Immunomodulatory assays to study structure-activity relationships of thalidomide.

Thalidomide, which has a long history of tragedy because of its ability to cause severe birth defects, is very effective in alleviating erythema nodosum leprosum in leprosy patients and aphthous ulcers in AIDS patients. The causes of these inflammatory diseases and the mechanism by which thalidomide diminishes them are unknown. It has been suggested that modulation of the immune response plays an important role. We found that thalidomide exerts immunomodulatory activity in three bioassays. It suppresses an IgM plaque forming cell response in mice injected with sheep erythrocytes: it inhibits TNF-alpha production by LPS stimulated human mononuclear cells: and it enhances IL-2 production by Con-A stimulated human mononuclear cells. We employed these bioassays to compare the activity of 15 analogs of thalidomide with thalidomide itself. Eight of the compounds were derivatives of the glutarimide moiety of thalidomide and the others were phthalimide or derivatives of the phthalimide moiety of thalidomide. N-hydroxyphthalimide, a simple derivative of phthalimide, was more effective than thalidomide and was also the most effective of the compounds assayed in suppressing the IgM plaque and TNF-alpha responses, but it did not enhance the IL-2 response, instead, it significantly suppressed it.

[The reaction of cultured mononuclear cells from the peripheral blood of patients with lepromatous leprosy to rabbit interleukin-1]. / Reaktsiia kul'tiviruemykh mononuklearov perifericheskoi krovi bol'nykh lepromatoznoi leproi na interleikin-1 krolika.

Interleukin-1 (IL-1) obtained from leukocytes of rabbit peritoneal exudate was added to concanavalin A-stimulated cultures of peripheral blood mononuclear cells obtained from healthy donors and patients with lepromatous lepra at the active stage of the process. The comitogenic effect of IL-1, observed in the process of its action on mononuclear donor cells, has been found to be linked with the presence of the state of leprous reaction in the patient and is not manifested in the absence of such state. This indicates that intercellular cooperation processes mediated by IL-1 play some role both in the mechanisms of immunodepression in lepra and in the pathogenesis of leprous reactions connected with the activation of cell immunity.

In vitro suppression of interleukin 2 production by Mycobacterium leprae antigen.

The suppressive activity of three different lots and sources of Mycobacterium leprae (M. leprae) was studied by measuring the inhibitory effect on interleukin 2 (IL-2) production in normal subjects. All three M. leprae preparations had suppressive activity on IL-2 production when peripheral blood mononuclear leucocytes (PBML) were stimulated with the mitogens PHA-P or Con A in a dose response. M. leprae also had suppressive activity on IL-2 production when PBML were stimulated with the specific antigen, PPD. The inhibitory activity of M. leprae on IL-2 was not due to the direct interaction of M. leprae and IL-2 because direct mixing of IL-2 with different concentrations of M. leprae did not alter the activity of IL-2. Incorporation of M. leprae for 0, 6 and 12 h in PHA-P and PBML cultures had no inhibitory effect on IL-2 production; however, after 14, 16 and 18 h of M. leprae incorporation, significant inhibitory effects were noted on IL-2 production. The suppressive mechanism of M. leprae was studied by incorporating M. leprae into PBML or adherent cells. The suppressive activity could be detected in both M. leprae-stimulated PBML and M. leprae-stimulated monocyte supernatant fluids. The suppressive mechanism of M. leprae was further evaluated by incorporating 1 and 2 micrograms/ml of indomethacin in PBML containing PHA-P and M. leprae. The suppressive activity of M. leprae was significantly diminished by indomethacin, suggesting that the inhibitory effect of M. leprae may result from the induction of PBML and adherent cells to produce the immunosuppressive activity of prostaglandin(s).

