenty-nine patients with MG were included in the study. The diagnosis was confirmed by a typical case history, a positive response to the acetylcholinesterase inhibitors, a decremental response to repetitive nerve stimulation, an increased jitter in single-fiber eiectromyography examination, and the presence of antibodies against the acetylcholine receptor. The mean age of the patients was 56 years (range 25-83 years). Clinical stage was determined according to the Osserman-Oosterhuis classification (31). Eighteen of the patients were in stage IIA (mild, generalized myasthenia) and were considered as having mild disease. Ten patients in stages IIB (severe, generalized myasthenia) and one single patient in stage III (acute fulminant myasthenia) were considered as having severe disease. Clinical features of the patients are summarized in Table 1. Eleven healthy individuals from the laboratory staff served as controls for the study. The mean age of the controls was 34 years (range 24-44 years). Preparation

of Cells

PBMC were isolated from fresh heparinized blood by centrifugation on a FicollPaque gradient (Pharmacia, Uppsala, Sweden). Cells from the interface were

T CELL

CLINICAL

FEATURES

FUNCTION

IN

TABLE 1 OF THE PATIENTS WITH __.-

95

MG

MYASTHENIA -~

GRAVIS

No.

of

patients Sex

Female Male

18 II

Disease severity

Mild

18

Age of onset

Severe ~36 >35

II 7 21 17 5 12 5 8 4 28 17 3

Thymectomy Years after thymectomy Thymus histology Treatment

1 Thymoma Hyperplasia Normal Cholinesterase inhibitor Azathioprine” Prednisoneb

Percentage

62 38 62 38 25 75 59 29 71 29 47 24 97 59 10

U 75-150 mg/day. ’ 15-25 mg every second day.

washed and resuspended in complete medium consisting of RPM1 1640 supplemented with L-glutamin (2 mM), penicillin (100 IU/ml), streptomycin (100 pg/ml), and heat-inactivated fetal calf serum (FCS) (10%) or serum from individuals belonging to blood group AB (15%). Determination

of the Production

of IL-1 and TNF-a in Cell Culture

PBMC (5 x 105/well in 1 ml) were incubated at 37°C in a humidified atmosphere of 5% CO2 in 24-well plates. After 1 hr, the nonadherent cells were discarded by repeated washing with warm complete RPM1 1640 medium. Adherent cells were incubated for 20 hr at 37°C in 5% COZ in 1 ml of complete RPM1 1640 medium. Supernatants were collected and stored at -20°C until analyzed. The supernatants were analyzed using the interleukin- 1p enzyme-linked immunosorbent assay (ELISA) kit (Cistron) and a radioimmunoassay kit for human TNF-cx (Genzyme, Boston). The sensitivity of the assays permits reliable measurement down to 100 pg/ml of TNF-a and 20 pg/ml of IL-l p. Determination

of IL-2, ZFN--y, and IL-2 Receptor

PBMC (I x lO’lfm1) in complete medium (10% FCS) were incubated for 42 hr at 37°C in cell culture tubes (final volume: 2 ml/tube), with or without Con A (2.5 or 20 kg/ml). The supernatants were stored at - 20°C. The cells were used immediately for IL-2 receptor surface analysis. The level of IL-2 in the supernatants was measured using a murine IL-Zdependent cytotoxic T cell line (CTLL) as an indicator line (34). Supernatants in serial dilutions were added in triplicate to microtiter wells containing 5 x lo3 CTLL cells. After incubation for 24 hr, the cells were incubated for an additional 24 hr with 1 &i of [3H]thymidine per well,

