Immunologic unresponsiveness in leprosy is mediated by modulation of E-receptor

Immunology Letters, 15 (1987) 199-204 Elsevier

IML 00897

Immunologic unresponsiveness in leprosy is mediated by modulation of E-receptor V e e r a p p a n M u t h u k k a r u p p a n , H r i s h i k e s h R. C h a k k a l a t h a n d M a t h e w M. J a m e s Department of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai, India (Received 20 March 1987) (Accepted 3 April 1987)

1. Summary By using an indirect immunofluorescence technique with OKT 3 and OKTll monoclonal antibodies, the percentage of CD 2 positive cells was found to be reduced in the peripheral blood of bacterial index positive lepromatous leprosy patients; however, in these patients, CD 3 positive cells were at the normal level. Further, CD 2 positive cells attained the normal proportion in lepromatous patients when mycobacterial load was reduced. Both CD 2 and CD 3 receptors were expressed at the normal level in tuberculoid leprosy patients. Prior treatment of peripheral blood mononuclear cells from healthy controls with Mycobacterium leprae significantly decreased the percentage of CD 2 but not CD 3 positive cells. Such a modulation of CD 2 on T cells also resulted in blocking the lymphoproliferative response induced by mitogen and antigen. These results suggest that there is a strong correlation between CD 2 modulation and immunologic unresponsiveness in leprosy. 2. Introduction Leprosy is a chronic infectious disease caused by Mycobacterium leprae. It exhibits a spectrum of Key words: E-receptor; Mycobacterium leprae; Leprosy; Unresponsiveness; Immunofluorescence;Lymphoproliferation Correspondence to: V. Muthukkaruppan, Department of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, India.

well-defined clinical symptoms, correlating with the immune status of these patients. One form, referred to as lepromatous leprosy is associated with disseminated infection with M. leprae, reduced cellmediated immunity (CMI) and a high level of serum antibody to mycobacterial antigens in contrast to the tuberculoid form of leprosy [1, 2]. During the past two decades several approaches have been made to understand the mechanism of anergy in leprosy [3-7]. Nevertheless, it has not been possible to fully explain the underlying immune defect. Earlier investigators, by using sheep erythrocyte rosetting assay, showed that the proportion of T cells (E-receptor positive cells) was reduced in the peripheral blood of lepromatous patients [8-10]. However, after the advent of monoclonal antibodies, when OKT 3 was used as a pan T cell marker the number of T cells was found to be at the normal level in these patients. It is proposed to study the nature of the E-receptor (recently designated as CD2) in leprosy, since a large number of monoclonal antibodies to CD 2 are available [11] and since this receptor is now known to be associated with the regulation of T cell functions [12-14]. The present study indicates that the E-receptor is modulated in lepromatous leprosy and that this condition is strongly associated with the impaired CMI noted in this disease. Further, it is also possible to bring about CD 2 modulation on T cells from normal subjects by treatment in vitro with M. leprae. (These results were briefly presented at the 6th International Congress of Immunology, Toronto, 1986.)

0165-2478 / 87 / $ 3.50 © 1987 Elsevier Science Publishers B.V. (BiomedicalDivision)