Inmunosupresión en lepra / Immunosuppression in leprosy

Son varios los trabajos que tratan de demostrar que en la formal lepromatosa (LL) de la lepra, existirían mecanismos supresores de la respuesta inmune. Al emplear un método de inducción inespecífico, como el sistema de la Concavalina A (ConA), observamos que los pacientes LL tenían disminuida la función supresora, valorada sobre un sistema de proliferación celular T; dicha función tendía a normalizarse durante el episodio de eritema nudoso lepromatoso (ENL). En este sistema demonstramos además, que las células CD8+ (Leu 2a+) eram capaces de interferir en la generación de supresión. Observamos además, que un alto porcentaje de pacientes LL tenían una supresión espontánea elevada; en este tipo de supresión hallados. Por otra parte, hemos demostrado que en este sistema tendría un papel importante el sistema supresor de los monocitos, a través de la liberación de factores solubles (PGE2). Al evaluar la capacidad que tiene el M. leprae para inducir especificamente supresión in vitro la proliferación célular T, hallamos que en los pacientes LL el M. leprae no inducía supresión. Paralelamente, determinamos en sangre periférica, el número de células que tenían el antígeno Leu8+. Observamos que este antígeno estaba marcadamente disminuido, en la fracción de células T, en los pacientes LL y tendía a normalizarse durante el receptores para IL-2 (Tac) durante la etapa de inducción era similar tanto en los TT com LL. La capacidad de las células para proliferar frente a los mitógenos empleados...

Inmunosupresión en lepra / Immunosuppression in leprosy

Son varios los trabajos que tratan de demostrar que en la formal lepromatosa (LL) de la lepra, existirían mecanismos supresores de la respuesta inmune. Al emplear un método de inducción inespecífico, como el sistema de la Concavalina A (ConA), observamos que los pacientes LL tenían disminuida la función supresora, valorada sobre un sistema de proliferación celular T; dicha función tendía a normalizarse durante el episodio de eritema nudoso lepromatoso (ENL). En este sistema demonstramos además, que las células CD8+ (Leu 2a+) eram capaces de interferir en la generación de supresión. Observamos además, que un alto porcentaje de pacientes LL tenían una supresión espontánea elevada; en este tipo de supresión hallados. Por otra parte, hemos demostrado que en este sistema tendría un papel importante el sistema supresor de los monocitos, a través de la liberación de factores solubles (PGE2). Al evaluar la capacidad que tiene el M. leprae para inducir especificamente supresión in vitro la proliferación célular T, hallamos que en los pacientes LL el M. leprae no inducía supresión. Paralelamente, determinamos en sangre periférica, el número de células que tenían el antígeno Leu8+. Observamos que este antígeno estaba marcadamente disminuido, en la fracción de células T, en los pacientes LL y tendía a normalizarse durante el receptores para IL-2 (Tac) durante la etapa de inducción era similar tanto en los TT com LL. La capacidad de las células para proliferar frente a los mitógenos empleados... (AU)

Reduced suppressor cell response to Mycobacterium leprae in lepromatous leprosy.

We have previously shown that concanavalin A (ConA) induction of suppressor cell activity is impaired in patients with lepromatous leprosy (LL). In this study, we demonstrated that the proportion of cells bearing the Leu8 antigen (associated with suppressor-inducer cells) is low in LL patients and tends to normalize during the erythema nodosum leprosum (ENL) episode. Antigen-induced suppressor cell function was evaluated by a two-stage assay. In the first stage, peripheral blood mononuclear cells (PBMC) were cultured for 5 days either in the presence of gamma-irradiated Mycobacterium leprae or in tissue culture medium as a control. In the second stage, mitomycin C-treated suppressor or control cells were added to phytohemagglutinin (PHA)- or ConA-stimulated autologous PBMC. The results indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in patients with tuberculoid (TT) and intermediate clinical (BB, BL, BT) forms and Mycobacterium bovis BCG-immunized normal controls. In ENL patients, the percent suppression was between that of TT and normal individuals. M. leprae-induced suppression was more effective on ConA- than on PHA-triggered T-cell proliferation in all groups. In contrast, normal PBMC cultured for 5 days in RPMI 1640 medium (N-C) and cells from patients with leprosy (TT-C and LL-C) had effects of their own on PHA- or ConA-induced proliferation. LL-C depressed the response to ConA and enhanced PHA-induced proliferation of autologous cells. Conversely, TT-C reduced PHA-induced proliferation and increased the ConA response. Suppression of proliferation could not be overcome with exogenous interleukin-2 and was not related to the induction of the Tac antigen. The abilities of LL, TT, ENL, and normal cells to proliferate upon PHA or ConA stimulus were similar, indicating that the defect in the generation of in vitro suppression by M. leprae in LL patients occurred during the induction period (step 1 of assay).

