Superoxide production from macrophages of leprosy patients after stimulation withMycobacterium leprae

The macrophages from peripheral blood of normal healthy individuals respond to live or killedMycobacterium leprae by producing superoxide. On the other hand, the macrophages from bacteriologically positive (B +LL) or long term treated bacteriologically negative (B -LL) and tuberculoid leprosy patients are unable to produce superoxide when stimulated with liveMycobacterium leprae. While killedMycobacterium leprae induce superoxide with the cells from tuberculoid andB(-)LL patients, cells fromB(+)LL patients fail to respond. The deficiency inB(-)LL patients to produce superoxide appears to be specific withMycobacterium leprae and the defect can be counteracted by the addition of colchicine. These observations indicate a preexisting membrane disposition which does not favour superoxide production. A similar situation is seen in the cells from tuberculoid leprosy patients. Thus it appears that both cured and active lepromatous leprosy patients have defective macrophages, unable to respond to liveMycobacterium leprae to produce superoxide anion, in contrast to the situation with the cells from normal healthy individuals.     

Mycobacterium leprae mediated stimulation of macrophages from leprosy patients and hydrogen peroxide production

Macrophages cultured from the peripheral blood of normal individuals, tuberculoid leprosy patients and long-term-treated, bacteriologically negative lepromatous leprosy patients are able to release hydrogen peroxide on stimulation withMycobacterium leprae. Macrophages from lepromatous leprosy patients who are bacteriologically positive produce considerably lower levels of hydrogen peroxide, even though stimulation of these cells withMycobacterium leprae is definitely demonstrable. This differential stimulation of macrophages appears to be largely specific toMycobacterium leprae. There is also a good indication that decreased stimulation of macrophages from positive patients could be due to an after-effect of infection. It is possible that while other factors aid survival ofMycobacterium leprae in the macrophages, hydrogen peroxide may not be as effective in the killing of the bacteria in infected patients as it would be, perhaps, in other infections.     

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.     

Biochemical characterization of a factor released by macrophages.

Peritoneal macrophages from the mouse are able to release a factor, which inhibits the incorporation of [³H]thymidine into the DNA of lymphocytes. The biochemical characterization of this factor reveals that this factor is thymidine, a degradation product of cells dying in culture.     

An erythropoietic stimulating factor similar to erythropoietin released by macrophages after treatment with silica

An erythropoietic stimulating factor (ESF) can be detected in the supernatant from fetal liver and adult bone marrow and spleen cells when preincubated with the macrophage-specific cytotoxic agent, silica. Stimulation is observed in 12-day fetal liver CFU-E cultures in the absence of added erythropoietin (Ep). The concentration of ESF in the supernatant added to CFU-E cultures is dependent on the preincubated cell dose and the volume added. The stimulating activity is abolished when mice are hypertransfused and increased above normal values when mice are bled. A concentrated silica-treated spleen supernatant was able to stimulate erythropoiesis in the polycythemic mouse bioassay. It is concluded that the ESF is similar, if not identical, to Ep.     

Binding, internalization and degradation of colony-stimulating factor by peritoneal exudate macrophages

Iodinated colony-stimulating factor produced by L-cells (¹²⁵I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37°C, the cell-bound ¹²⁵I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0°C, the cell-bound ¹²⁵I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37°C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH₄Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added ¹²⁵I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37°C but not at 0°C in culture medium containing fetal calf serum.     

Detection of Toll-like receptor 2 (TLR2) mutation in the lepromatous leprosy patients

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections and the mutations in the TLR2 have been shown to confer the susceptibility to severe infection with mycobacteria. To define this, we screened the intracellular domain of TLR2 in 131 subjects. Groups of 45 lepromatous and 41 tuberculoid leprosy (TT) patients and 45 controls were investigated. Ten subjects among the lepromatous leprosy (LL) patients had a band variant detected by single-stranded conformational polymorphism. DNA sequencing detected a C to T substitution at nucleotide 2029 from the start codon of the TLR2. The mutation would substitute Arg to Trp at amino acid residue 677, one of the conserved regions of TLR2. In our results, the mutation was involved in only LL, not TT and control. Thus, we suggest that the mutation in the intracellular domain of TLR2 has a role in susceptibility to LL.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

Inhibition by rapamycin of the lipoteichoic acid-induced granulocyte-colony stimulating factor expression in mouse macrophages

Granulocyte-colony stimulating factor (G-CSF) is a cytokine which involves in anti-inflammation and inflammation as well. Rapamycin is an inhibitor of mTOR which also plays a role in innate immunity. This study investigated the effect of rapamycin on the lipoteichoic acid (LTA)-induced expression of G-CSF in macrophages and its underlying mechanism. Our data show that LTA induced G-CSF expression in RAW264.7 and bone marrow-derived macrophages and that this effect was inhibited by rapamycin. Analysis of the G-CSF 5′ flanking sequence revealed that the −283 to +35 fragment, which contains CSF and octamer elements, was required for maximal promoter activity in response to LTA stimulation. Western blot analyses of proteins that bind to the CSF and octamer element show that LTA increased protein levels of NF-κB, C/EBPβ and Oct-2, and that rapamycin inhibited the LTA-induced increase in Oct-2 protein levels, but not the others. Knockdown of Oct-2 by RNA interference resulted in a decrease in LTA-induced G-CSF mRNA levels. Moreover, forced expression of Oct-2 by transfection with the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LTA-induced increase in G-CSF mRNA levels and promoter activity. This study demonstrates that rapamycin reduces G-CSF expression in LTA-treated macrophages by inhibiting Oct-2 expression.     

