In vitro studies on extracellular matrix production by M.leprae infected murine neurofibroblasts.

Fibroblasts and a host of macrophage secretory products have been implicated in a number of diseases where excess extracellular matrix (ECM) deposition is the main pathological feature. Fibrosis characterized by excessive deposition of collagen also contributes to the irreversible nerve damage observed in leprosy. Since M. leprae are seen within neurofibroblasts (Nf) in the advanced stages of the disease and macrophages form a common infiltrating cellular constituent of leprous nerves at all stages, secretion of ECM proteins by Nf was studied, in vitro following infection with M. leprae and in the presence of macrophage secretory products. These studies were compared in cells derived from two strains of mice, Swiss White (SW) and C57BL/6, as they differ in their response to M. leprae infection and parallel those observed in lepromatous and tuberculoid patients, respectively. On infection with M. leprae, Nfs showed a decrease in secretion of collagen type IV in SW and type I in C57Bl/6 strain. Macrophages caused a further decrease in the secretion of collagen types affected by M. leprae infection per se, while the other collagen types, viz. I and III in SW strain and III and IV in C57Bl/s strain, were unaffected. This study indicates that neural collagenization in nerves in advanced leprosy may be of Nf origin. However, unlike other diseases with excess collagen deposition, ECM proteins produced by Nfs in response to nerve damage may not be of prime importance in the progression of leprous neuropathy and occur as a general response to loss of cellular content in leprous nerves.

Differential in vitro modulation of Schwann cell proliferation by Mycobacterium leprae and macrophages in the murine strains, Swiss white and C57Bl/6.

The special susceptibility of Schwann cells (SCs) to parasitization by M. leprae and of macrophages to M. leprae-induced defects implicates them in leprous nerve pathogenesis. SC proliferation is an important prerequisite for peripheral nerve regeneration and is regulated by a number of secretory factors. Several of these factors are secreted by SCs themselves as well as by the macrophages which are recruited at the site of lesion to assist in regeneration. SC proliferation, as indicated by 3H-thymidine incorporation, was therefore studied in response to M. leprae infection and in the presence of macrophages in order to determine the role of SC in leprous neuropathy. Cells derived from two strains of mice, Swiss White (SW) and C57Bl/6 were used, as macrophages from these strains have been shown to differ in their response to M. leprae; such differences are similar to those observed in macrophages from lepromatous and tuberculoid leprosy patients, respectively. Infection with M. leprae for a duration of 9 days resulted in reduced proliferation of SCs from SW strain, while SCs from C57Bl/6 remained unaffected. However, in the presence of macrophages, SCs from both strains not only showed enhanced proliferation, but SW SCs also overcame the M. leprae-induced suppression of their proliferation. Altered SC proliferation, therefore, can be implicated as a factor in leprous nerve pathogenesis. The strain variation observed in the response of SCs indicate different nerve damage mechanisms in lepromatous and tuberculoid patients.

Schwann cell extracellular matrix protein production is modulated by Mycobacterium leprae and macrophage secretory products.

Extracellular matrix (ECM) protein deposition is an important feature of leprous nerves, where Schwann cells (SCs) and macrophages are the main hosts for Mycobacterium leprae. Since, SCs are involved in the synthesis of ECM proteins and its production is regulated by macrophage secretory factors, the present study aimed to determine in vitro, the effect of M. leprae infection and macrophage secretory products on secretion of ECM proteins by SCs in two strains of mice, Swiss White (SW) and C57BL/6, that are known to differ in their nerve pathology and macrophage functions in response to infection. Following six days of M. leprae infection, SCs from SW mice responded with increased secretion of 14C-leucine radiolabelled proteins and a concomitant increase in laminin and collagens type I, III and IV, as determined by enzyme-linked immunosorbent assay. In contrast infected C57BL/6 SCs responded with decreased secretion of total proteins and fibronectin. Exposure of SCs to macrophage conditioned medium resulted in decreased ECM protein secretion in both strains of mice. This decrease was a function of protein breakdown by macrophage derived proteases and also active regulation by macrophage secreted cytokines. A similar effect of M. leprae and macrophage secretory products on SC metabolism in leprous nerves would have major ramifications on damage and repair activities. In addition ECM proteins would also influence the composition of the infiltrating cell population in lepromatous and tuberculoid nerves.