[Immunosuppression in leprosy]. / Immunosupresión en lepra.

Many authors tried to show that in lepromatous leprosy (LL), suppressor mechanisms are involved in the immune response. We have previously shown that non-specific suppression (Con A induced) was impaired in LL patients and tends to normalize during the erythema nodosum leprosum episode (ENL). In this system we have shown that CD8+ cells (Leu 2a+) can interfere with the generation of Con A-induced suppression. We also observed that a high percentage of LL patients had an increased spontaneous suppression. In these patients, the number of Leu 2a+ cells added in the assay did not correlate with the suppression values. On the other hand, we had demonstrated that the monocyte suppressor system may have an important role, due to the release of soluble factors (PGE2). We evaluated M. leprae-induced suppressor cell function using a two step assay, on T cell proliferation. The results of this study indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in tuberculoid (TT), intermediate clinical forms (BB, BL, BT) and BCG-immunized controls. On the other hand, we determined that the proportion of peripheral blood mononuclear cells (PBMC) bearing the Leu 8+ antigen (associated to suppressor inducer cells) was low in LL and tends to normalize during the ENL episode. Suppression of proliferation could not be overcome with exogenous IL-2 and was not related to the induction of Tac antigen. The ability of LL, TT, ENL and normal cells to proliferate upon PHA or Con A stimulus was similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the immune deficit in leprosy patients and the effect of recombinant IL-2 in vitro.

Although the mechanism of immunologic unresponsiveness in lepromatous leprosy remains unknown, it has been shown that interleukin-2 (IL-2) production is defective in these patients. Peripheral blood mononuclear cells (PBMC) were isolated from treated (less than 16 months) and untreated leprosy patients as well as household contacts; age, sex, ethnically matched control subjects; and laboratory staff. PBMC were cultured for 6 days with sonicated Mycobacterium leprae (1-10 micrograms/ml), Dharmendra lepromin (1:10), or phenolic glycolipid-I (PGL-I) (0.05-5.0 micrograms/ml) in medium supplemented with various concentrations of recombinant IL-2 (rIL-2) or cultured for 3 days with one of the three mycobacterial antigens in the presence of concanavalin A (ConA). TT/BT patients and household control subjects had a robust response to M. leprae and lepromin, but were unresponsive to PGL-I delivered in liposomes. PBMC from LL patients did not respond to any of the three antigen preparations. rIL-2 induced proliferation of PBMC both in leprosy patients and control subjects regardless of the presence or absence of the three leprosy antigen preparations. This antigen nonspecific augmentation of proliferation by the wide range of doses of rIL-2 employed makes difficult the interpretation of the enhanced thymidine incorporation noted when rIL-2 is added in the presence of antigen to cultures of lymphocytes from LL patients. Our studies are at variance with reports that leprosy antigens, specifically PGL-I, induce immunological suppression, in that mycobacterial antigens did not cause significant suppression of the ConA-induced proliferations of PBMC from patients.

Mycobacterium leprae antigen-induced suppression of T cell proliferation in vitro.

The extent to which M. leprae and its products induced suppression of T lymphocyte proliferation in vitro was evaluated. M. leprae antigens suppressed T cell proliferation in response to mitogens and antigens in both lepromatous and tuberculoid patients, as well as controls never exposed to M. leprae or M. leprae endemic areas. Both soluble and particulate fractions of M. leprae were found to suppress proliferation in a dose-dependent manner. The extent of suppression was inversely related to the proliferative response of the donors mononuclear cells to M. leprae. Evidence indicates that M. leprae contains both stimulatory and suppressive molecules for T cells. One such suppressive antigen, Lipoarabinomannan (LAM)-B of M. leprae, also suppressed the proliferative response of tuberculoid patients. Suppression was also observed with the LAM-B of M. tuberculosis. The suppressive effects observed were not due to the toxicity of the antigen. Some of the suppressive activity was mediated by T8+ suppressor cells and was expressed in both lepromatous and tuberculoid patients. We suggest that previous sensitization to M. leprae and other cross-reactive mycobacterial antigens determines the sensitivity of T cells to the suppressive effects of M. leprae antigens.

