Progress of the Art of Macrophage Polarization and Different Subtypes in Mycobacterial Infection.

Mycobacteriosis, mostly resulting from Mycobacterium tuberculosis (MTb), nontuberculous mycobacteria (NTM), and Mycobacterium leprae (M. leprae), is the long-standing granulomatous disease that ravages several organs including skin, lung, and peripheral nerves, and it has a spectrum of clinical-pathologic features based on the interaction of bacilli and host immune response. Histiocytes in infectious granulomas mainly consist of infected and uninfected macrophages (Mφs), multinucleated giant cells (MGCs), epithelioid cells (ECs), and foam cells (FCs), which are commonly discovered in lesions in patients with mycobacteriosis. Granuloma Mφ polarization or reprogramming is the crucial appearance of the host immune response to pathogen aggression, which gets a command of endocellular microbe persistence. Herein, we recapitulate the current gaps and challenges during Mφ polarization and the different subpopulations of mycobacteriosis.

Selective killing of human M1 macrophages by Smac mimetics alone and M2 macrophages by Smac mimetics and caspase inhibition.

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Immune Complex-Driven Generation of Human Macrophages with Anti-Inflammatory and Growth-Promoting Activity.

To maintain homeostasis, macrophages must be capable of assuming either an inflammatory or an anti-inflammatory phenotype. To better understand the latter, we stimulated human macrophages in vitro with TLR ligands in the presence of high-density immune complexes (IC). This combination of stimuli resulted in a broad suppression of inflammatory mediators and an upregulation of molecules involved in tissue remodeling and angiogenesis. Transcriptomic analysis of TLR stimulation in the presence of IC predicted the downstream activation of AKT and the inhibition of GSK3. Consequently, we pretreated LPS-stimulated human macrophages with small molecule inhibitors of GSK3 to partially phenocopy the regulatory effects of stimulation in the presence of IC. The upregulation of DC-STAMP and matrix metalloproteases was observed on these cells and may represent potential biomarkers for this regulatory activation state. To demonstrate the presence of these anti-inflammatory, growth-promoting macrophages in a human infectious disease, biopsies from patients with leprosy (Hanseniasis) were analyzed. The lepromatous form of this disease is characterized by hypergammaglobulinemia and defective cell-mediated immunity. Lesions in lepromatous leprosy contained macrophages with a regulatory phenotype expressing higher levels of DC-STAMP and lower levels of IL-12, relative to macrophages in tuberculoid leprosy lesions. Therefore, we propose that increased signaling by FcγR cross-linking on TLR-stimulated macrophages can paradoxically promote the resolution of inflammation and initiate processes critical to tissue growth and repair. It can also contribute to infectious disease progression.

A Negative Feedback Loop Between Autophagy and Immune Responses in Mycobacterium leprae Infection.

The obligate intracellular bacterium Mycobacterium leprae is the causative agent of leprosy and primarily infects macrophages, leading to irreversible nerve damage and deformities. So far, the underlying reasons allowing M. leprae to persist and propagate in macrophages, despite the presence of cellular immunity, are still a mystery. Here, we investigated the role of autophagy, a cellular process that degrades cytosolic materials and intracellular pathogens, in M. leprae infection. We found that live M. leprae infection of macrophages resulted in significantly elevated autophagy level. However, macrophages with high autophagy levels preferentially expressed lower levels of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, IL-12, and tumor necrosis factor-α, and preferentially primed anti-inflammatory T cells responses, characterized by high IL-10 and low interferon-γ, granzyme B, and perforin responses. These anti-inflammatory T cells could suppress further induction of autophagy, leading to improved survival of intracellular M. leprae in infected macrophages. Therefore, these data demonstrated that although autophagy had a role in eliminating intracellular pathogens, the induction of autophagy resulted in anti-inflammatory immune responses, which suppressed autophagy in a negative feedback loop and allowed the persistence of M. leprae.

Mycobacterium indicus pranii mediates macrophage activation through TLR2 and NOD2 in a MyD88 dependent manner.