96

AHLBERG,

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LEFVER’I

harvested, and counted in a liquid scintillation counter. The results were expressed as units of IL-2/ml (U/ml) by comparison with the mitogenic activity of serial dilutions of one single batch of a reference supernatant (Mitogen-stimulated rat spleen cells). One unit was defined as the amount of IL-2 which causes half maximal incorporation of [3H]thymidine in 5 x IO3 CTLL cells. The levels of IFN-y in the supernatants were assayed using an immunoradiometric assay kit (MEDGENIX, Belgium). The level of the soluble IL-2 receptor in supernatant and plasma was determined by ELISA using the cell free IL-2R test kit (T-cell Sciences Inc., Cambridge, MA), according to the method of Rubin et (11. (33). The sensitivity of the assays permits reliable measurements down to 50 U/ml of sIL-2R and 1 IU/ml of IFN-y. Enumeration of cells bearing IL-2 surface receptors was done by direct immunofluorescence using phycoerythrin-conjugated anti-CD25 antibodies. This cell labeling was performed on cells previously washed in mannoside containing medium (RPM1 1640) after 42 hr of incubation with or without Con A (2.5 and 20 idmU. Determination

of Proliferation

and lmmunoglobulin

Production

The lymphocyte proliferation was measured using the following technique. PBMC, suspended in RPM1 1640 medium with 15% heat-inactivated human sera from individuals belonging to blood group AB, were macrophage depleted (iron depletion technique). The cells were cultured in microtiter wells (1 x lO’/well) at 37°C in 5% CO? for 66 hr. Tritiated thymidine was added during the last 20 hr of culture. The mitogens used were Con A (10, 20, 40, or 80 pg/ml), pokeweed mitogen (PWM) (1 or 10 p&ml), and PPD (2.5 &ml). The spontaneous proliferation was analyzed by an immediate 20 hr of incubation of cell cultures in triplicate with tritiated thymidine. The plates were stored at - 20°C until harvested and counted using a liquid scintillation counter. The concentration of IgG and IgM in cell culture supernatants was determined by ELISA (34) after 6 days of culture of PBMC (1 x lO?ml) in complete medium (10% FCS) in culture tubes (final volume 2 ml), with and without 10 p-g/ml PWM. Analysis of Data

All data are presented as median value and percentile 10 and 90. Comparison between groups was carried out using the nonparametric Mann-Whitney test and the Spearman rank correlation coefficient. The level of significance was defined as P < 0.05. The P values within the range of 0.05-O. 10 are presented in parentheses. RESULTS Production of IFN-y

and IL-2

The spontaneous production of IFN-7 was significantly reduced in patients (Table 2) and the reduction was more pronounced in nonthymectomized patients. The IFN-y production increased in response to Con A but to a lesser extent in

T CELL

FUNCTION

97

IN MG

patientscomparedto controls. The reducedproductionof IFN-y in responseto Con A was statistically significant when nonthymectomizedpatients were compared to controls (Table 2, Fig. 1) The IL-2 production in cell cultures stimulatedwith 20 @ml Con A was markedly reduced in patients (Table 2). The production of this cytokine was the lowest in nonthymectomized patients. There was a positive correlation between the production of IL-2 and IFN-y in response to Con A (P < 0.01). There was no detectable spontaneous IL-2 production in the majority (37/40) of the cultures. The decreased production of IL-2 and IFN-y did not correlate with clinical severity, age of onset, or treatment with azathioprine or prednisone. No significant correlation (P > 0.05) was found between the level of IL-2 or IFN-y secretion and the age of the patients, as measured by Spearman rank correlation test. Surjhce-Bound

and Soluble IL-2 Receptors

There was a positive correlation between the amount of sIL-2R and the percentage of IL-2R positive cells in the patients and both increased in response to increasing Con A concentrations. Patients with severe disease had a slightly higher level of sIL-2R and a higher percentage of IL-2R positive cells in nonstimulated cultures than patients with mild disease (Table 3). There was no difference between patients and controls or between patients with different treatments (azathioprine, prednisone, thymectomy), age of onset, or clinical stage. No difference was found in the plasma levels of sIL-2R of patients and controls. There was no correlation between the production of sIL-2R or the percentage of IL-2R positive cells and the IL-2 production. Spontaneous

Production

of TNF-a

and IL-1

Patients with severe disease had a higher spontaneous production of TNF-a than controls (Table 4). Treatment with azathioprine, prednisone, or thymectomy

MEDIAN CULTURE

TABLE 2 (P,,) AND PERCENTILE 10 AND 90 (P,,-P,) VALUES FOR IFN-y AND IL-2 IN CELL SUPERNATANTS AFTER A Z-DAY CULTURE WITH OR WITHOUT CON A STIMULATION IFN-7 Con A (0)

No. Controls Patients NT T

10 28 12 16

Significance P-C NT-T NT-C Note.