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3. Materials and Methods

3.1. Patients A total of 33 lepromatous and tuberculoid patients were studied. All patients were from an endemic area, 55 km from the campus and were graded on the basis of the Ridley-Jopling [15] classification using bacillary smears and clinical symptoms. Volunteers from the university were used as controls. 3.2. Indirect immunofluorescence Peripheral mononuclear cells were isolated from heparinized blood, using Ficoll-Conray gradient, washed by centrifugation in RPMI 1640 (Sigma) and resuspended in RPMI containing 5070 foetal calf serum (Gibco). To 1 x 106 peripheral blood mononuclear cells (PBMC) in 100/~1 of medium, mouse monoclonal antibodies (OKT 3, OKTll , T 3 and TII , Orthodiagnostics and Coulter Electronics) were added at a final dilution of 1/40 and tubes were incubated at 4°C for 30 min with frequent mixing. After washing the cells in RPMI, 50/~l of a 1/20 dilution of either goat anti-mouse IgG-FITC (Coulter Electronics) or rabbit anti-mouse IgGFITC (Cappel) was added and the tubes were incubated at 4 °C for 30 min. After washing, the cells were mounted in 10070 glycerol-carbonate buffered saline (pH 8.4) and observed in a Carl Zeiss Jena Fluoval microscope. The percentage of fluorescent cells was determined by phase-contrast fluorescence microscopy, counting 200 cells per tube. Ascites (BRL) was used as control for monoclonal antibodies. To study the effect of M. leprae on the modulation of the CD 2 receptor, 1 × 106 PBMC were incubated at 37°C for 24 h with Mitsuda (WHO) or Dharmendra lepromin (JALMA Institute for Leprosy, India) containing 3 x 105 bacilli. For controls, the same number of BCG (Guindy, India) was used. After washing, the cells were subjected to immunofluorescence assay for CD 2 and CD 3 receptors. 3.3. Lymphocyte transformation assay PBMC were suspended (2 × 106 per ml) in RPMI 1640 supplemented with 20070 human AB serum (heat inactivated), penicillin (100 U/ml) and streptomycin (100 #g/ml) (Gibco). Triplicate cul200

tures of 2 x 105 cells per well were prepared in a flat-bottom 96-well microculture plate (Nunc). PHA-M (Bacto) and PPD (U.K. Ministry of Agriculture) were added to the cultures at the final concentration of 80 and 20/~g/ml respectively. The culture plates were incubated for 3 days for mitogen and 6 days for antigen at 37 °C in a humidified atmosphere of 507o CO 2 and 95070 air. Sixteen hours before harvesting, 0.5 t~Ci [3H]thymidine (Bhabha Atomic Research Centre, India, spec. act. 6.7 Ci/mmol) was added to each well. Cells were harvested onto a glass fibre filter and the radioactivity incorporated was counted in a scintillation counter (LKB, Wallac). In parallel cultures, PBMC (2 x 105) were incubated with M. leprae or BCG, 1 × 105 bacilli per well at 37 °C for 24 h before adding mitogen or antigen.

4. Results

4.1. Proportion of CD2 and CD3 positive cells in

PBMC of leprosy patients PBMC from 21 lepromatous, 10 tuberculoid patients and 12 normal subjects were studied, using OKT 3 and OKTH monoclonal antibodies. As shown in Table l, OKT 3 positive cells were at the normal level (64-69070) in all the groups. However, the proportion of OKTll positive cells was significantly reduced in bacterial index positive (BI+) lepromatous patients. Further, when the bacillary load was reduced ( B I - ) after prolonged treatment of lepromatous patients, the percentage of OKTll positive cells attained the normal level. These results indicate that the modulation of the CD 2 receptor on T cells is specifically associated with lepromatous leprosy. 4.2. Modulation of the CD 2 receptor on T cells of normal subjects by M. leprae On the basis of the above findings, it was of interest to see whether M. leprae could modulate CD 2 expression on normal T cells. Data presented in Table 2 clearly demonstrate that Dharmendra lepromin reduced the percentage of OKT H positive cells, while the number of OKT 3 positive cells was not altered, In other words, CD 2 modulatory effect of M. leprae found in lepromatous patients could

Table 1 Proportion of CD 3 and CD 2 positive cells in the peripheral blood of leprosy patients. P B M C from

Mean % O K T 3 + cells _+ SE

Mean 07o OKT11+ cells ± SE

P

Normal subjects (12) Lepromatous B I + (10)* Lepromatous B I - (11) Tuberculoid (10)

69.1 +_0.78 68.5_+ 1.20 67.0_+ 1.38 64.4 _+ 1.91

68.4 _+0.95 38.9_+3.89 63.0+ 1.39 64.2 + 2.10

> 0.1 <0.001 >0.05 > 0.1

N u m b e r s in parentheses are the numbers of subjects studied. * P B M C from lepromatous leprosy patients with bacterial index (BI) ranging from 1.0 to 4.1, or from bacterial index negative ( B I - ) patients.