Mycobacterium indicus pranii (MIP) is a non-pathogenic strain of mycobacterium and has been used as a vaccine against tuberculosis and leprosy. Here, we investigated the role of different pattern recognition receptors in the recognition of heat-killed MIP by macrophages. Treatment of macrophages with MIP caused upregulation of pro-inflammatory cytokines (like TNFα and IL-1ß) which was mediated through both TLR2 and NOD2, as revealed by our knockdown and/or knockout studies. Mechanistically, MIP-induced macrophage activation was shown to result in NF-κB activation and drastically abrogated by MyD88 deficiency, suggesting its regulation via an MyD88-dependent, NF-κB pathway. Interestingly, the IFN-inducible cytokine, CXCL10, which is known target of the TRIF-dependent TLR pathway was found to be upregulated in response to MIP but, in an MyD88-dependent manner. Collectively, these results demonstrate macrophages to recognize and respond to MIP through a TLR2, NOD2 and an MyD88-dependent pathway. However, further studies should clarify whether additional TLR-dependent or -independent pathways also exist in regulating the full spectrum of MIP action on macrophage activation.

Macrophage polarization and bacterial infections.

PURPOSE OF REVIEW: Macrophages are the first line of defense against pathogens, and the mode of their activation will determine the success or failure of the host response to pathogen aggression. Based on limited numbers of markers, activated macrophages can be classified as classically activated (M1) macrophages that support microbicidal activity or alternatively activated (M2) macrophages that are not competent to eliminate pathogens. The development of high-throughput gene expression methods affords a reappraisal of the concept of macrophage activation in human infectious diseases. RECENT FINDINGS: By combining microarray data and conventional approaches, it is becoming clear that the M1 polarization program is associated with gastrointestinal infections (e.g. typhoid fever and Helicobacter pylori gastritis) and active tuberculosis. An M2 signature is observed in lepromatous leprosy, Whipple's disease, and localized infections (keratitis, chronic rhinosinusitis). However, these findings could not be predicted from the analysis of the M1/M2 programs of macrophages stimulated in vitro. SUMMARY: The reappraisal of macrophage polarization by high-throughput methods is critical to understanding the role of macrophage polarization in infectious diseases. Only the identification of individual profiles will support promising therapeutic approaches based on target determination.

Lipid droplet formation in leprosy: Toll-like receptor-regulated organelles involved in eicosanoid formation and Mycobacterium leprae pathogenesis.

A hallmark of LL is the accumulation of Virchow's foamy macrophages. However, the origin and nature of these lipids, as well as their function and contribution to leprosy disease, remain unclear. We herein show that macrophages present in LL dermal lesions are highly positive for ADRP, suggesting that their foamy aspect is at least in part derived from LD (also known as lipid bodies) accumulation induced during ML infection. Indeed, the capacity of ML to induce LD formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human peripheral monocytes and murine peritoneal macrophages. Furthermore, infected cells were shown to propagate LD induction to uninfected, neighboring cells by generating a paracrine signal, for which TLR2 and TLR6 were demonstrated to be essential. However, TLR2 and TLR6 deletions affected LD formation in bacterium-bearing cells only partially, suggesting the involvement of alternative receptors of the innate immune response besides TLR2/6 for ML recognition by macrophages. Finally, a direct correlation between LD formation and PGE(2) production was observed, indicating that ML-induced LDs constitute intracellular sites for eicosanoid synthesis and that foamy cells may be critical regulators in subverting the immune response in leprosy.

Application of a viability-staining method for Mycobacterium leprae derived from the athymic (nu/nu) mouse foot pad.

Mycobacterium leprae cannot be cultured, so ascertaining viability of the organism remains a major obstacle, impeding many avenues of investigation. This study tested a two-colour, Syto9 and propidium iodide, fluorescence assay, which scores for membrane damage in individual bacilli, to determine if a rapid direct-count viability-staining technique can be reliably applied to M. leprae. A variety of experimental conditions were employed to validate this technique. This technique was also used to correlate the viability of M. leprae with the course of athymic mouse foot pad infection to optimize the provision of viable M. leprae as a research reagent. The data show that in untreated suspensions of M. leprae there is a good correlation between the metabolic activity of leprosy bacilli and their membrane damage. Fixation of M. leprae with ethanol, paraformaldehyde and gluteraldehyde completely suppressed their metabolic activity but showed little effect on their membrane integrity. The present study also showed that the metabolic activity of M. leprae declines more than the extent of membrane damage at 37 degrees C within 72 h, but that they are not significantly affected at 33 degrees C. Irradiation at 10(4) Gy showed high numbers of dead bacilli by the staining method. The results show that the reliability of metabolic-activity data as well as viability-staining data is dependent on the method by which M. leprae is killed. This staining method helped us predict reliably that the smaller M. leprae-infected athymic mouse foot pad seen early in infection, between 4 and 5 months, yields markedly better quality leprosy bacilli than older, larger foot pad infections, as defined by their metabolic activity and membrane integrity.

