Recovery of IFN-gamma levels in PBMCs from lepromatous leprosy patients through the synergistic actions of the cytokines IL-12 and IL-18.

The shift to the production of a Th1 cytokine profile during an intracellular infection has been shown to depend on antigen presenting cells-derived IL-12 and T-cell-derived IFN-gamma production. IL-18 facilitates Th1 priming in synergy with IL-12 through the stimulation of IFN-gamma production by T cells, B cells, NK cells, macrophages and DCs. A low level of IFN-gamma production in PBMC cultures from lepromatous leprosy patients (LL) has been previously reported by several groups. We evaluated the synthesis of this cytokine after exogenous addition of recombinant IL-12 and IL-18 (IL12/IL18) in order to induce recovery of the IFN-gamma levels with Mycobacterium leprae antigenic stimulation. The aim of this study was to investigate if exogenous addition of IL12/IL18 to PBMC cell cultures in the presence of M. leprae antigens could induce recovery of IFN-gamma levels. We found that IFN-gamma levels in PBMCs cultured from LL patients were reestablished after exogenous addition of exogenous IL12/IL18 and we also observed a diminished IL-18R expression. Although the molecular mechanisms of IL12/IL18 synergy have not been clearly elucidated, we assume that recombinant cytokines can activate several transcription factors that induce IFN-gamma synthesis.

Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset.

The efficacy of thalidomide (alpha-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor alpha. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2-mediated T cell proliferation and interferon gamma production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset.

Proliferative responses of T cells from the skin and nerve lesions of leprosy patients.

In the current study we compared the mitogenic responses of T cells from skin and nerve biopsies of leprosy patients with those of peripheral blood mononuclear cells (PBMC). Lymphocytes from these sources were cultured at < or = 100 cells/well in the presence of PHA, irradiated autologous feeder cells, and IL-2, and proliferation was assessed after 6 to 12 days. Whereas PBMC were capable of vigorous responses, the growth of cells from skin and nerve was markedly reduced. The diminished response was independent of the clinical status of leprosy patients and was also observed in skin-infiltrating lymphocytes from patients suffering from other disorders. Analysis of proliferative responses at 1 cell/well suggested both a reduction in precursor frequency and a decrease in mean burst size. Analysis of lymphokine production suggested that cultured cells from skin lesions had reduced IL-w and IL-4 production relative to PBMC generated under similar conditions. Equal numbers of CD3+ cells were present in each source, but lesion cells were enriched in CD45RA- "memory" T cells, as well as CD3+CD28+ T cells. However, these alterations in subpopulation distribution could not account for the substantial differences in proliferative potential. We conclude that significant differences exist in the activation potential of cells from different tissue sources.

Analysis of cytokine production by Mycobacterium-reactive T cells. Failure to explain Mycobacterium leprae-specific nonresponsiveness of peripheral blood T cells from lepromatous leprosy patients.

Recent analyses of antimycobacterial T cells clones from a small number of individuals indicate that mycobacteria preferentially induce Th cells that produce high levels of IFN-gamma and no or little IL-4 in Mycobacterium leprae-resistant tuberculoid leprosy (TT) patients and healthy subjects, whereas in one study M. leprae-induced Ts clones from polar lepromatous leprosy (LL) patients showed a reciprocal cytokine secretion profile and mediated their suppressive activity via the release of high levels of IL-4. We have evaluated these findings in peripheral blood T cells from a larger panel of TT and LL patients as well as healthy individuals. Mycobacterium-reactive T cell lines generated from the PBMC of these individuals were tested for cytokine secretion and proliferative capacity in response to M. leprae, Mycobacterium tuberculosis, and various individual mycobacterial Ag. The lepromatous pole of the leprosy spectrum was additionally investigated by analyzing the cytokine-secretion profile of M. leprae-induced (suppressor) T cell clones as well as primary ex vivo PBMC. All T cell lines from healthy individuals and TT patients responding to M. leprae, M. tuberculosis, or individual Ag, produced high levels of IFN-gamma and TNF-alpha but little or no IL-4 and IL-6. At the lepromatous pole, T cell lines failed to proliferate upon stimulation with M. leprae but in some cases produced significant levels of IFN-gamma. No IL-4 or IL-6 secretion was observed in response to M. leprae. These lines displayed strong proliferation and Th1-like cytokine production upon stimulation with M. tuberculosis. Similarly, stimulation of primary PBMC from LL patients with M. leprae or M. tuberculosis resulted in the release of IFN-gamma but no detectable IL-4 production. Control tetanus toxoid-reactive T cell lines from the same individuals instead produced large amounts of IL-4 and low levels of IFN-gamma. The analysis of M. leprae-induced T cell clones, including those with known suppressive activity, revealed that all lepromatous T cell clones produced large amounts of IFN-gamma. Most of these clones released no or little IL-4, but some clones produced higher levels of IL-4 in addition to IFN-gamma. Most clones tested produced IL-10 as well. The suppressor activity of suppressor T cell clones could not be inhibited by a neutralizing anti-IL-4 antibody and only in one case by neutralizing anti-IL-10 antibody. Anti-IL-4 and anti-IL-10 could not overcome the M. leprae-specific unresponsiveness observed in primary PBMC from LL patients.(ABSTRACT TRUNCATED AT 400 WORDS)