Table 2 Effect o f prior treatment of normal lymphocytes with M. leprae on CD 3 and CD 2 positive cells. Prior treatment

Mean percent O K T 3 + cells _+ SE

None (6)

67.5_+0.72

Lepromin* (6)

63.7+2.27 --

BCG (4)

66.3 _+ 1.48

P

Mean percent P O K T l l + cells _+ SE

<0.001 - -

46.2_+ 1.71

67.7_+0.89

70.0_+ 1.97

)

<0.001 <0.001

N u m b e r s in parentheses are the numbers of subjects studied. * 1 × 106 P B M C were treated with D h a r m e n d r a lepromin or BCG preparation containing 3 × 105 bacilli as described in Materials and Methods.

be brought about in vitro on T cells o f normal subjects as well. Additional experiments revealed that the modulation of CD 2 was specifically mediated in P B M C by M. leprae, but not by BCG (Table 2) and P P D (data not shown). The possibility of neutralization of Tll activity by M. leprae was also excluded (Table 3) and this indicates that the reduction of CD 2 positive cells was not due to direct interaction between M. leprae and the monoclonal antibody. 4.3. Effect of lepromin on the proliferative re-

sponse o f lymphocytes In order to elucidate the relationship between the CD 2 modulation and immunosuppression, P B M C were treated with lepromin for 24 h, prior to the addition of stimulants. In the presence o f M . leprae (1 × l0 s bacilli for 2 × 105 PBMC per well), the

Table 3

M. leprae does not neutralize the activity of Tll monoclonal antibody. Prior incubation of M o A b (T 3 or Tll ) with M. leprae None

1 × 106 Bacilli

3 × 105 Bacilli

6 × 105 Bacilli

°7o Fluorescence positive cells with T 3

73.2

66.1

62.0

75.5

07o Fluorescence positive cells with T11

71.1

69.3

67.0

76.7

Monoclonal antibody T 3 or TII was incubated with the given number of M. leprae at 37 °C for 30 min. After spinning, the supernatant was used for immunofluorescence assay with normal P B M C .

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Table 4 Effect of prior treatment of normal lymphocytes with Dharmendra lepromin on their response to P H A and PPD. Normal subject no.*

CPM with PHA alone

1 40921 2 34872 3 47428 4 58474 5 39170 Mean ± SEM

% Suppression***

CPM with

% Suppression

P H A with lepromin**

P H A with BCG

With lepromin

With BCG

PPD alone

PPD with lepromin

PPD with BCG

With lepromin

With BCG

139 216 9410 2592 7350

42268 33164 N.D. N.D. N.D.

99.6 99.3 80.0 95.6 81.2 91.1 ±4.4

- 3.4 4.9 -

1465 5199 23249 38285 8947

130 177 210 2250 306

1027 5888 N.D. N.D. N.D.

91.1 96.5 99.1 94.1 96.5 96.5± 1.3

29.8 - 13.0 -

* PBMC used in this study were from healthy persons who were not in contact with leprosy patients. ** Cultures with lepromin alone showed low stimulation (CPM ranging from 222 to 1251). Mean CPM of cultures with lepromin or BCG and stimulant *** o70 Suppression = 1 Mean CPM of culture-----~with-----~ti--mulan-----~a-~one ] x 100.

proliferative response of lymphocytes from normal subjects to P H A and PPD was significantly reduced, whereas BCG at the same concentration did not alter this response (Table 4). Thus, a strong correlation exists between the M. leprae-mediated changes in the CD 2 receptor and the suppression of lymphocyte proliferation induced by mitogen and antigen.

4.4. Recovery of proliferative response of leproma-

tous lymphocytes by trypsin treatment It was demonstrated earlier that E-rosette formation [16] and OKT u binding onto T cells [17] were eliminated after mild trypsin treatment. Similarly, by prior treatment of T cells with trypsin, mitogen and antigen induced proliferative response could not be inhibited by OKT u antibody [12]. There-

Table 5 Effect of trypsin treatment on the proliferative response of lymphocytes from lepromatous patients. Patient no.