Effects of thalidomide on intracellular Mycobacterium leprae in normal and activated macrophages.

Thalidomide is an effective drug for the treatment of erythema nodosum leprosum (ENL). ENL is an inflammatory reaction that may occur in multibacillary leprosy patients. Its cause(s) as well as the mechanism of thalidomide in arresting this condition are not fully understood. It has been suggested that ENL is an immune complex-mediated hypersensitivity precipitated by the release of Mycobacterium leprae from macrophages. The released antigen may complex with precipitating antibodies, initiating complement fixation and the production of inflammatory cytokines like tumor necrosis factor alpha (TNF-alpha). Thalidomide has been shown in vitro to reduce antigen- or mitogen-activated macrophage production of TNF-alpha. We investigated if thalidomide could also influence the viability of intracellular M. leprae. Mouse peritoneal macrophages were infected with M. leprae, activated with gamma interferon and endotoxin, or nonactivated, and treated with thalidomide. Intracellular bacilli were recovered, and metabolic activity was assessed by a radiorespirometric procedure. Thalidomide did not possess antimicrobial action against M. leprae in normal and activated host macrophages. This suggests that thalidomide does not retard the release of mycobacterial antigens, a possible prelude or precipitating factor for ENL. A distinct sequence of events explaining the mechanism of action for thalidomide's successful treatment of ENL has yet to be established.

An in vitro model for the lepromatous leprosy granuloma: fate of Mycobacterium leprae from target macrophages after interaction with normal and activated effector macrophages.

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mphi) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mphi harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-gamma-activated (ACT) effector Mphi. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mphi possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mphi incubated with normal effector Mphi exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mphi cocultured with ACT effector Mphi displayed a markedly decreased metabolic activity. Inhibition by ACT Mphi required an E:T ratio of at least 5:1, a coculture incubation period of 3-5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-gamma nor TNF-alpha were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mphi was necessary for augmentation of M. leprae metabolism by normal effector Mphi as well as for inhibition of M. leprae by ACT effector Mphi. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mphi were acquired by the effector Mphi. Thus, the state of Mphi infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mphi in the lepromatous lesion.

Comparison of multidrug therapy treatment results between multibacillary leprosy patients in hyperendemic and hypoendemic areas in Gowa Regency, South Sulawesi, Indonesia.

We studied 88 multibacillary (MB) leprosy patients, who received multidrug therapy (MDT) treatment in hyperendemic (44 persons) and hypoendemic (44 persons) areas in Gowa Regency, South Sulawesi, Indonesia. Bacteriological examinations were carried out (bacteria index and morphology index), immunological examinations (MLPA and TNF-alpha) and genetic variation in blood and ear lobe slit skin smears of MB leprosy patients, which had been treated by MDT, were performed. The collected data were analyzed with the chi-square test and discriminant analysis. Eight persons (9.1%) had a positive bacteria index and 2 persons (2.4%) had a morphology index. All patients (100%) increased their TNF-alpha concentration. All the samples (100%) showed an increase in the TNF-alpha concentration, compared to controls. MLPA positive conversion was found in 4 persons (47.7% samples) and TNF-alpha gene genetic variation at the position of -308, occurred in 12 persons (13.6% samples). Statistic test results showed a significant difference (p<0.01) in the TNF-alpha concentration between hyperendemic and hypoendemic areas. On the basis of a positive conversion by bacteriological results and its association with immunological results, it can be concluded that multibacillary leprosy patients with MDT treatment did not become a source of leprosy transmission.

DDT inhibits the functional activation of murine macrophages and decreases resistance to infection by Mycobacterium microti.

