The effect of antigen presenting cells on the cytokine profiles of stable and reactional lepromatous leprosy patients.

In view of varied reports on the Th1/Th2 paradigm in leprosy, we used a novel real time (RT) fluorogenic reverse transcriptase based PCR (RT-PCR) to measure cytokine expression in peripheral blood cells from lepromatous leprosy patients with stable disease and those suffering from erythema nodosum leprosum (ENL/Type II) reactions. To evaluate the role of accessory cells in Th cell differentiation, co-expression of Th cytokines interferon gamma (IFNgamma) and interleukin (IL) 4 and regulatory cytokines IL 10 and IL 12 was compared in antigen stimulated peripheral blood mononuclear cells (PBMC), cultures containing T cells reconstituted with autologous monocytes (MO) and cultures containing T cells reconstituted with autologous dendritic cells (DC). 7/8 stable lepromatous leprosy patients showed co-expression of both IFNgamma and IL 4, suggesting a Th0 or a combination of Th1 + Th2 subsets in PBMC. The RT-PCR demonstrated that stable lepromatous patients and patients in ENL had significantly higher levels of IFNgamma mRNA molecules compared to IL 4. In fact, 5/8 ENL patients had undetectable levels of IL 4 mRNA, with a skewing of the cytokine response towards a Th1-like profile. Consistent with this. IL 12p40 mRNA molecules were significantly higher in the PBMC of ENL patients compared to stable lepromatous patients (P < 0.01). Reconstitution of purified T cells with autologous DC and MO from the stable lepromatous group resulted in down regulation of IL 4 (P < 0.03 for DC and P < 0.02 for MO) and IL 10 (P < 0. 01 for DC and P < 0.02 for MO), and a consequent skewing towards a Th1 profile similar to that seen in ENL patients. The fact that accessory cells could alter the cytokine profile in the reconstituted cultures suggests that they may play a role in determining Th subset differentiation in chronic diseases, and may influence the immunological stability of such diseases.

Dysregulation of IL-4 expression in lepromatous leprosy patients with and without erythema nodosum leprosum.

In order to increase our understanding of the immunological basis of erythema nodosum leprosum (ENL), we studied Th-like cytokine profiles in 130 leprosy patients, employing both the conventional and a novel, real-time, fluorogenic reverse transcriptase-based PCR (RT-PCR). The concomitant expression of both Th-like cytokines, interferon-gamma and IL-4, and the regulatory cytokines, IL-10 and IL-12, was studied in the peripheral blood cells of leprosy patients with and without ENL. In the conventional RT-PCR, varied cytokine profiles were observed in individual patients of all clinical types. Fifty-three percent of lepromatous patients without ENL and 59% of tuberculoid leprosy patients showed co-expression of IFN gamma and IL-4, indicating a non-polarized Th 0 pattern. Of the 36 patients with ENL, 58% demonstrated a polarized Th 1 pattern, with only 30% expressing both cytokines. Semiquantitative RT-PCR indicated a lower expression of IL-4 compared to that of IFN gamma in the lepromatous patients without ENL; the difference was even greater among those with ENL. The sensitive, real-time PCR confirmed the down-regulation of IL-4 and IL-10, with absence of IL-4 in half of the patients, resulting in skewing of the cytokine response toward a Th 1-like profile.

Lepromatous leprosy patients show T helper 1-like cytokine profile with differential expression of interleukin-10 during type 1 and 2 reactions.