18 3 2 6

C, controls;

P, patients:

IL-2

Con A

Con A (20 (&ml)

(2.5 *g/ml)

-_

(2-888) (O-29) (O-7) (W7)

P < 0.05 NS(P = 0.09) P < 0.01

(IUlml)

111 52 38 68

(211388) (14-311) (11-130) (H-346)

232 109 103 243

NS (P = 0.08) NS(P = 0.09) P < 0.05

T, thymectomized;

NT,

(72-1294) (36-789) (34-300) (361012)

NS NS P < 0.05

nonthymectomized;

(U/ml)

Con A (20 t&ml) 16.1 3.4 3.4 6.1

(3.3-67.9) (o-18.5) (O-10.6) (O-25.9)

P < 0.01 NS P< 0.005 NS, nonsignificant.

98

AHLBERG.

PIRSKANEN,

AND LEFVERT

IFNgamma IlJ/ml 250 -

=

Thymactomized

v

Non-thymectomized

-+-

control

ConA 0

10

20

FIG. 1. Median value for the concentration culture with or without Con A stimulation.

kg/ml

of IFN-y in cell culture supematants after a 2-day

did not influence the production. There was no difference in the spontaneous production of IL-1 between patients and controls or between the different groups of patients. Spontaneous

and Mitogen-Induced

Immunoglobulin

Production

There was no difference in the spontaneous production of IgG and IgM between patients and controls (Table 5). Cells from thymectomized patients produced higher amounts of IgG but the same amount of IgM as cells from nonthymectomized patients (Table 5). The PWM-induced immunoglobulin production was lower in patients than in controls, although the difference was significant only for IgG. It was not influTABLE 3 MEDIAN (P,,) AND PERCENTILE 10 AND 9O(P,,,-P&VALUES FORTHE LEVELS OF sIL-2R IN CULTURE SUPERNATANTANDFORTHEPERCENTAGEOF IL-2R POSITIVE CELLSINTHE SAMECULTURES sIL2-R No. Controls Patients significance

10 28

Mild Severe Significance

18 10

No&.

NS, nonsignificant.

Con A (0) 58 (O-126) 68 (a-196) NS 37 (O-139) 97 (O-272) NS (P = 0.08)

(U/ml)

Con A (2.5 &ml)

_---Con A (20 LLg/ml)

360 (234-601) 326 (147-544) NS

990 (65~1385) 875 (446-1493) NS

300 (147-588) 418 (160-484) NS

921 (446-1251) 814 (462-2016) NS - _-

% IL-2R Con A (0) 3 (2-5) 3 (l-7) NS 3 (I-5) 4 (2-22) P < 0.05

positive

Con A (2.5 &ml)

cells Con A (20 &ml)

12 (5-22) 10 (5-20) NS

36 (9-45) 30 (13-W NS

12 G-27) 10 (7-20) NS

30 (1449) 25 (l&431 NS -_. -..

T CELL

FUNCTION

IN

MG

TABLE 4 MEDIAN (P,,) AND PERCENTILE 10 AND 90 (P,,,-Pm)VALUES FOR THE PRODUCTION

No. Patients Controls Severe Mild Significance P-C S-M s-c Note.

SPONTANEOUS

TNF-a

OF

TNF (&ml) 2.2 0.9 3.2 2.0

29 11 11 18

(011.9) (04.5) (O-12.5) (0.2-3.0)

NS (P = 0.09) NS (P = 0.09) P < 0.05

-

P. patients; C, controls; S, severe; M, mild; NS, nonsignificant.

enced by treatment, age of onset, or clinical stage. A positive correlation was found between the PWM-induced IgG and IgM production and the spontaneous and 2.5 t&ml Con A-induced production of IFN-7; that is, patients with low IFN--y production produced low amounts of immunoglobulins in response to mitogen. Spontaneous and Mitogen-Induced Blood Lymphocytes

