Effect of recombinant interferon-gamma on hydrogen peroxide-releasing capacity of monocyte-derived macrophages from patients with lepromatous leprosy

Monocyte-derived macrophages from 14 patients with lepromatous leprosy respond to rIFN-gamma with an enhanced secretion of H2O2 in a fashion similar to that of cells obtained from normal donors. The activation is not dependent on the cutaneous bacterial index, the length of treatment, or the stage and activity of the disease. H2O2 release can be triggered in these cells both by phorbol myristate acetate and by intact irradiated Mycobacterium leprae. Uptake of M. leprae by both normal donors' and patients' macrophages is proportional to the number of bacilli added. Prior ingestion of M. leprae does not interfere with the ability of macrophages to respond to IFN-gamma by the production of oxygen intermediates. We conclude that the immune defect in lepromatous leprosy probably results from a lack of response to M. leprae by the patients' T cells rather than an inability of mononuclear phagocytes to respond to IFN-gamma.     

Emergence of an effective adaptive cell mediated immune response to Mycobacterium leprae is not impaired in reactive oxygen intermediate-deficient mice

Cytokine-activated macrophages (MPhi) employ reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to combat pathogens. The requirement for ROI for an effective host response to experimental leprosy using mice which have a disruption in the 91-kD subunit of the NAPDH oxidase cytochrome b (phox91-/-) was examined. Mycobacterium leprae multiplication in phox91-/- foot pads (FP) was elevated early in infection but subsequently arrested similarly to control mice within a noninvasive granuloma. Using a modified lepromin test model, a similar cellular composition in the M. leprae-induced FP granuloma in both strains with lymphocyte infiltration consisting primarily of CD4+CD44(hi)CD62L(lo) effector cells was found. Of great interest was the disparity in the T cell population between the granuloma and the draining lymph node which contained predominantly na?ve CD4+CD44(lo)CD62L(hi) cells and was, therefore, not representative of the infection site. TH1 cytokines, chemokines and inducible nitric oxide synthase were comparably expressed in the FP of both strains. When infected in vitro, normal MPhi from B6 and phox91-/- mice supported bacterial viability, whereas IFNgamma-activated MPhi killed M. leprae in a RNI-dependent manner, emphasizing that ROI was dispensable. These data show that phox91-/- mice generate a strong adaptive immune response and control long-term infection with M. leprae.     

Activation of human monocytes in leprosy

In leprosy, the common etiologic agent is the same Mycobacterium leprae, but the clinical manifestations are various, including the tuberculoid and lepromatous types. In tuberculoid type leprosy, macrophages in the granuloma differentiate into epithelioid cells; in the lepromatous type, in contrast, they differentiate into lepra cells containing multiple M. leprae. Thus host factors, which regulate macrophage activities, determine the type of leprosy. To understand such regulation of macrophage activities, we assayed superoxide production, hydrogen peroxide production and glucose consumption in monocytes in vitro. Glucose consumption spontaneously increased, with lymphokine enhancing the consumption rate. Superoxide production increased spontaneously and decreased from the 4th day; lymphokine added on the 4th day supressed the decrease of superoxide production. Hydrogen peroxide production increased until the 3rd day of culture. Twenty-four hour incubation with lymphokine, from day 0 to the 1st day, had no effect on hydrogen peroxide production, while from the 2nd to 3rd day it enhanced such production. Supernatants of lymphocytes incubated with M. leprae were prepared from tuberculoid and lepromatous patients. Tuberculoid supernatant enhanced reactive oxygen production and glucose consumption, while that from lepromatous patients had no remarkable effect on glucose consumption or reactive oxygen production. The range of spontaneous increase and decrease of reactive oxygen production was greater than the regulatory effect of lymphokine on these activities. These data show that rapid provision of new monocytes to the granuloma is one of the important factors in the defense mechanism, that lymphocytes separated from lepromatous patients are not activated in response to M. leprae antigen, and that they do not secrete corresponding lymphokines.     

An indomethacin sensitive suppressor factor released by macrophages of leprosy patients

Reduction inF _c receptor expression as assayed by ‘erythrocyte’ rosetting of macrophage cultures from long term treated lepromatous leprosy patients (bactereologically negative) was seen in the presence of viableMycobacterium leprae. Macrophages with and without intracellular bacilli demonstrated this reduction. On the basis of this observation the conditioned medium ofMycobacterium leprae infected macrophage cultures of lepromatous patients, were tested on macrophages from normal individuals for [³H]-leucine incorporation and antigen specific physical interaction with lymphocytes. Both these parameters showed decreased values as compared to the controls which were not exposed to this conditioned medium. Lymphocyte transformation toMycobacterium leprae in leucocyte cultures of normal individuals was also reduced in the presence of the conditioned medium from lepromatous patients’ macrophages. The indication that this factor may be a prostaglandin was suggested by the observation that its synthesis was inhibited by indomethacin. Its importance in the non-specific depression in cell-mediated immunity seen in lepromatous patients is discussed.     