[Immunologic indices in a varying course of lepromatous leprosy]. / Immunologicheskie pokazateli pri razlichnom techenii lepromatoznoi lepry.

The study of the proliferative and regulatory functions of lymphocytes in patients with lepra of the lepromatous type has shown that at the active stage of the disease both the response of lymphocytes to mitogens and their suppressor functions are decreased. During the regression of the disease these characteristics are restored to the normal level only in patients with the relapse-free course of the disease, while patients with relapses in their medical history retain the low level of such characteristics. It is expedient to use these cell-mediated immunity characteristics as signs permitting the formation of risk groups of patients who may expect the relapse of the disease.

Leprosy in a mangabey monkey--naturally acquired infection.

Naturally acquired leprosy was detected in an otherwise normal "sooty" mangabey monkey (Cercocebus atys). This animal was imported from West Africa in 1975 and developed clinical symptoms of leprosy in 1979. Histopathologic findings were those of subpolar-lepromatous to borderline-lepromatous leprosy in the Ridley-Jopling classification. The disease was progressive, with crippling neuropathic deformities of the hands and feet. The disease regressed under specific therapy. The etiologic agent was identified as Mycobacterium leprae by the following criteria: invasion of nerves of host, staining properties, electron microscopic findings, noncultivable on mycobacteriologic media, DOPA-oxidase positive, lepromin reactivity, infection patterns in mice and armadillos, sensitivity to sulfone, and DNA homology. We believe the animal acquired the disease from a patient with active leprosy. The mangabey monkey offers promise as a primate model for leprosy, and adds a third reported species to animals with naturally acquired leprosy.

Serum inhibitory factor in lepromatous leprosy: its effect on the pre-S-phase cell-cycle kinetics of mitogen-stimulated normal human lymphocytes.

The sera of ten Egyptian man with long-standing lepromatous leprosy (LL) (mean duration 17.4 years) that had failed to respond to dapsone treatment were shown to inhibit mitogen stimulation responses of normal human lymphocytes. When first tested, the sera partly inhibited the response to phytohaemagglutinin (PHA) and pokeweed mitogen and virtually abolished that to concanavalin A (Con A): after repeated freezing and thawing, the Con A inhibition had disappeared, whereas the PHA response was still partly inhibited. The inhibitory serum factor(s) had similar actions on lymphocytes from each of six normal donors. Although the sera varied in potency, they showed similar dose-response curves when tested against lymphocytes from a single donor. The principal action of the sera was to reduce the number of cells responding to mitogen, without modifying the kinetics of recruitment or rate of volume growth during G1-phase in those cells that were unaffected by the inhibitory substances(s). Study of PHA dose-response curves and of the effect of delayed addition of LL serum suggested that the serum factor(s) act by diminishing the responsiveness of the cells, rather than by reducing the concentrations of free mitogen or by blocking cell membrane mitogen receptors. The serum from one apparently healthy attendant, who had nursed leprosy patients for 30 years but who did not have leprosy or other chronic infective disease, inhibited completely stimulation by all three mitogens in a manner different from that of LL sera. Serum from the other 13 control patients did not modify the response of normal lymphocytes to stimulation by any of the three mitogens studied. It was concluded that the inhibitory factor(s) in the serum of patients with LL were a consequence of the disease and not of the environment in which the patients lived. Microscopy confirmed that the techniques used for recovery of the cultured cells did not introduce bias into the volume spectroscopy measurements.