Some leprosy patients suffer from clinical episodes associated with tissue damage which are designated as Type 1 (reversal reaction) when localized to the lesions and Type 2 (erythema nodosum leprosum, ENL) when accompanied by systemic involvement. We had reported earlier that stable, non-reaction lepromatous leprosy subjects show T helper 2 (Th2)- and Th0- but not Th1-like responses in the peripheral blood. To further understand the development of Th-like responses during disease, 32 lepromatous patients undergoing reactions were studied using cytokine-specific reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in peripheral blood and some skin biopsies. Of interest was the evidence of a Th1-like response with presence of interferon-gamma (IFN-gamma) and absence of interleukin-4 (IL-4) mRNA in the peripheral blood mononuclear cells (PBMC) of 85 and 64% of Type 1 and 2 reaction patients, respectively, and in all reaction sites. Whereas a Th0- was seen in some, a Th2-like response was absent. IL-12p40 mRNA was seen in 21/25 ENL and all Type 1 reaction subjects irrespective of the Th phenotype. IL-12p40 and IFN-gamma were detectable in unstimulated PBMC suggesting an in vivo priming during reactions. IL-10 was mainly associated with adherent cells and showed a differential expression in the two reactions. It was present in the PBMC of ENL but not in reversal reaction patients. Moreover, it was not detectable in the skin lesions of either type of reactions. A Th1-like cytokine profile was associated with immunopathology and persisted up to 6-7 months after the onset of reactions.

Expression and functional characterisation of the clpC gene of Mycobacterium leprae: ClpC protein elicits human antibody response.

This paper reports the expression of a previously described gene [Nath and Laal, Nucleic Acids Res. 18 (1990) 4935], currently identified as the clpC gene of Mycobacterium leprae, using an in vitro rabbit reticulocyte lysate-coupled transcription/translation system. The produced protein moved as a 95-kDa band on SDS-PAGE. An additional band of 79 kDa was seen which may have resulted from a GTG codon downstream to the initiating ATG in the clpC sequence. A threefold increase in synthesis of the 95-kDa protein was achieved by altering the translation codon context sequence of the ATG start codon. The ClpC (caseinolytic protease C) amino acid sequence, which contained two nucleotide-binding sites, exhibited in vitro ATP binding. Of functional significance was its immunoreactivity in human subjects with mycobacterial infection. Leprosy and tuberculosis patients with active disease had antibodies which recognised ClpC in dot ELISA.

Monocyte derived IL 10 and PGE2 are associated with the absence of Th 1 cells and in vitro T cell suppression in lepromatous leprosy.

Our previous studies had shown that the clinicopathological spectrum in leprosy was associated with discrete T cell subsets in circulation, with tuberculoid patients having antigen-induced Th 1, whereas lepromatous leprosy patients with antigen-specific T cell anergy possessed Th 2 cells. The present study shows that infected monocytes from lepromatous but not tuberculoid leprosy patients released soluble factors (MoF(s)) containing IL-10 and PGE2 which inhibited M. leprae induced in vitro lymphoproliferation of previously sensitised healthy or tuberculoid leprosy subjects. A strong negative correlation was observed between adherent cell derived IL-10 and IL-2 at the level of both the product and cytokine mRNA. Moreover, anti-IL-10 antibodies and indomethacin partially reversed the suppressor effects of MoF(s). Taken together these studies indicate that infected monocytes contribute to the development of T cell anergy by releasing factors that affect regulatory cytokines and T cell subset differentiation in lepromatous leprosy.

Cytokine profile of circulating T cells of leprosy patients reflects both indiscriminate and polarized T-helper subsets: T-helper phenotype is stable and uninfluenced by related antigens of Mycobacterium leprae.

Cytokine profiles of circulating mononuclear cells were studied with the aim of delineating T-cell subsets in leprosy patients with active disease. Using reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokine mRNA and enzyme-linked immunoassay (ELISA) for the secreted products, interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied. Three antigens, native Mycobacterium leprae, a recombinant antigen LSR/A15 of M. leprae and peptide 624 spanning 58-77 amino acids of the latter, were used to induce cytokine expression and release. Half of the subjects, irrespective of the clinical type or antigen used, showed a mixed T-helper type 0 (Th0)-like cytokine pattern, with evidence of the concomitant presence of IFN-gamma and IL-4. The remainder showed a polarized pattern based on the type of leprosy. Lepromatous patients with disseminated disease had Th2-type cytokines, with IL-4 but not IFN-gamma. In contrast, tuberculoid leprosy patients with localized disease showed a Th1-like profile, with the presence of IFN-gamma but not IL-4. Of interest was the stability of the Th phenotype for M. leprae-related antigens. Both the recombinant and the peptide antigens induced the same phenotype as the natural M. leprae bacillus in all except four of 45 leprosy patients.