Glycyrrhizin inhibits neutrophil-associated generation of alternatively activated macrophages

Patients with severe burn injuries are extremely susceptible to infection, and the host's antibacterial responses are frequently suppressed by alternatively activated macrophages (M2Mphi), commonly demonstrated in these patients. An immunosuppressive subset of neutrophils (PMN-II), demonstrated in the peripheral blood of thermally injured patients, has been described as an inducer of M2Mphi. In the present studies, the inhibitory effect of glycyrrhizin (GL) on M2Mphi generation stimulated by PMN-II was examined. M2Mphi were generated from resident Mphi (R-Mphi, lower chamber) after cultivation with PMN-II (upper chamber) in a dual-chamber transwell. However, M2Mphi were not generated from R-Mphi when the same transwell cultures were performed in the presence of GL. M2Mphi were not generated from R-Mphi after cultivation with PMN-II previously treated with GL, while R-Mphi previously treated with GL converted to M2Mphi after they were cultured with PMN-II in transwells. Interleukin-10 and CCL2 released from PMN-II were shown to be effector molecules responsible for the generation of M2Mphi. However, these soluble factors were not produced by PMN-II treated with GL. These results indicate that GL inhibits PMN-II-stimulated M2Mphi generation through the inhibition of CCL2/interleukin-10 production by PMN-II.     

L-arginine-dependent macrophage effector functions inhibit metabolic activity of Mycobacterium leprae

Recently, L-arginine has been shown to be a necessary substrate for murine-activated macrophage-mediated tumor cytostasis and microbiostasis of certain fungi, bacteria, and intracellular protozoa. We report here the effects of the L-arginine-dependent pathway of activated mouse macrophages (MO) on the obligate intracellular prokaryote, Mycobacterium leprae. Due to the inability to culture M. leprae in vitro, a simple, quantitative assay was employed to measure the metabolism/viability of M. leprae released from MO: the metabolic capacity of M. leprae to oxidize 14C-palmitic acid to 14CO2. Murine normal MO or MO activated in vitro with IFN-gamma or in vivo by injection with Corynebacterium parvum were infected with viable M. leprae freshly harvested from the footpads of nu/nu mice. Activated MO strikingly inhibited the metabolism of M. leprae; however, in L-arginine-free medium or in medium containing L-arginase, the inhibitory effects of activated MO on M. leprae metabolism were abolished. The competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, also blocked the inhibitory effects of activated MO for M. leprae, but the addition of supplemental L-arginine overcame the NG-monomethyl-L-arginine-induced block. Furthermore, in the culture supernatants, the levels of NO2-, an end product of L-arginine degradation, were directly proportional to the ability of the activated MO to inhibit M. leprae metabolism. These data present five lines of evidence that suggest that activated MO utilize the L-arginine-dependent pathway to cope with M. leprae.     

Mechanisms of Mycobacterium leprae-specific T-cell deficiency in lepromatous leprosy

Patients suffering from lepromatous leprosy fail to develop an efficient cell-mediated immunity towards Mycobacterium leprae, the causative agent. The mechanism of such a specific T-cell tolerance to the bacillus remains a key question in the pathophysiology of leprosy. Macrophages do not show any intrinsic defect in phagocytizing and killing M. leprae or in presenting antigen to helper T-cells. On the other hand, M. leprae-reactive helper T-cells do persist in lepromatous patients, but their activation appears to prevented by active suppressor mechanisms, involving both suppressor T-cells and macrophages. The target of this specific suppression could be the interleukin 2-producing T-cell subset. A better molecular definition of M. leprae antigens, both by monoclonal antibodies and T-cell clones, should open new perspectives for further analysis of the regulation of immune responses to M. leprae.     