Proliferation of Peripheral

Spontaneous proliferation was slightly higher in patients than in controls (Table 6). Peripheral blood lymphocytes from nonthymectomized patients had a reduced proliferative response to the different mitogens compared to peripheral blood lymphocytes from thymectomized patients and controls (Table 6, Fig. 2). The proliferative response was not influenced by clinical stage or treatment with azathioprine and prednisone. There was a negative correlation between the spontaneous proliferation and the TABLE MEDIAN

(P,,)

AND PERCENTILE PWM-INDUCED

5

10 AND 90 (P,,P,) VALUES FOR THE SPONTANEOUS PRODUCTION OF TOTAL IgG AND IgM ____

I& (rig/ml) Controls Patients NT T Significance P-C NT-T NT-C T-C

No.

Spontaneous

11 29 12 17

114 (52-209) 137 (58-242) 102 (46-177) 155 (79-250) NS

-

IgM (rig/ml)

PWM (10 I.Lg/m) 267 245 248 241

AND

(247-385) ( 178-435) (151-534) (171281)

Spontaneous

PWM (10 kg/m)

57 (24-249) 45 (14-531) 47 (15-310) 34 (14-596)

1018 948 966 894

NS NS

(608-1525) (194-1324) (130-1752) (264-1253)

P < 0.05

P < 0.05 NS

NS NS

NS

NS

NS

P < 0.05

NS

NS (P = 0.09)

(P = 0.06)

Note. C, controls; P, patients; T, thymectomized:

NT, nonthymectomized;

NS NS

NS, nonsignificant.

12

17

Significance Nonthymectomized

Thymectomized

Note.

NS.

29

Patient

Significance

275 (176-418l 377 (203-500)

nonsignificant.

NS

340 (205-517) 392 (205-503)

P i 0.05

Spontaneous

11

^~..

NO.

RESPONSE

Control

PROLIFERATIVE

TABLE

PWM .--~~

P < 0.05

4866 (409-22,008) 11868 (495Cb26.938)

( 1676-26.665) NS

15032 (3572-23,985) 10037

10 &g/ml

MITOGENS MEASURED VALUES AFTER THYMIDINE

P < 0.05

t1065-19.307)

4468

2432 (489-6.651)

NS

3316 ~1017-15.495) 3634 (541-12,064J

1 b&d

TO DIFFERENT

6

NS

702 1259-2.985) 1301 (3W.344)

NS

(309-3.697)

NS

2244 (38%11,181) 4495 (483-18.163)

NS

4931 (673-21.989) 3724 (455-17.265)

1066

10 &ml

2809 (405-13.268)

AND

2.5 &ml

PPD

AS MEDIAN (P,,) INCORPORATION

P < 0.05

NS 5190 1wL12.227) 10878 (1702-36288)

-~

Con

IO AND

86% t1562-+1.013) 7500 (635-29.553)

20 &ml ..- . ..__-.

PERCENTILE

40 &ml -

(P,,-P,,)

(P ="s.w,

24160 t324SS6.633)

NS 10310 1513-32.073)

33704 (5017-57.280) 13605 (73b50.251)

A

90

cpm

IP = 0.091

9793 1Y61-25.4.56J 13331 (4Y42-58.17Yl NS

NS

14132 (463lL25,491) 13331 tl108-38.0801

80 pghl

T CELL FUNCTION

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IN MG

n Non-thymectomized 20000

H

Thymectomized Control

10000

0 10

20

40

80

1

10

2.5

py”“’

PPD 2. Proliferative response to different mitogens measured as median cpm values after thymidine incorporation. COtlA

PWM

FIG.

spontaneous (P < 0.05) and the 2.5 I&ml Con A-induced (P < 0.01) IFN-y production and a negative correlation to the Con A-induced production of IL-2 (P = 0.01). A positive correlation between the proliferative response to all mitogens and the IL-2 production was evident, although significant only for Con A at the concentrations of 10 and 80 pg/ml (P < 0.05). Thus, patients with a reduced production of IL-2 and IFN-y demonstrate an increased spontaneous proliferation and a reduced proliferative response to the different mitogens. DISCUSSION

