cord-factors and Granuloma

There is great need for a therapeutic that would limit tuberculosis related pathology and thus curtail spread of disease between individuals by establishing a "firebreak" to slow transmission. A promising avenue to increase current therapeutic efficacy may be through incorporation of adjunct components that slow or stop development of aggressive destructive pulmonary pathology. Lactoferrin, an iron-binding glycoprotein found in mucosal secretions and granules of neutrophils, is just such a potential adjunct therapeutic agent. The focus of this review is to explore the utility of lactoferrin to serve as a therapeutic tool to investigate "disruption" of the mycobacterial granuloma. Proposed concepts for mechanisms underlying lactoferrin efficacy to control immunopathology are supported by data generated based on in vivo models using nonpathogenic trehalose 6,6'-dimycolate (TDM, cord factor).

Topics: Animals; Cord Factors; Granuloma; Humans; Lactoferrin; Mice; Tuberculosis

Tuberculosis, once thought to have been controlled, is now resurgent in many parts of the world. Many gaps exist in understanding the pathogenesis of tuberculosis, especially secondary and cavitary disease. Evidence presented here suggests that cord factor (trehalose 6,6'-dimycolate, TDM) is a key driver of these processes. It is the most abundant lipid released by virulent M. tuberculosis (MTB) and can switch between two sets of activities. On organisms, TDM is non-toxic and protects them from killing by macrophages. On lipid surfaces, it becomes antigenic and highly toxic. Caseating granulomas, the hallmark of primary tuberculosis, develop from interaction of TDM with lipid within granulomas. New evidence indicates that secondary tuberculosis begins as a lipid pneumonia that accumulates mycobacterial antigens and host lipids in alveoli before developing conditions for activation of the toxicity and antigenicity of TDM. This rapidly produces caseation necrosis that leads to cavities. Finally, virulent MTB release large amounts of TDM during growth as a pellicle within cavities. We propose that such growth results in activation of the toxicity and antigenicity of TDM at the air interface and that presence of the activated TDM perpetuates the cavity.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Disease Models, Animal; Granuloma; Humans; Macrophages; Mice; Mycobacterium tuberculosis; Necrosis; Tuberculosis

Mycolic acids are characteristic fatty acids of Mycobacteria and are responsible for the wax-like consistence of these microorganisms. Decades of research revealed that mycolic acid-containing glycolipids, in particular trehalose-6,6'-dimycolate (TDM, cord factor) as their best-studied representative, exert a number of immunomodifying effects. They are able to stimulate innate, early adaptive and both humoral and cellular adaptive immunity. Most functions can be associated with their ability to induce a wide range of chemokines (MCP-1, MIP-1alpha, IL-8) and cytokines (e.g., IL-12, IFN-gamma, TNF-alpha, IL-4, IL-6, IL-10). This review tries to link well-known properties of mycolic acid-containing glycolipids, e.g., stimulation of cellular and humoral immunity, granuloma formation and anti-tumor activity, with recent findings in molecular immunology and to give an outlook on potential practical applications.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Cord Factors; Cytokines; Granuloma; Humans; Mice; Mycobacterium tuberculosis; Neovascularization, Pathologic

Primary infection with

Topics: Animals; Cord Factors; Female; Fluoroquinolones; Granuloma; Humans; Inflammation; Lactoferrin; Mice; Mice, Inbred C57BL; Mycobacterium; Recombinant Proteins

Murine models of Mycobacterium tuberculosis (Mtb) infection demonstrate progression of M1-like (proinflammatory) and M2-like (anti-inflammatory) macrophage morphology following primary granuloma formation. The Mtb cell wall cording factor, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant and useful molecule for modeling early macrophage-mediated events during establishment of the tuberculosis-induced granuloma pathogenesis. Here, it is shown that TDM is a major driver of the early M1-like macrophage response as seen during initiation of the granulomas of primary pathology. Proinflammatory cytokines tumor necrosis factor-α, IL-1β, IL-6, and IL-12p40 are produced in lung tissue after administration of TDM to mice. Furthermore, CD11b

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Female; Granuloma; Inflammation Mediators; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium; Pneumonia

In this issue of Cell Host & Microbe, Walton et al. (2018) uncover the mycobacterial factors that activate VEGF signaling and promote aberrant angiogenesis in the tuberculous granuloma. Preventing abnormal angiogenesis in the granuloma represents a potential therapeutic approach for tuberculosis.