Expression of mycobacterial antigens on murine dissociated Schwann cells infected with Mycobacterium leprae.

Mycobacterial antigens were detected in a cell surface ELISA on murine dissociated Schwann cells infected with Mycobacterium leprae. The time kinetics of expression and its refractoriness to modulation with monensin suggests that the antigens are likely to be integrated into the membrane during bacterial entry. This may be partially responsible for the defective Schwann cell functions leading to subsequent peripheral nerve damage.

Nerve growth factor production and expression of p75 by Schwann cells and neurofibroblasts in response to M. leprae infection and macrophage secretory products.

This study describes the changes occurring in vitro in nerve growth factor (NGF) production and expression of p75 by murine Schwann cells and neurofibroblasts, following infection with Mycobacterium leprae and in the presence of macrophage secretory products, using a semiquantitative ELISA. These parameters are compared in two strains of mice, Swiss White (SW) and C57B1/6, as they differ in their response to M. leprae infection; C57B1/6 is the 'resistant' strain. On infection, NGF levels remained unaltered in Schwann cells from both strains, while fibroblasts from C57B1/6 strain showed an increase in NGF production. Expression of p75 by Schwann cells was decreased on infection in both strains of mice. In vivo, this opposing effect of infection on NGF production and p75 expression by Schwann cells and neurofibroblasts may result in suboptimal amounts of NGF reaching neurons of the affected leprous nerves. Macrophage secretory products suppressed the production of NGF by infected neurofibroblasts from SW strain mice and the expression of p75 in Schwann cells from both strains. These results indicate that macrophages do not assist in nerve repair in leprosy and the differences in response to macrophage secretory products in the two strains suggests that different mechanisms of nerve repair operate in SW and C57B1/6 mice and presumably in lepromatous and tuberculoid patients.

Effect of T-cell depletion on bacterial multiplication and pattern of nerve damage in M. leprae-infected mice.

Various mechanisms for nerve damage in tuberculoid leprosy have been proposed. A common feature amongst them is the crucial role played by T-cells. Therefore, the present study was designed to determine the role of T-cells in the induction of nerve damage in leprosy using two different protocols for obtaining graded levels of T-cell depletion: (i) Cyclosporine A, for depletion of T-helper cells and (ii) Anti Thy 1.2, for total depletion of T-cells. The findings indicate that the early changes seen in the unmyelinated fibres may not involve T-cells. However, the later stages of nerve damage associated with demyelination are dependent on T-cell responses.

Correlation between macrophage activation and bactericidal function and Mycobacterium leprae antigen presentation in macrophages of leprosy patients and normal individuals.

The killing of Mycobacterium leprae by resting and gamma interferon (IFN-gamma)-activated macrophages in normal subjects and leprosy patients was assessed. Resting macrophages from normal individuals demonstrated the ability to kill M. leprae. For macrophages from tuberculoid patients, killing of M. leprae was only achieved in the presence of IFN-gamma, suggesting that initial T-cell activation occurs prior to the killing of M. leprae. In contrast, though activation with IFN-gamma rendered the lepromatous macrophages microbicidal, it failed to induce lymphocyte proliferation, suggesting a defect at either the antigen-presenting cell or the lymphocyte level or both. The concept that T-cell anergy is primarily due to lack of lymphokine generation was ruled out by our results, since responsiveness was restored in only a small proportion of lepromatous patients after exogenous lymphokine addition. In conclusion, this study demonstrated that killing and antigen presentation are two independent events. It appears that the ability of the macrophages per se to kill M. leprae may be of greater importance than lymphocyte-mediated activation for protection against M. leprae infection.

Effect of Mycobacterium leprae-infected Schwann cells and their supernatant on lymphocyte neuroglia interaction.

Since the resolution of neural lesions and subsequent nerve damage in leprosy must inevitably involve the participation of immune cells sensitized to Mycobacteria, we have used the dissociated Schwann cell culture model to study the relationship between M. leprae-infected Schwann cells and sensitized immune cells. Our earlier study on light and ultrastructural observations showed that on infection with M. leprae, the cytomorphology of Schwann cells remains unaffected, while degenerative changes suggestive of apoptosis are seen in extraneous lymphocytes which are subsequently phagocytosed by the Schwann cells. We now present additional evidence confirming that the phagocytosis of splenic cells by Schwann cells is indeed a two-step process. The first involves M. leprae-dependent cytotoxicity to splenic cells. This is followed by phagocytosis of these cells, which is a secondary and M. leprae-independent phenomenon. This finding has implications particularly on the weak inflammatory response observed in nerve lesions of a majority of lepromatous patients.