T cell defect in lepromatous leprosy is reversible in vitro in the absence of exogenous growth factors

T lymphocytes from patients with lepromatous leprosy (LL) characteristically fail to respond to Mycobacterium leprae. This specific immunologic defect is thought to contribute to the aggressive clinical course that typifies patients with LL. We report that although fresh CD4+ (helper) T cells from most LL patients are specifically unresponsive to M. leprae, after culture in medium alone for 48 hr the same cells respond to M. leprae antigens. The recovery of T cell function is specific for M. leprae, occurs at the level of responder CD4+ T cells, and is not affected by monocytes or CD8+ (suppressor) T cells. Recovery of T cell reactivity is blocked by the presence of M. leprae bacilli in the preculture medium. These findings indicate that despite the apparent specific anergy seen in patients with LL, the T cells of most LL patients can respond to M. leprae. Their failure to do so, in vivo, may be due to the persistence of antigen, which renders antigen-reactive T cells nonresponsive either directly or via activation of CD4+ suppressor cells.     

A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy

The interaction of CD40 ligand (CD40L) expressed by activated T cells with CD40 on macrophages has been shown to be a potent stimulus for the production of IL-12, an obligate signal for generation of Th1 cytokine responses. The expression and interaction of CD40 and CD40L were investigated in human infectious disease using leprosy as a model. CD40 and CD40L mRNA and surface protein expression were predominant in skin lesions of resistant tuberculoid patients compared with the highly susceptible lepromatous group. IL-12 release from PBMC of tuberculoid patients stimulated with Mycobacterium leprae was partially inhibited by mAbs to CD40 or CD40L, correlating with Ag-induced up-regulation of CD40L on T cells. Cognate recognition of M. leprae Ag by a T cell clone derived from a tuberculoid lesion in the context of monocyte APC resulted in CD40L-CD40-dependent production of IL-12. In contrast, M. leprae-induced IL-12 production by PBMC from lepromatous patients was not dependent on CD40L-CD40 ligation, nor was CD40L up-regulated by M. leprae. Furthermore, IL-10, a cytokine predominant in lepromatous lesions, blocked the IFN-gamma up-regulation of CD40 on monocytes. These data suggest that T cell activation in situ by M. leprae in tuberculoid leprosy leads to local up-regulation of CD40L, which stimulates CD40-dependent induction of IL-12 in monocytes. The CD40-CD40L interaction, which is not evident in lepromatous leprosy, probably participates in the cell-mediated immune response to microbial pathogens.     

Surface membrane changes in lepromatous macrophages affecting the adherence ofMycobacterium leprae

Macrophages from lepromatous leprosy patients showed poor adherence toMycobacterium leprae. The phagocytic activity of the macrophages was not correlated to the influence on the adherence ability. Based on the phagocytic behaviour of macrophages from normal individuals and from lepromatous leprosy, patients as well as the action of neuraminidase in reversing the extent of adherence, it is suggested that macrophages from lepromatous leprosy patients differ from those from normal individuals in regard to their surface properties. There was no relationship between the degree of adherence and the concentration of Fc receptors of the macrophages. It was also shown that an extract of lysed macrophages from lepromatous leprosy patient was able to reduce the adherence ofMycobacterium leprae to normal macrophages. This study shows that adherence is a good indicator of the surface property of macrophages which in turn could play an important role in the cell mediated immunity of the patient. The observations suggest altered macrophage membrane structure in the long term-treated, otherwise normal, lepromatous leprosy patients.     

A lipopeptide facilitate induction of Mycobacterium leprae killing in host cells

Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells.     

Experimental and Clinical Studies on Rifampicin in Treatment of Leprosy

Rifampicin showed high activity against experimental leprosy, inhibiting the multiplication of dapsone-sensitive and dapsone-resistant strains of Mycobacterium leprae in mice fed 5 mg./kg. body weight. In a formal pilot-type trial on six previously untreated patients with active lepromatous leprosy, rifampicin (600 mg. daily by mouth) was as effective as standard treatment with dapsone. Myco. leprae, however, appeared to be killed more rapidly by rifampicin than by dapsone or other antileprosy drugs so far studied. This was confirmed on a further 10 patients, including two with dapsone resistance, and from the infectivity in mice of bacilli recovered from patients during treatment with rifampicin or dapsone. These results are consistent with the bactericidal activity of rifampicin against other micro-organisms, which could be important to the chemotherapy of leprosy, since all antileprosy drugs in current use are bacteriostatic.     

Antibodies against BCG antigen 60 in mycobacterial infection.