DDT is still widely used in several parts of the world to control malaria, typhoid and dengue vectors, even though its use was banned in many countries based on toxicity data in wild life species. DDT has been shown to have immunotoxic effects in mice and to increase susceptibility to intracellular pathogens such as Mycobacterium leprae. However, little is known about the mechanisms underlying this effect. Activated macrophages play an important defensive role against intracellular pathogens, therefore our objective was to evaluate the effect of in vitro exposure to technical grade DDT (a mixture of three forms: 1,1,1-thricloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) (85%), o,p'-DDT (15%) and o,o'-DDT (trace amounts)), p,p'-DDT, 1,1-dicloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane on the functional activation of J774A.1 macrophages and their capability to limit growth of intracellular pathogens, using Mycobacterium microti as a model. We evaluated cytotoxicity and the effect on cell proliferation of 2.5, 5.0 and 10 microg/ml of DDT compounds. Functional macrophage activity (NO(*) and O(2)(-) production, and mRNA expression of TNF-alpha, IL-1beta and iNO synthase) and the ability of treated cells to limit infection by M. microti in IFN-gamma-activated macrophages were evaluated in cells exposed to 2.5 microg/ml of DDT compounds. Doses of 5 and 10 microg/ml induced direct cytotoxic effects precluding meaningful analysis of the above parameters, whereas 2.5 microg/ml of all DDT compounds inhibited macrophage activity and reduced their ability to limit the intracellular growth of M. microti without inducing cytotoxicity. Technical grade DDT and p,p'-DDE were the more potent compounds. Therefore, exposure to DDT compounds could represent an important risk for infection development by those intracellular pathogens against which NO(*) and/or O(2)(-) production represent the main immune protective mechanism.

Immunomodulatory potential of hydrophobic analogs of Rigin and their role in providing protection against Plasmodium berghei infection in mice.

Here, we report the immunomodulating potential of N-palmitoyl-amino-ethyl-rigin amide (PR) and N-cholestanyl-amino-ethyl-rigin amide (CR), the two new structural analogs of rigin (an IgG-derived tetrapeptide). Their activity profiles are compared with native tuftsin (NT) and/or N-palmitoyl-amino-ethyl-tuftsin amide (PT) taken as positive control. To explore the possibility of their use as targeting molecules, they are incorporated into the liposome bilayer and, subsequently, interacted with macrophages in an in vitro study. The new analogs of rigin with the hydrophobicity introduced at the C-terminus are found to considerably improve both the cell-mediated and the humoral immune responses in mice. However, unlike tuftsin and its analog, which mainly activate polymorphonuclear leukocytes and macrophages, the rigin analogs appear to manifest their response more through lymphocytes. When administered prophylactically to a group of mice, at the dose of 100 micrograms/0.5 ml/mouse/day for 2 days (i.v.), followed by a challenge presented with 1 x 10(6) rbcs parasitised with Plasmodium berghei on day 0, substantial reduction in parasitaemia and rate of mortality is observed. This led to increase the median survival time (MST) of the treated group in comparison to the control group. The response is found to be more prominent in CR-treated mice possibly because of the presence of steroid moiety, which is likely to have more productive interaction with cell membranes. Incorporation of these peptides into the bilayer of liposomes does not alter the permeability behavior of vesicles and, in fact, enhances their uptake by the macrophages in an in vitro study. The effect, however, is dependent on both, the concentration of peptide liposomes and the time of incubation. Present study, thus, establishes the possible use of these analogs not only as adjuvant in chemotherapy, but also as a prophylactic supplement to boost the natural immune status. The activity response of rigin analogs is manifested through lymphocytes, they can also find use in the chemotherapy of diseases, like leishmaniasis, tuberculosis and leprosy, where macrophage activity is either tamed or impaired by pathogens.

Regulación por células de la inmunidad innata y citoquinas tipo 1 de la respuesta T antígeno-específica en lepra / Regulation by innate immunity cells cytokines type 1 of antigen-specific T response in leprosy

La lepra es una enfermedad infecciosa crónica causada por la infección con M.leprae. Tiene como carcterística la de presentar un espectro clínico e inmunológico que se correlaciona estrechamente con el nivel de inmunidad mediada por células inducido por los distintos antígenos del M.leprae. Los pacientes paucibacilares (PB) son capaces de restringir el crecimiento del bacilo presentando fuertes respuestas celulares a antígenos del M.leprae. Por el contrario, los pacientes multibacilares (MB) no logran restringir la multiplicación del bacilo y tienen anergia específica para antígenos del M.leprae. El Mycobacterium leprae (patógeno intracelular) enfrenta en su interacción con el huésped dos sistemas de inmunidad: la respuesta inmune innata y la adaptativa. La respuesta innata involucra la activación de células preprogramadas para responder a estímulos no propios (monocitos/macrofágos, células "natural killer" (NK) y linfocitos T gamma delta). La respuesta adaptativa involucra la selección y diferenciación de linfocitos T alfa beta y linfocitos B de memoria. La activación de los macrófagos representa uno de los primeros eventos en la resistencia innata a infecciones intracelulares. Las micobacterias fagocitadas por el macrófago inducen la activación y la producción de citoquinas proinflamatorias que a su vez activan las células NK y T gamma delta... (TRUNCADO)(AU)