Presence of Mycobacterium leprae-reactive lymphocytes in lymph nodes of lepromatous leprosy patients.

A critical problem in leprosy is the relative deficiency of antigen-specific T cell-mediated immunity. We were successful in detecting a significant response to viable M. leprae in mononuclear cells isolated from the lymph nodes of lepromatous leprosy patients in contrast to the apparent M. leprae-specific energy seen in the peripheral blood. This observation suggests that antigen-reactive lymphocytes are generated in the lymph nodes of lepromatous patients but the inability to detect them in the circulation may be due either to a different processing and presentation of mycobacterial antigens within the peripheral blood and lymph node compartments or to a selective sequestration of lymphocytes within the lymph node.

Localization and retention of mycobacterial antigen in lymph nodes of leprosy patients.

Although leprosy, a chronic disease caused by M. leprae, primarily affects skin and peripheral nerves, pathological changes and granulomas have been observed in lymph nodes which are: (a) present in tuberculoid lymph nodes in the absence of acid-fact bacilli and (b) persistent in lepromatous patients even after prolonged treatment. We detected substantial amounts of mycobacterial antigen in 16 leprous lymph nodes using anti-BCG by the peroxidase anti-peroxidase method. The load and distribution of antigen varied along the spectrum and with the duration of treatment. Tuberculoid and long-term treated lepromatous lymph nodes had a similar distribution of antigen in clusters of cells giving a 'speckled' appearance. The untreated lepromatous had a 'diffuse' staining of antigen in foamy histiocytes whereas lepromatous lesions with a lower bacillary load had a mixed pattern of 'diffuse' and 'speckled'. Antigen was also detected in a number of plasma cells along the spectrum but predominantly in lepromatous lymph nodes. Our observations indicate that: (a) antigen exists in lymph nodes despite prolonged chemotherapy which may be responsible for the persistent granuloma and (b) antigen is not confined to any particular anatomical compartment of the lymph node.

M. leprae phagocytosis and its association with membrane changes in macrophages from leprosy patients.

Abnormal phagocytosis of Mycobacterium leprae by macrophages of lepromatous patients was demonstrated under various conditions. The largest proportion of macrophages with an excessive bacterial load belonged to the lepromatous group of patients. Lepromatous macrophages treated with Cytochalasin B, an inhibitor of phagocytosis, exhibited a significantly lower degree of ingestion of heat-killed organisms whereas uptake of 'viable' organisms was not affected to the same extent. Regulation of phagocytosis was studied by noting the rate of phagocytosis of M. leprae after the ingestion of a primary particle viz carbonyl iron. Solely in lepromatous macrophages, phagocytosis of carbonyl iron did not result in a decreased uptake of M. leprae implying aberrant phagocytic activity. Lastly, excessive phagocytosis was always noted in macrophages of familial contacts of leprosy patients who displayed decreased Fc receptor expression after M. leprae ingestion. This is of interest since phagocytosis, like Fc receptor expression, is a membrane dependent event and other membrane associated defects have been recognized by us earlier in lepromatous macrophages.

Mycobacterium leprae-induced alterations in macrophage Fc receptor expression and monocyte-lymphocyte interaction in familial contacts of leprosy patients.

Macrophage Fc receptor expression and monocyte-lymphocyte interaction in the presence of Mycobacterium leprae were examined in familial contacts of leprosy patients. Defective M phi functions similar to those of borderline and lepromatous patients could be observed in approximately 71% of consanguineous contacts and 43% of spouses of index patients. Although the values in the latter group were markedly lower than those of the consanguineous contacts, they tended to be higher than those of normal individuals (20%). These in vitro M phi functions were independent of age, sex, and age at onset of exposure and were only weakly associated with duration of exposure. The outcome of the monocyte-lymphocyte interaction test paralleled to a large extent the in vivo Mitsuda lepromin response. Four contacts with defective M phi functions also showed signs of leprosy. The value of these in vitro tests as markers of 'susceptibility' could therefore prove significant.