TUNEL and limited immunophenotypic analyses of apoptosis in paucibacillary and multibacillary leprosy lesions

Some mycobacterial infections, such as tuberculosis, are characterized by apoptosis of infected or by-stander mononuclear immune cells. For localized (paucibacillary, PB) and disseminated (multibacillary, MB) leprosy, characterized by polarized Th1-like vs. Th2-like immune responses, respectively, little is known about lesional apoptosis. We analyzed sections of paraffin-embedded, untreated leprosy lesions from 21 patients by an indirect immunofluorescent terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Some TUNEL (+) PB sections were then reacted with phycoerythrin-conjugated (red) antibodies against T cells, monocytes, or antigen-presenting (Langerhans) cells. TUNEL (+) bodies were detected in 9 of 16 PB lesions (56%) and in 1 of 5 MB lesions (20%). Some TUNEL (+) bodies in PB disease were CD3+ (T cell), as well as CD4+ (T-helper) or CD8+ (T-cytotoxic). Apoptosis characterizes PB and MB leprosy lesions and may be more frequent in PB disease. In PB disease, some TUNEL (+) bodies may derive from T cells.     

Self-mortification and the stigma of leprosy in northern India

This article examines the biocultural dynamics of social discrimination and physical disfigurement among people with leprosy, or Hansen's disease (HD), in Banaras, northern India. Based on the narratives and observations ofpeople living in colony and street settings, I trace three destructive processes by which the social stigmata of leprosy become physically expressed. First, strategies of concealment further the progression and spread of HD through late detection and undertreatment. Second, the internalization of stigma can lead to bodily dissociation and injury through self-neglect. Finally, some people intentionally seek injuries under conditions of desperate poverty. As a result of such mortification processes, these people came to embody, quite literally, the prejudices that exacerbated their condition in the first place.     

Scavenging by alginate of free radicals released by macrophages

Failure to eradicate mucoid forms of P. aeruginosa has implicated bacterial alginate in a local evasion of host defence mechanisms within the lung of Cystic Fibrosis (CF) patients. We have found that purified bacterial alginate scavenges free radicals released by triggered macrophages as detected by lucigenin amplified chemiluminescence (CL) and reduction of cytochrome c. In agreement with this, alginate was also able to scavenge radicals generated by a chemical system (hydrogen peroxide and copper; detected by benzoate hydroxylation and chemiluminescence), and by an enzymatic system (hypoxanthine and xanthine oxidase; detected by chemiluminescence). All inhibitions were dose-related. Oxygen consumption by neutrophils (unlike that of macrophages) could be detected in a Clark electrode, and was not reduced by alginate, confirming that scavenging of radicals was responsible for the earlier observations. These data suggest that bacterial alginate by scavenging free radicals, may favour the survival of mucoid forms of P. aeruginosa, particularly in the CF lung.     

Mechanism of action of macrophage-derived suppressor factor produced by soluble immune response suppressor-treated macrophages

After a 2-hr incubation with soluble immune response suppressor (SIRS), a product of concanavalin A-activated murine T cells, macrophages release a factor, M phi-derived suppressor factor (M phi-SF), which nonspecifically suppresses immune responses in vitro. The mechanism(s) of action of M phi-SF and range of cell types affected by M phi-SF have been investigated. M phi-SF suppressed antibody responses to background levels if added at culture initiation and by 80 to 90% if added as late as 2 hr before assay. Primary and secondary IgM and IgG antibody responses, proliferative responses to T cell and B cell mitogens, antibody and protein secretion, and the division of several tumor cell lines in culture were inhibited by M phi-SF. Division of synchronized tumor cells was inhibited when M phi-SF was added at any point prior to and during mitosis; this inhibition could be reversed with 2-mercaptoethanol. In the presence of M phi-SF, asynchronous tumor cells accumulated in the cell cycle just prior to cell division and could be released into mitosis by 2-mercaptoethanol. These data indicate that M phi-SF inhibits cell division by causing a block at or in mitosis and suggest that M phi-SF may be a general inhibitor of cellular proliferation and possibly of protein secretion.     