Aberrations

of T Cell Function

IL-2 and IFN-71 modulate the immune response by the regulation of antigeninduced T cell proliferation and antibody production. The reduced production of IL-2 and IFN-y in MG patients could be caused by an intrinsic defect of the T lymphocyte. A more probable explanation is an inhibitory effect by a cell population with suppressor effects. Such a mechanism has been clearly shown in systemic lupus erythematosus (35). The reduced production of IL-2 and IFN-y is not due to an inability of the T cells to respond to mitogens since a reduced production of IFN-y was also found in unstimulated cultures. Moreover, the cells responded normally to increased mitogen concentrations with an increased expression of IL-2R. IL-2 is essential for the synthesis of IFN-y (12). The positive correlation between the levels of the two cytokines indicates that the reduced levels of IFN-y are secondary to a decreased production of IL-2. A positive correlation was also found between the production of IFN-y and the mitogen-induced production of

102

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IgG. Since PWM-induced B cell differentiation is a T ceil-dependent phenomenon (36, 37), it is reasonable to believe that the reduced IgG production in response to mitogen is secondary to alterations in T cell function. The increased spontaneous proliferation of cells from patients with MC indicates the presence of an increased number of activated cells. In unstimulated cultures from patients with severe disease, there was an increased percentage of CD 25 positive cells, a higher amount of sIL-2R (P = O.Og), and an increased production of TNF-a as compared to patients with mild disease. If a certain population of these activated cells have suppressor function the net result might be a decrease of the total in vitro production of IL-2 and IFN-y. Effect of Thymectomy The thymus is a central lymphoid organ responsive for the development and control of cell-mediated immunity (38). Data presented here indicate that thymectomy in MG has a significant effect on the T cell function. The reduced production of IL-2 and IFN-?I. as well as the reduced proliferative response to mitogens, was more pronounced in nonthymectomized patients. Earlier studies of the effect of thymectomy on lymphocytes in MG have given conflicting results, ranging from no difference in the percentage of B and T cells (39) to a prompt, small, and persistent decrease in the total number of circulating lymphocytes and in the percentage of peripheral blood T cells (40). This later study also showed a marked increase in the proliferative response to alloantigens after thymectomy which was attributed to a selective loss of T suppressor cells. An increased response to alloantigens following thymectomy has also been reported in mice (41). Such a genera1 decrease in the suppressor cell activity after thymectomy could explain the increased spontaneous production of IgG found among the thymectomized patients in the present study. Thymectomy is reported to lower the concentration of the anti-AChR antibody in MG patients with hyperplasia of the thymus and with normal thymus histology (42). In one study. a correlation was found between a very progressive decline of antibody titers and clinical improvement (43). In another study, the anti-AChR antibody titer was not significantly changed after thymectomy (44). The autosensitization of suppressor T cells in MG may be triggered by acetylcholine receptors on thymic cells. The reduced production of IL-2 and IFN-r was less pronounced and the mitogen-induced proliferation was normalized in the thymectomized patients, indicating that thymectomy partially removes the inhibitory influence on T cells. Thus, thymectomy removes an organ that might be a source of a certain autoimmune T cell population with a suppressive effect on normal T cells. Thymectomy would then prevent perpetuation of the autosensitization process. Cells previously primed would still be present which may account for the incomplete recovery often seen after thymectomy. To summarize, the T lymphocytes in MG have an impaired production of the cytokines IL-2 and IFN-y and a reduced proliferative response to mitogens. This suppressed T cell function was the most pronounced in the nonthymectomized patients and was partially reversed after thymectomy. The results give further support for the crucial role of the thymus in the pathogenesis of MG.

T CELL FUNCTION

IN MG

103

ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council, the Swedish Society of Physicians, the foundations of the Karolinska Institute, and the Swedish association of the neurologically disabled. The technical assistance of Mrs. Juta Andersson and Mrs. Margaretha Siiderqvist is gratefully acknowledged.

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and immunological