1 2 3 4 5

Disease classification*

LL LL LL LL LL

BI + BI+ BI + BI + BI+

2.0 1.0 2.0 4.0 1.2

CPM in controls with trypsin**

CPM to PHA***

CPM to PPD

Without trypsin

With trypsin

Without trypsin

With trypsin

921 1083 598 150 318

7090 3774 4342 1452 5129

22943 23074 18451 2772 11653 ~

168 165 358 126 1864

6472 26851 20062 16002 3959

* PBMC from lepromatous leprosy (LL) patients with bacterial index (BI) ranging from 1 to 4 were used. ** Trypsin treated cell without stimulants. *** 1 × 106 PMBC were incubated with trypsin (200/~g/ml, Sigma) for 45 min at 37 °C and washed twice with medium (RPMI with 20°7o AB serum) before initiating cultures with mitogen and antigen.

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fore, in order to see whether the proliferative response could be recovered, PBMC from BI+ lepromatous patients were treated with trypsin and then cultured with stimulants. As shown in Table 5, a significant recovery of the lymphoproliferative response was observed. Further, trypsin treated PBMC were found to be viable and having a normal level of OKT 3 positive cells; however, they were unable to form E-rosettes and were negative for OKT n binding (data not shown). These results indicate that the modulation of the CD 2 receptor is clearly associated with the impairment of CMI in leprosy.

5. Discussion Earlier studies showed that the number of sheep erythrocyte-rosetting ceils was reduced in the peripheral circulation of lepromatous leprosy patients [8-10]. However, with the use of OKT 3 monoclonal antibody the proportion of T cells was found to be at the normal level [18, 19]. The significance of the present study is the confirmation of both of these findings (Table 1). In other words, while the T cell proportion was not altered, the CD 2 receptor as detected by OKTll monoclonal antibody was modulated in BI+ lepromatous leprosy patients. Further, CD2 expression on T cells attained normalcy in lepromatous patients when they became bacteriologically negative after treatment, thus confirming the earlier report [10]. Another salient finding of the present study was the specificity of CD 2 modulation and the inhibition of lymphoproliferation by M. leprae, but not by BCG (Tables 2 and 4). This could be correlated with the conditions referred to as generalized and M. leprae specific anergy in leprosy. It has been demonstrated that the generalized unresponsiveness was rectified after effective chemotherapy; however, M. leprae specific anergy persisted even after the clearance of bacilli in lepromatous patients [20]. It appears, in the light of the present findings, that specific anergy is not due to the development of antigen (M. leprae) specific suppressor function [2, 20], but due to some specific component o f M. leprae, which has the ability to modulate the CD 2 receptor on T cells. This con-

tention, supported by reports from other laboratories that M. leprae suppressed the in vitro mitogenic response of all the spectrum of leprosy patients and healthy contacts [21], was discussed in detail elsewhere [22]. What is the functional significance of CD 2 modulation in leprosy? Recent studies [12-14] on the regulatory role of the CD 2 molecule in T cell functions provide a convincing answer to this question. By the use of monoclonal antibodies (OKTn, 9.6, Leu 5b) directed against certain epitopes of the CD 2 molecule, it was possible to down-regulate the proliferative response of T cells. Therefore, it is quite likely that an analogous situation exists in lepromatous leprosy patients due to the modulation of the CD2 receptor by M. leprae. The present study is clearly a new approach to understand the mechanism of immunologic unresponsiveness in leprosy. This approach would also be a useful experimental model, since CD2 modulation and immunosuppression can be brought about in PBMC from normal subjects. Using the model, it is possible to answer several questions, regarding the nature of the M. leprae specific component which modulates CD 2, the need for macrophage in this phenomenon and the interrelationship between CD 2 modulation and immunosuppression.

Acknowledgements This work was partly supported by Indian Council of Medical Research, New Delhi. Cooperation of Dr. P. Suresh, Mr. John Dalton and the staff of Arogya Agam, Andipatti, Madurai District in providing us with blood samples from leprosy patients is gratefully acknowledged.

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