Effects of microenvironment on local regulation of granulomas caused by Mycobaterium leprae

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INTRODUCTION
M ycobacterium leprae is an obligate intracellular pathogen and the causative agent of Hansen's Disease (HD), a disease characterized by a broad spec-trum of clinical, histopathological and immunological presentations that afford the opportunity to study immune regulation and the interplay of T cells and macrophages (MO) in a specific, nonfatal, human, chronic immunodeficiency disease.At one pole of the HD clinical disease spectrum is tuberculoid disease (TT), characterized by low levels of antibody, very few bacilli in the skin lesions and a fully competent cell mediated immunity (CMI) to M. leprae antigens.In sharp contrast, at the other pole of clinical HD is lepromatous disease (LL), characterized by an M. leprae -specific anergy in CMI, a potent antibody response and enormous numbers of M. leprae in the lesions.
At both poles M. leprae infection is local-ized to the cooler areas of the body, i.e. the skin and mucous membranes of the upper respiratory tract.HD granulomas across the spectrum are composed of numerous cell types, including an array of lymphocytes of the helper and sup-pressor phenotype, M at various stages of maturity, and other cells.The present report summarizes the interest from our laboratory in t h e l o c a l i z e d m e c h a n i s m s and lesional microenvironmental factors that regulate the interaction between T cells and M) in CMI to M. leprae at both poles of the HD spectrum.Our interest in control at the level of the granuloma microenvironment stems from our studies of macrophage success and failure in coping with viable HD bacilli 8 and our initial failure to demonstrate by adoptive transfer enhanced resistance to M. leprae in infected nu/nu mice infused with viable sensitized T cells.

M Success in Coping with M. leprae
Using radiorespirometry as an objective measure of M. leprae viability 4 , we demonstrated that, although viable bacilli appear to thrive in normal mouse peritoneal M) 10 , IFNy-activat-ed M were able to markedly kill or inhibit M. leprae in vitro by microbicidal mechanisms dependent on the arginine-dependent production of NO 1 .These findings confirmed the demonstration that in normal M phagosome-lysosome fusion was blocked by M. leprae.In activated M, phagosomes harboring M. leprae fused with secondary lysosomes 11 .

Function by M. leprae
To explore the ability of M from lepromatous granulomas to cope with the HD bacillus we isolated and cultured M from the enlarged foot pads of M. leprae -infected athymic (nu/nu mice).Foot pads of mice infected for nine to twelve months routinely yielded five to ten million macrophages engorged with M. leprae.These granuloma M were evaluated in parallel with peritoneal MCI) from the same animals for a number of phenotypic markers and IFNyinducible immune functions".Interestingly, except for containing enormous numbers of M. leprae, the granuloma M) were phenotypically indistinguishable from normal peritoneal MO: they were adherent to plastic, phagocytic, supported the intracellular growth of Toxoplasma gondii, were nonspecific esterase positive, and possessed Fc and C3Bi receptors.However, unlike the peritoneal M, the granuloma Mff) released prostaglandin E2 (PGE2) into supernatant medium.In addition, granuloma M) differed markedly from the autologous peritoneal cells in their responsiveness to IFNy (Table 1).
The M. leprae-engorged granuloma M) were refractory to IFNγ-mediated activation for In vitro studies shed more light on these ex vivo findings 13 .Normal peritoneal M infect-ed with large numbers (E:T=50:1) of viable bacilli and cultured > 48 hr also produced PGE2 and became refractory to IFNγ (Table 1) by an indomethacin reversible mechanism.Equally large numbers of killed bacilli failed to induce this effect.Additional studies have focused on an abundant mycobacterial cell wall compo-nent, lipoarabinomannan (LAM) as a potent constituent underlying the ability of M. leprae to down regulate M) effector function 7 .

Traffic of M. leprae into Lepromatous Lesions
To explore the turnover of M. leprae infect-ed M in the nu/nu mouse foot pad lesions we pulse labeled bone marrow promonocytes and followed their traffic into the foot pads by autora-diography 9 .Remarkably these studies showed that 25-30% of the M. leprae gorged M were less than five days old, suggesting that there is a continuous, very dynamic turnover and replacement of M in such lesions.There is ample evidence that cytotoxic CD8 and CD4 T cells can lyse target M infected with mycobacteria, although there is a paucity of these specific T cells in lepromatous HD (and nu/nu mice).However, we have shown that NK cells are able to lyse M. leprae infected M target cells and that the interaction of activated effector M and M. leprae infected target MED results in the killing or inhibition of the bacilli'.

Adoptive Transfer Experiments
To further explore the localized CMI to HD, adoptive transfer (AT) experiments were performed.Briefly, in our model nu/nu mice infected with M. leprae 6 months previously were infused i.v. with 2 x 10 7 sensitized lymph node T cells from immunocompetent donors immunized with M. leprae.As an objective indicator of successful AT, after 2 weeks we harvested the M. leprae from the foot pads of recipient nu/nu mice and determined their viability by radiorespirometry.
An initial series of AT experiments in M. leprae infected nu/nu mice yielded disappoint-ing results.As summarized in Table 2, AT of sensitized T cells failed to adversely effect the viability of M. leprae from the foot pads of recipient nu/nu mice.Because of the potent down regulating effects of PGE2 in CM! in general and its role in our in vitro and ex vivo studies of down regulation of the effector function in M. leprae infected M, subsequent AT experiments were designed to focus on the role of PGE2 in modulating local CMI to M. leprae.
PGE2 is synthesized from arachidonic acid (AA), one of the major phospholipids found in cell membranes, via the cyclooxygenase (COX) pathway.AA is generated from linoleic acid, a plant derived, essential fatty acid (EFA) provided by diet.Feeding mice a defined diet containing all necessary nutrients except linoleic acid greatly reduces the levels of AA in the tis-suess, thus depriving COX of its substrate and blocking COXmediated biosynthesis of PGE2.To test the importance of localized PGE2 pro-duction in AT infected nu/nu mice were fed EFA sufficient (S) or deficient (D) diets for 3 months prior to infusion with normal or sensitized T cells.The results (Table 2) clearly showed that the AT of M. leprae sensitized T cells resulted in killing or inhibition of HD bacilli in the foot pads of recipient mice on EFAD diets in comparison to mice on EFAS diets, presumably by activated M.Obviously, the immunoregulatory mechanisms operative in CMI to HD are more complex than presented here; numerous monokines, cytokines, chemokines and other cell products are likely involved.
Collectively, our findings that lepromatous MO are refractory to IFNy activation accomodate the results of experiments that showed the upgrading of LL lesions in HD patients treated intralesionally with human IFNγ6.We have demonstrated that M can be activated before infection with M. leprae and although we have shown that M production of PGE2 may under-lie the refractory state to IFNγ activation in heavily infected MO there is a delay before exogenous PGE2 down regulates M function.Since the LL granuloma is a very dynamic lesion with active turnover of infected MO, we suggest that the human IFNγ immunotherapy findings are a consequence of the activation of new M before or shortly after their traffic into the lesion.

Table 1 .
IFNγ-induced Effector Function in M) Infected with M. leprae or Treated with LAM both microbicidal and tumoricidal activity.In addition, there was no IFNγ-induced augmentation of Class ll MHC expr ession or PMAinduced superoxide production in the M. lepra eengorged granuloma M.These findings show that M. leprae can be a potent modulator of M effector and also demonstrate that this effect is confined to the microenvironment of the granuloma.

Table 2 .
Viability of M. leprae isolated from nu/nu foot pads two weeks after adoptive transfer of T cells.