The putative tumor suppressor Zc3h12d modulates toll-like receptor signaling in macrophages

Toll-like receptors (TLR) are pivotal in macrophage activation. The molecular mechanisms controlling TLR signaling and macrophage activation are not completely understood. Zc3h12d is originally identified as a possible tumor suppressor gene. However, its function remains unknown. We here report that Zc3h12d negatively regulates TLR signaling and macrophage activation. Zc3h12d was enriched in spleen, lung and lymph node. In macrophages, the expression of Zc3h12d was remarkably induced by TLR ligands through JNK and NF-κB signal pathways. On the other hand, overexpression of Zc3h12d significantly inhibited TLR2 and TLR4 activation-induced JNK, ERK and NF-κB signaling as well as macrophage inflammation. Similar to Zc3h12a/MCPIP1, Zc3h12d also decreased the global cellular protein ubiquitination. These findings suggest that Zc3h12d is a novel negative feedback regulator of TLR signaling and macrophage activation and thus may play a role in host immunity and inflammatory diseases.     

A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin

Using the luminescent protein polynoidin, present in the bioluminescent system isolated from the marine annelid Harmothoe lunulata, we have developed a new method to measure, specifically, superoxide anion (O2-) released by macrophages or neutrophils. A small quantity of an aqueous crude extract of polynoidin is used to detect O2- released by stimulated cells. Light emission is linearly dependent on the number of cells over a wide range (10(3) to 10(7) cells), and the assay is thus more sensitive than either luminol or ferricytochrome c reduction. Luminescence is enhanced 20% by mannitol, 80% by catalase, and is totally quenched by superoxide dismutase. For the same number of cells, neutrophils showed a threefold higher release of O2- and a twofold faster first-order light decay than stimulated macrophages, in accordance with data obtained by other methods.     

Activation of macrophages for tumor cell cytotoxicity: identification of indomethacin sensitive and insensitive pathways

Macrophages can be activated for tumor cell cytotoxicity by endotoxin- or lymphokine-containing solutions, and in both cases activation can be blocked by the addition of indomethacin. In contrast, the activation of macrophages by nonadherent or inflammatory peritoneal cells or antibody-coated tumor cells is not affected by indomethacin. These results demonstrate that there are at least 2 distinct pathways of macrophage activation, only 1 of which is affected by the addition of the drug. Activation by endotoxin that is indomethacin sensitive requires 2 steps. At present, the first is undefined and not blocked by indomethacin, but the second is inhibited by the drug and appears to require the production of E series prostaglandins. Our results also suggest that neither step alone is sufficient to activate macrophages for cytotoxicity.     

Microparticles released from Mycobacterium tuberculosis‐infected human macrophages contain increased levels of the type I interferon inducible proteins including ISG15

Microparticles (MPs) are small membranous particles (100–1000 nm) released under normal steady‐state conditions and are thought to provide a communication network between host cells. Previous studies demonstrated that Mycobacterium tuberculosis (M. tb) infection of macrophages increased the release of MPs, and these MPs induced a proinflammatory response from uninfected macrophages in vitro and in vivo following their transfer into uninfected mice. To determine how M. tb infection modulates the protein composition of the MPs, and if this contributes to their proinflammatory properties, we compared the proteomes of MPs derived from M. tb‐infected (TBinf‐MP) and uninfected human THP‐1 monocytic cells. MP proteins were analyzed by GeLC‐MS/MS with spectral counting revealing 68 proteins with statistically significant differential abundances. The 42 proteins increased in abundance in TBinf‐MPs included proteins associated with immune function (7), lysosomal/endosomal maturation (4), vesicular formation (12), nucleosome proteins (4), and antigen processing (9). Prominent among these were the type I interferon inducible proteins, ISG15, IFIT1, IFIT2, and IFIT3. Exposure of uninfected THP‐1 cells to TBinf‐MPs induced increased gene expression of isg15, ifit1, ifit2, and ifit3 and the release of proinflammatory cytokines. These proteins may regulate the proinflammatory potential of the MPs and provide candidate biomarkers for M. tb infection.     

Inhibition of lymphocyte mitogenesis by factor(s) released from macrophages isolated from ascitic fluid of advanced ovarian cancer patients

Summary Ascitic fluid from women with advanced ovarian carcinomas was shown to contain factor(s) which inhibit(s) T lymphocyte mitogenesis. The factor(s) was (were) demonstrated to be associated with the infiltrating macrophages. The inhibition was reversible and inhibited mitogenesis at some late event in the cell cycle. The inhibitory substance(s) was (were) noncytotoxic, dialyzable, heat-stable at 70° C for 10 min (but unstable at 100° C for 15 min), and partially resistant to protease treatment (55%–70%). Further experiments demonstrated that macrophages isolated from the ascitic fluid of patients with cirrhosis of the liver also released factor(s) which inhibit(s) T lymphocyte mitogenesis. On the basis of our data and data from other investigators, we propose that in advanced human ovarian cancer of epithelial origin, macrophages which infiltrate the ascitic fluid elaborate nonspecific inhibitors of T lymphocyte blastogenesis within the proximal environment, resulting in localized immunosuppression and the subsequent enhancement of metastasis within the peritoneal cavity, the tumor cells themselves being resistant to the cytocidal action of the macrophages due to genetic selection and/or their inherent biochemical ability to circumvent normal immunosurveillance mechanisms. This may account, at least in part, for the rapid metastasis and poor prognosis of human ovarian adenocarcinomas.