A sensitive specific radioimmunoassay was developed to measure antibodies against BCG antigen 60, a prominent antigenic component of BCG bacilli which cross-reacts with similar components in many mycobacterial species including Mycobacterium leprae and M tuberculosis. A lepromatous serum pool had anti-BCG-60 activity with a titre of 10(5) and the tuberculoid pool a titre of 10(4). Testing of individual sera showed striking variations within groups of patients with lepromatous and tuberculoid leprosy. In five of the 20 tuberculoid leprosy sera the anti-BCG-60 activity was above the median for the lepromatous group. The current view that antibody formation against mycobacterial antigens is very low in tuberculoid leprosy thus no longer appears to be tenable. Sera from eight patients with active pulmonary tuberculosis also showed a striking variation in anti-BCG-60 content, and the median value of this group was even higher than in those with lepromatous leprosy.     

Suppressor T lymphocytes from lepromatous leprosy skin lesions

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.     

Purine metabolism in Mycobacterium leprae grown in armadillo liver

Abstract Mycobacterium leprae organisms generally incorporated purines more rapidly than pyrimidines into nucleic acids from the incubation medium. Purine synthesis de novo took place at a very slow rate suggesting a preference of the organism for preformed purines. In cell-free extracts of leprosy bacilli, enzyme for scavenging and interconversion of purines were detected. The results are discussed in the light of the failure to cultivate M. leprae in vitro, and the use of labelled substrates to determine the viability of suspensions of leprosy bacilli and their sensitivity to anti-leprosy drugs.     

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.     

Subclinical Infection in Leprosy

Lymphocyte transformation has been used to study the immune response to Mycobacterium leprae among contacts and non-contacts of leprosy patients. Of 26 subjects living in a leprosy endemic area for less than two months none responded to M. leprae; 24% of subjects who had lived in an endemic area for more than a year gave a positive response to M. leprae; more than 50% of individuals with occupational contact of leprosy for more than a year responded; and about 50% of contacts of tuberculoid and treated lepromatous patients responded to M. leprae, while only 22% (4/18) of contacts of lepromatous patients treated for less than six months responded.It seems that leprosy is more highly infectious than is indicated by the prevalence of the disease and that a subclinical infection commonly follows exposure to M. leprae. The relatively low response found in contacts of active lepromatous patients suggests that in these contacts “superexposure” to M. leprae can bring about a decrease in host resistance.     

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.     

ELECTRON MICROSCOPE STUDY OF MYCOBACTERIUM LEPRAE AND ITS ENVIRONMENT IN A VESICULAR LEPROUS LESION.

Imaeda, Tamotsu (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela) and Jacinto Convit. Electron microscope study of Mycobacterium leprae and its environment in a vesicular leprous lesion. J. Bacteriol. 83:43-52. 1962.-Biopsied specimens of a borderline leprosy lesion were observed with the electron microscope. In this lesion, the majority of Mycobacterium leprae were laden with cytoplasmic components. The bacilli were separated from the cytoplasm of host cells by an enclosing membrane, thus differing from the environment of well-developed lepra cells in lepromatous lesions.The cell wall is composed of a moderately dense layer. A diffuse layer is discernible outside the cell wall, separated from it by a low density space. It is suggested that the cell wall is further coated by a low density layer, although the nature of the outermost diffuse layer has not yet been determined.The plasma membrane consists of a double layer, i.e., dense inner and outer layers separated by a low density space. The outer layer is closely adjacent to the cell wall. In the region where the outer layer of the plasma membrane enters the cytoplasm and is transformed into a complex membranous structure, the inner layer encloses this membranous configuration. Together they form the intracytoplasmic membrane system.In the bacterial cytoplasm, moderately dense, presumably polyphosphate bodies are apparent. As neither these bodies nor the intracytoplasmic membrane system are visible in the degenerating bacilli, it seems probable that these two components represent indicators of the state of bacillary activity.     

Infection and elimination of Mycobacterium leprae in SCID C.B.-17 mice (severe combined immunodeficiency)]

Previous studies documented that T-cell deficient nude mice failed to control M. leprae infection. In the present investigation we monitored the growth of M. leprae for up to 15 months in the SCID C.B.-17 mouse, a host deficient in both T and B lymphocytes. At 8 months post-infection 10(8) organisms/foot-pad were recovered from SCID mice vs 5 x 10(6) in normal BALB/c mice. Thereafter the number of bacilli decreased rapidly in mice infected with high-dose inoculum (10(7)); however, at all doses SCID mice eventually cleared M. leprae. During infection both T and B cells as well as serum Ig remained as low as in uninfected mice; however, in the spleen MAC-1+ cells which include macrophages and NK cells were substantially increased. These results suggest that MAC-1+ cells are involved in the anti-mycobacteria-1 defence mechanisms adopted by SCID mice to compensate their deficiency in T and B cells.