Regulación por células de la inmunidad innata y citoquinas tipo 1 de la respuesta T antígeno-específica en lepra / Regulation by innate immunity cells cytokines type 1 of antigen-specific T response in leprosy

La lepra es una enfermedad infecciosa crónica causada por la infección con M.leprae. Tiene como carcterística la de presentar un espectro clínico e inmunológico que se correlaciona estrechamente con el nivel de inmunidad mediada por células inducido por los distintos antígenos del M.leprae. Los pacientes paucibacilares (PB) son capaces de restringir el crecimiento del bacilo presentando fuertes respuestas celulares a antígenos del M.leprae. Por el contrario, los pacientes multibacilares (MB) no logran restringir la multiplicación del bacilo y tienen anergia específica para antígenos del M.leprae. El Mycobacterium leprae (patógeno intracelular) enfrenta en su interacción con el huésped dos sistemas de inmunidad: la respuesta inmune innata y la adaptativa. La respuesta innata involucra la activación de células preprogramadas para responder a estímulos no propios (monocitos/macrofágos, células "natural killer" (NK) y linfocitos T gamma delta). La respuesta adaptativa involucra la selección y diferenciación de linfocitos T alfa beta y linfocitos B de memoria. La activación de los macrófagos representa uno de los primeros eventos en la resistencia innata a infecciones intracelulares. Las micobacterias fagocitadas por el macrófago inducen la activación y la producción de citoquinas proinflamatorias que a su vez activan las células NK y T gamma delta... (TRUNCADO)

[Protective immunity against intracellular parasitic bacteria].

Facultative intracellular bacteria are resistant to the killing mechanism inside macrophages by virtue of various escape mechanisms. Activation of macrophages by cytokines is the key event to overcome of bacterial escape in macrophages of the infected host. Generation of TH1 type of antigen-specific T cell is the essentially required for the macrophage activation. This short review summarizes the escape mechanism of activated macrophages and the mechanisms involved in the generation of TH1 cells.

The Nramp1 protein and its role in resistance to infection and macrophage function.

Susceptibility to infectious diseases is under genetic control in humans. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. The Nramp1 gene was isolated by positional cloning the host resistance locus Bcg/Ity/Lsh, and mutations at this locus impair the resistance of mice to infections with intracellular parasites, such as Salmonella, Leishmania, and Mycobacterium. Allelic variants at the human Nramp1 homologue have recently been found to be associated with susceptibility to tuberculosis and leprosy in humans. The Nramp1 protein is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication. Although the biochemical mechanism of action of Nramp1 at that site remains unknown, Nramp homologues have been identified in many other animal species and actually define a protein family conserved from bacteria to humans. Some of these homologues have been shown to be divalent cation transporters. Recently, a second member of the mammalian Nramp family, Nramp2, was discovered and shown to be mutated in animal models of iron deficiency. The Nramp2 protein was subsequently shown to be the major transferrin-independent iron uptake system of the intestine. Together, these results suggest that Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space.

Genetic regulation of macrophage activation: understanding the function of Nramp1 (=Ity/Lsh/Bcg).