Clinical utility of LSR/A15 gene for Mycobacterium leprae detection in leprosy tissues using the polymerase chain reaction.

Skin biopsy and slit-skin smears from 46 leprosy patients and 4 nonleprosy patients were tested for the presence of Mycobacterium leprae by the polymerase chain reaction (PCR) using primers based on the sequence of the LSR/15 kD gene. The PCR was found to be specific and sensitive, with a detection level of 10 and 100 bacilli. PCR using skin biopsies gave a higher detection rate than did slit-skin smears, probably due to the higher density of bacilli in a 4-mm punch biopsy. Dot blot hybridization with radioactive probes was 10-fold more sensitive than the ethidium bromide staining. Eight patients who did not show acid-fast bacilli in tissues by the conventional methods were shown to have PCR-amplified M. leprae DNA. False-negative results were obtained in 3 cases even though formal evidence for tissue inhibitors was absent.

Critical residues of the Mycobacterium leprae LSR recombinant protein discriminate clinical activity in erythema nodosum leprosum reactions.

We reported earlier (S. Singh, N. P. Shanker Narayan, P. J. Jenner, G. Ramu, M. J. Colston, H. K. Prasad, and I. Nath, Infect. Immun. 62:86-90, 1994) that polyclonal antibodies directed against selective sequences in the Mycobacterium leprae recombinant protein designated LSR were present in lepromatous leprosy patients undergoing erythema nodosum leprosum (ENL) reactions (type 2 reactions). In this study using peptides with single-residue deletions from positions 6 to 24, we define three distinct regions, GVTY, NAA, and RGD, which were important for antibody recognition and for the discrimination of clinically silent and active ENL reactions. Antibodies against NAA were found only in patients undergoing active reactions. This is in contrast to the results for the RGD motif, which was recognized in all ENL patients, irrespective of the clinical status. Though GVTY was recognized in both groups of patients, its recognition was masked by the flanking glutamic acid. These findings point towards a specific molecular recognition pattern that emerges when a lepromatous leprosy patient undergoes immune perturbations leading to ENL reactions. Moreover, the fine specificity of immunological recognition changes during the natural evolution of the host-parasite interaction.

Sera of leprosy patients with type 2 reactions recognize selective sequences in Mycobacterium leprae recombinant LSR protein.

Type 2 reactions (erythema nodosum leprosum [ENL]) are episodic, reactional states causing significant morbidity in lepromatous leprosy patients. With a view to defining the immunological differences between the stable and reactional forms of lepromatous leprosy, we determined antibody responses to LSR, a recombinant protein of Mycobacterium leprae previously described by us (S. Laal, Y.D. Sharma, H.K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. S. Mishra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991), as well as to 10- to 15-mer overlapping peptides synthesized on the basis of the LSR amino acid sequence. We report here the selective recognition of B cell epitopes by sera from patients with ENL as compared with a control group with nonreactional lepromatous leprosy. Peptides 2 and 3, with the sequences GVTYEIDLTNKNAA and IDLTNKNAAKLRGD, respectively, were recognized by > 95% of sera from patients with active ENL. Peptide 3 in addition showed reactivity with sera taken from 91.6% of lepromatous leprosy patients who were apparently stable but who were recorded to have had ENL several weeks before or after the sample collection. The core sequence IDLTNKNAA common to both these peptides may be a major target of humoral responses in ENL. In addition, the RGD motif at the C terminus appeared to influence the antigenicity of peptide 3 in enzyme-linked immunosorbent assay. It would appear that humoral responses during ENL are directed to selective antigenic determinants of the leprosy bacillus. The use of such serological markers to identify lepromatous leprosy patients with a high risk for developing ENL would be of clinical and predictive value.

Effect of recombinant interferon gamma administration on lesional monocytes/macrophages in lepromatous leprosy patients.