Lepromin-induced suppressor cells in lepromatous leprosy.

The presence or absence of suppressor cells in leprosy patients was investigated by measuring peripheral blood lepromin-induced suppression of the Con A response. Significant suppressor activity was measured in 15 of 15 untreated or recently treated patients with lepromatous leprosy and 3 of 5 patients with borderline lepromatous leprosy. In addition, in patients with lepromatous leprosy, suppressor cell activity was found in 10 of 14 patients that had been under treatment for more than 1 year but in only 2 of 27 patients who had active or thalidomide controlled erythema nodosum leprosum. Suppression was observed in only 5 of 29 tuberculoid leprosy patients, 1 of 6 patient contacts, and 0 of 11 normal controls. The differences between the lepromatous or borderline lepromatous group as compared with the tuberculoid group were statistically significant (P less than 0.001). Our findings confirm the presence of lepromin-triggered suppressor cells in the peripheral blood of patients with lepromatous leprosy. These suppressor cells may contribute to the selective unresponsiveness of lepromatous patients to the antigens of Mycobacterium leprae.

Suppressor response in lepromatous leprosy patients: role of Leu 2a cells.

The contribution of non-specific suppressor mechanisms to the overall immunoregulatory defect observed in lepromatous leprosy was evaluated. Con A-induced suppression was assayed using the standard two-stage test in 27 lepromatous leprosy patients, 19 of them during the quiescent stage (LL) and eight during erythema nodosum lepromatosum (ENL). Lymphocytes from normal individuals react in this assay, yielding higher suppression as the numbers of Con A-induced suppressor cells (Leu 2a+ cells) increase. In contrast, two patterns of response were observed in both LL and ENL patients: those giving lower suppression as the number of suppressor cells increased (LL-A and ENL-A) and those responding with the normal pattern (LL-B and ENL-B). The abnormal dose-response profile was not related to the disease stage, as both ENL and LL patients were included in groups with normal or atypical response. Reaction of the potential suppressor cells with anti-Leu 2a antibody abolished suppression in LL-B and normals, whereas Con A-induced suppression was unchanged or higher in ENL-A, ENL-B and LL-A, indicating that in these patients Leu 2a+ cells interfered with the generation of Con A-induced suppression. The contribution of spontaneous suppression was examined and it was shown that suppressor activity in the absence of Con A stimulus was higher in ENL (both ENL-A and ENL-B) and LL-A. Thus, it appears that the occurrence of high spontaneous suppressor activity, probably related to in vivo activation, is associated with a relative inability to generate de novo suppression after Con A stimulation in these patients.

Phenolic glycolipid-I of Mycobacterium leprae induces general suppression of in vitro concanavalin A responses unrelated to leprosy type.

Using a costimulant assay, in vitro Con A responses of patients across the leprosy spectrum were found to be markedly suppressed by phenolic glycolipid-I (PGL-I), a unique antigen of M. leprae. The degree of inducible suppression as well as the number of leprosy patients showing suppression of mitogenic responses was higher with PGL-I as compared with integral M. leprae (p less than 0.05 to less than 0.01). Both untreated lepromatous (60%) as well as tuberculoid leprosy (67%) patients showed significant suppression ranging from 13 to 64% and 12 to 79%, respectively. Thus, PGL-I appears to have a universal suppressive effect on Con A responses and is unlikely to play a central role in determining the leprosy spectrum.

An analysis of in vitro T cell responsiveness in lepromatous leprosy.