The Nramp1 gene was originally described as Ity/Lsh/Bcg, a single gene controlling resistance and susceptibility of inbred mice to a range of intramacrophage pathogens. Functional studies demonstrated that Ity/Lsh/Bcg had multiple pleiotropic effects on macrophage activation pathways, broadening interest in the gene to include its candidacy as an autoimmune disease susceptibility gene. In 1993 the gene was positionally cloned and found to encode a polytopic integral membrane protein of unknown function. Subsequent studies have localized the protein to late endosomal and lysosomal compartments, and demonstrated that it functions as an iron transporter. Precisely how this function influences macrophage activation pathways is still under investigation, but is likely to include direct effects on pathogen survival in the endosomal/lysosomal compartment as well as influences on intracellular signalling pathways and in regulating mRNA stability. Several studies now provide evidence for a role for NRAMP1 in determining human susceptibility to autoimmune (rheumatoid arthritis. juvenile rheumatoid arthritis, diabetes, Crohn's disease) and infectious (tuberculosis, leprosy) diseases. Amongst these. data are accumulating to support the hypothesis that a functional Z-DNA forming repeat polymorphism in the promoter region of human NRAMP1 contributes directly to disease susceptibility. Four alleles have been observed, alleles 1 and 4 are rare (gene frequencies approximately equal to 0.001), alleles 2 and 3 occur at gene frequencies approximately 0.25 and approximately 0.75, respectively. In the absence of exogenous stimuli, alleles 1, 2 and 4 are poor promoters of gene expression in a luciferase reporter gene system; allele 3 drives high expression. Allele 3 shows allelic association with autoimmune disease susceptibility, allele 2 with infectious disease susceptibility. Hence, balancing selection is likely to be maintaining these two alleles in human populations. Although the association of NRAMP1 with autoimmune disease susceptibility may be related to any one of the multiple pleiotropic effects associated with macrophage activation, the function of NRAMP1 as an iron transporter now prompts more interesting speculation that regulation of iron transport may contribute directly to the disease phenotype in arthritic disease. Patients suffering from rheumatoid arthritis show increased deposition of iron in the synovial membrane, which may contribute to free radical generation and local inflammation. Further analysis of NRAMP1 function will continue to be of importance in understanding the molecular basis to autoimmune and infectious disease susceptibility.

Down-regulatory effect of Mycobacterium leprae cell wall lipids on phagocytosis, oxidative respiratory burst and tumour cell killing by mouse bone marrow derived macrophages.

The authors have previously demonstrated that lipids from Mycobacterium leprae cell walls inhibit macrophage functions and are endowed with anti-inflammatory properties in vivo. To investigate these observations further, the authors describe here the influence of dead M. leprae or of the lipids extracted from the cell wall of the mycobacterium, enclosed in liposomes, on the phagocytic, oxidative respiratory burst and tumouricidal ability of bone marrow derived macrophages in vitro. Dead M. leprae or its cell wall lipids abrogated the oxidative respiratory burst and phagocytic ability of mouse bone marrow derived macrophages. A dose-dependent inhibitory effect of the bacterial lipid extract on tumour cell killing by lipopolysaccharide (LPS)-activated bone marrow derived macrophages was demonstrated. However, when delipidated M. leprae was added to cultures of bone marrow derived macrophages, immune phagocytosis and superoxide production was up-regulated. Mycobacterium leprae or its lipids did not appear to be toxic to those cells assayed by the MTT (methyl thiazol tetrazolium) test. These data, added to our preceding observations, support the hypothesis that the down-regulatory activity of M. leprae wall lipids on macrophage function might be one of the evasive mechanisms of the bacterium to enable it to perpetuate itself in the host tissues.

Nonspecific resistance to Mycobacteria: the role of the Nramp1 gene.

The genus Mycobacteria consists of over 50 species that include two of the best-known human pathogens, M. tuberculosis and M. leprae, the causes of tuberculosis (TB) and leprosy, respectively. Whereas the spread of leprosy currently appears to be under control, there are presently about 30 million active cases of TB worldwide, with an alarming increase in the number of multidrug resistant case of M. tuberculosis. As strategies for antibiotic intervention against TB become more limited, it is imperative to develop new therapeutic approaches against this oppressive disease. One promising avenue is to characterize the host genes and gene products which regulate resistance to mycobacterial infections. In the mouse, resistance and susceptibility to intracellular growth of Mycobacteria in macrophages is controlled by the Bcg (Nramp1) gene, which has now been cloned and shown to encode a macrophage transmembrane protein with a putative transporter function. Sequencing of Nramp1 revealed that susceptibility to infection is associates with a single, nonconservative glycine to aspartic acid substitution at position 169 (G169D). Although the intracellular location of the Nramp1 protein in macrophages has not yet been determined, a phagosomal site has been postulated. Consistent with the proposed role of Nramp1 in macrophage activation, recent studies of the Nramp1 promoter region have revealed consensus sequences associated with responsiveness to IFN-gamma and LPS. Finally, a total of 11 polymorphisms have been identified within the human NRAMP1 gene which are being used to test for linkage of NRAMP1 alleles with human susceptibility to TB and leprosy.