Mitogen-induced DNA synthesis in various mouse strains infected with a large or small dose of murine leprosy bacilli.

Mice of the C57BL strain have been shown to be rather resistant to infection with Mycobacterium lepraemurium, whereas C3H mice are highly susceptible. Accordingly, it seemed to be somewhat paradoxical that enhanced antibody formation coupled with a depressed state of cell-mediated immunity as expressed by negative macrophage migration inhibition tests was observed not in C3H but in C57BL mice when they were inoculated with a large dose of murine leprosy bacilli, as reported in our previous studies. In the present study mitogen-induced DNA synthesis by lymph node cells was examined in 16 strains of mice which had been infected with a large or small dose of M. lepraemurium. According to the response to two kinds of T-cell mitogens, these mouse strains could be roughly divided into three groups consisting of two polar groups represented by C57BL/6J and C3H/HeN, respectively, and one intermediate between them. Furthermore, both humoral and cellular immune responses so far observed in C57BL and C3H mice were substantiated by DNA synthesis by lymph node cells harvested from these strains of mice and then exposed in vitro to B-cell and T-cell mitogens, respectively. However, no correlation was found between mitogen-stimulated DNA synthesis by these 16 strains of mice and their H-2 specificity.

Concanavalin A induced suppressor activity in human leprosy.

Peripheral blood lymphocytes from nine normal subjects and 40 patients with leprosy were pretreated in vitro with Concanavalin A (Con A). Cells from normal subjects pretreated for 24 hours showed consistent and effective generation of suppressive activity which inhibited mitogen induced transformation of autologous lymphocytes. Prolongation of Con A pretreatment to 40 hours resulted in maximal suppressive activity. Tuberculoid leprosy patients had lymphocytes in their blood which on 24 hour pretreatment with Con A exerted suppressive effects markedly greater than the maximal suppression noted with 40 hour pretreated normal lymphocytes. In contrast, untreated patients with polar lepromatous leprosy showed a decrease in suppressive activity which could not be altered by prolongation of Con A pretreatment: the loss of suppressive activity noted in this form of leprosy was restored during erythema nodosum leprosum.

A placebo controlled clinical trial of transfer factor in lepromatous leprosy.

The effects of repeated injections of transfer factor over a period of 20 weeks were investigated in fourteen bacteriologically positive patients at the lepromatous side of the leprosy spectrum. All patients showed negative (0 mm induration) skin tests to M. leprae antigens (i.e. leprolin and lepromin). Of these patients, seven were treated with transfer factor with a total of 9 units (1 unit being equivalent to 5 x 10(8) lymphocytes) and seven with a placebo. Maintenance treatment with clofazimine was continued. Transfer factor was prepared from the lymphocytes of donors who showed positive skin tests to M. leprae antigens (i.e. leprolin greater than or equal to 12 mm induration, average 15.5 mm or lepromin greater than or equal to 8 mm induration, average 13.6 mm), as well as a positive lymphocyte transformation in vitro to M. leprae (the average transformation being higher than the average transformation of lymphocytes of tuberculoid leprosy patients). No differences were found between the two groups as regards the clinical course of the disease, the histopathological and bacteriological evaluation of skin biopsies, changes in skin test reactivity to various antigens (i.e. lepromin, leprolin, PPD, Mumps, C. albicans, Tr. rubrum and Varidase), as well as the lymphocyte transformation in vitro to various mitogens (i.e. PHA, PWM, Con A) and antigens (i.e. M. leprae, leprolin, PPD, BCG, Mumps, C. albicans, Trichophyton and Varidase). No evidence was found to suggest that transfer factor is a valuable adjuvant in the treatment of lepromatous leprosy patients or that it increases cell-mediated immune reactivity towards M. leprae.