In lepromatous leprosy, there is extensive replication of Mycobacterium leprae (M. leprae) within dermal macrophages. This lack of microbial resistance has been attributed to a defective cell-mediated immune response to M. leprae antigens. We have examined the in vitro response of T cells to M. leprae to determine if hyporesponsiveness could be reversed. The study included 40 unselected patients from New York and from Colombia, most with the severe lepromatous form of the disease. We first noted that lepromatous leprosy patients were of two types: those unable to respond, as assessed by T cell proliferation and immune (gamma) interferon (IFN-gamma) release, and a second group, exhibiting low but detectable responses relative to tuberculoid controls. When the effect of exogenous recombinant interleukin-2 (IL-2) on the response to M. leprae antigens was compared in the two groups, many of the low responders, but not the nonresponders, showed enhanced proliferation and IFN-gamma release. To evaluate a possible suppressive effect of monocytes, these cells were eliminated with a cell-specific monoclonal antibody and complement. Depletion of monocytes often expanded preexisting weak responses but did not reverse the anergy of the M. leprae nonresponders. The enhancement was not M. leprae-specific, since it was also observed when bacillus Calmette-Guerin was the antigenic stimulus for proliferation and IFN-gamma production. Removal of the suppressor T cell subset, with OKT8 antibody and complement, also did not restore responses in nonresponder patients. We conclude that a sizable number of lepromatous leprosy patients exhibit a low degree of responsiveness to M. leprae and that the responses can be enhanced in vitro with IL-2 or with monocyte depletion. Nonresponsiveness, however, cannot be reversed. Since currently available assays measure the function of previously sensitized T cells, suppressor mechanisms may yet contribute to defective cell-mediated immunity by impairing the initial sensitization to M. leprae antigens.

Lepra lepromatosa: estudio de algumas subpoblaciones de linfocitos y su análisis funcional / Lepromatous leprosy: study of some subpopulations of lymphocytes and its functional analysis

Se investigaron en un grupo de pacientes de lepra lepromatosa y en otro de sujetos clínicamente sanos los niveles de linfocitos T totales por medio de formación de rosetas E, determinación de linfocitos T supresores Fc-gamma y de la capacidad de transformación blastoide de sus células linfoides cuando se estimularon con concanavalina A. La determinación de linfocitos T por medio de rosetas E mostró que los pacientes tenían disminución en comparación con el grupo testigo de sujetos normales. La determinación de linfocitos T supresores Fc-gamma evaluados por la formación de rosetas con eritrocitos de pollo sensibilizados con IgG, demostró en los enfermos un incremento significativo en los niveles de dichos linfocitos T supresores. En el cultivo de linfocitos de sangre periférica, la transformación blastoide inducida por estimulación con concanavalina A y medida por incorporación de timidina tritiada presentó niveles bajos en los pacientes leprosos. Los estudios mencionados demostraron que los enfermos de lepra lepromatosa presentan un número significativamente superior de linfocitos T supresores que los mostrados en sujetos clínicamente sanos, y que aquéllos pueden ser los causantes de la transformación insuficiente de sus células linfoides tras el estímulo mitogénico de linfocitos T

Accessory cell function of cells of the mononuclear phagocyte system isolated from mycobacterial granulomas.

Epithelioid cells from BCG-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas were examined for their ability to act as accessory cells for T-cell proliferation to mitogen (Con A) and antigen (PPD). The granuloma cells were separated on a FACS using monoclonal antibody specific to guinea pig macrophages. Epithelioid cells (which are Ia negative) were able to support proliferation to Con A but not to antigen. Cultures containing Ia positive granuloma macrophages from M. leprae sensitized animals did not show responsiveness to Con A or to PPD. Oil-induced peritoneal exudate macrophages from BCG or M. leprae immunized animals were able to act as accessory cells for both mitogen and antigen proliferation. The nonresponsiveness of cultures containing epithelioid cells stimulated with PPD or M. leprae granuloma macrophages stimulated with Con A was not due to suboptimal or supraoptimal accessory cell:lymphocyte ratios.

Cell-mediated immunity in mice treated with Mycobacterium leprae or with macrophages harbouring M. leprae.

Following treatment of BALB/c or C3H/HeN mice in the hind footpads with irradiated Mycobacterium leprae, a marked enhancement of natural killer (NK) activity was observed in cells from the draining popliteal lymph node or from the spleen. NK activity was further enhanced when the treatment consisted of killed M. leprae which had been incorporated into mouse peritoneal macrophages. This effect was noted as early as 2 weeks after treatment and persisted for at least 9 weeks. Lymphoblastic transformation in response to suboptimal doses of the T-cell mitogen transformation in response to suboptimal doses of the T-cell mitogen concanavalin A or to M. leprae antigen was assayed in parallel in cells from the draining popliteal lymph node and from the spleen. In contrast to NK assays, treatment with M. leprae alone moderately altered the response to mitogen. However, there was a prominent enhancement of the T-cell response when treatment consisted of M. leprae-laden macrophages.