Hydrogen peroxide (H2O2) and superoxide anion (O2-) were estimated in lesional cells from 10 lepromatous leprosy patients injected intralesionally with recombinant interferon-gamma (rIFN-gamma). Clinically similar contralateral lesions injected with excipient served as controls. Lesional esterase-positive cells (suggestive of monocytes/macrophages) from rIFN-gamma-injected sites of many subjects showed net increments in the H2O2 and O2 levels compared to controls. When these cells were exposed to Mycobacterium leprae in vitro, there was a down-regulation of O2- in 4 of 5 subjects. Such inhibition was not observed in rIFN-gamma-injected sites. From the present studies it was not possible to determine whether the above effects of rIFN-gamma were primarily on lesional mature macrophages or on newly migrated young monocytes. Erythema and induration were observed at the cytokine-injected site but not at the control site between 24 and 72 hr. A monthly slit-skin smear examination of the former site showed a bacterial index (BI) reduction compared to the controls in 4 of 10 patients, this reduction occurring as early as 4 to 8 weeks. Histopathology of the biopsies of 6 of 10 subjects between 9 and 10 months showed a further BI decrease attributable to rIFN-gamma and not to the subsequently instituted chemotherapy.

Immunomodulation of human T cell responses with receptor selective enkephalins.

The delta-opioid receptor selective [2-D-penicillamine-5-D-penicillamine] enkephalin (DPDPE) and the mu receptor selective Tyr-D-Orn-Phe-Asp-NH2 (TOPA) were found respectively, to have marked immunostimulant and immunosuppressant activities in both normal subjects and patients suffering from leprosy and tuberculosis. Antigen specific lymphoproliferation and numbers of rosette forming T cells were significantly (P < 0.05) enhanced on in vitro treatment with Met-enkephalin. This was further increased (P < 0.001) in the presence of the delta selective DPDPE. In contrast, treatment with mu selective TOPA inhibited lymphoproliferation substantially (P < 0.01) and rosette formation to a lesser extent.

Recombinant fusion protein identified by lepromatous sera mimics native Mycobacterium leprae in T-cell responses across the leprosy spectrum.

Pooled polyvalent sera from lepromatous leprosy patients were used to screen a lambda gt11 recombinant DNA expression library of Mycobacterium leprae in order to identify the relevant antigens recognized by the human immune response. Of the 300,000 phages screened, 4 clones were identified that coded for fusion proteins of the same molecular mass. The fusion protein from clone LSR2 was tested for immunoreactivity in assays using peripheral blood cells and sera from 11 laboratory personnel and 105 patients across the leprosy spectrum. LSR2 protein appears to be predominantly a T-cell antigen. It evokes similar lymphoproliferative responses as the native bacillus both at the individual level and in the leprosy spectrum as a whole. Though only 50% of patient sera with anti-M. leprae antibodies reacted with the fusion protein, the pattern of reactivity in the antibody responses was also similar for the various clinical types. The coding regions of clones LSR1 and LSR2 are identical. They show no homology with sequences stored in data banks and encode a protein of 89 amino acids with a calculated molecular mass of approximately 10 kDa.

Uptake of purine and pyrimidine nucleosides by macrophage-resident Mycobacterium leprae: 3H-adenosine as an indicator of viability and antimicrobial activity.

Freshly extracted human- and armadillo-derived Mycobacterium leprae maintained within murine macrophages incorporated significant levels (p less than 0.05 to p less than 0.001) of 3H-adenosine and 3H-hypoxanthine by 6 and 9 days of the culture period. The incorporation of 3H-adenosine was twofold or more higher than 3H-thymidine in 10 out of 15 human-derived M. leprae isolates. Macrophage-adapted bacilli incorporated 10-14-fold higher levels of 3H-adenosine compared to the same bacilli maintained in axenic cultures. The incorporation of these two labels was inhibited by dapsone and rifampin, indicating the utility of in vitro radiometric assays for screening antileprosy drugs and drug sensitivity/resistance in patients.