Topics: Cord Factors; Cyclopropanes; Granuloma; Humans; Trehalose; Vascular Endothelial Growth Factor A

Trehalose 6'6-dimycolate (TDM) is the most abundant glycolipid on the cell wall of Mycobacterium tuberculosis (MTB). TDM is capable of inducing granulomatous pathology in mouse models that resembles those induced by MTB infection. Using the acute TDM model, this work investigates the effect of recombinant human and mouse lactoferrin to reduce granulomatous pathology. C57BL/6 mice were injected intravenously with TDM at a dose of 25 μg·mouse

Topics: Administration, Oral; Animals; Cord Factors; Cytokines; Female; Granuloma; Humans; Lactoferrin; Lung Diseases; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Recombinant Proteins; Tuberculosis

In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

Topics: Animals; Cell Line; Cells, Cultured; Cord Factors; Cytokines; Eukaryotic Translation Initiation Factor 5A; Gene Expression; Granuloma; Immunoblotting; Inflammation; Lectins, C-Type; Lysine; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mycobacterium tuberculosis; NIH 3T3 Cells; Nitric Oxide; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Peptide Initiation Factors; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Toll-Like Receptors

Trehalose 6,6'dimycolate (TDM) is a glycolipid found in nearly pure form on the surface of virulent Mycobacterium tuberculosis (MTB). This manuscript investigated the production of TDM, growth rate and colony morphology of multiple strains of MTB, each of which had been isolated from both pulmonary (sputum) and extrapulmonary sites of multiple patients. Since sputum contains MTB primarily from cavities and extrapulmonary biopsies are typically granulomas, this provided an opportunity to compare the behavior of single strains of MTB that had been isolated from cavities and granulomas. The results demonstrated that MTB isolated from pulmonary sites produced more TDM (3.23 ± 1.75 μg TDM/mg MTB), grew more rapidly as thin spreading pellicles, demonstrated early cording, and climbed culture well walls. In contrast, extrapulmonary isolates produced less TDM (1.42 ± 0.58 μg TDM/mg MTB) (p < 0.001) and grew as discrete patches with little tendency to spread or climb. Both Beijing pulmonary isolates and the non-Beijing pulmonary isolates produced significantly more TDM (1.64 ± 0.46 μg TDM/mg MTB) and grew faster than the Beijing and non-Beijing extrapulmonary isolates (1.14 ± 0.63 μg TDM/mg MTB) (p < 0.001 and p < 0.005 respectively). These results indicate that MTB from pulmonary sites (cavities) grows faster and produces more TDM than strains isolated from extrapulmonary sites (granulomas). This report suggests a critical role for TDM in cavitary TB.

Topics: Biopsy; Cord Factors; Granuloma; Humans; Mycobacterium tuberculosis; Sputum; Time Factors; Tuberculosis, Pulmonary; Virulence

Trehalose 6,6'-dimycolate (TDM) is a cell wall glycolipid and an important virulence factor of mycobacteria. In order to study the role of TDM in the innate immune response to Mycobacterium tuberculosis, microarray analysis was used to examine gene regulation in murine bone marrow-derived macrophages in response to 90-μm-diameter polystyrene microspheres coated with TDM. A large number of genes, particularly those involved in the immune response and macrophage function, were up- or downregulated in response to these TDM-coated beads compared to control beads. Genes involved in the immune response were specifically upregulated in a myeloid differentiation primary response gene 88 (MyD88)-dependent manner. The complexity of the transcriptional response also increased greatly between 2 and 24 h. Matrix metalloproteinases (MMPs) were significantly upregulated at both time points, and this was confirmed by quantitative real-time reverse transcription-PCR (RT-PCR). Using an in vivo Matrigel granuloma model, the presence and activity of MMP-9 were examined by immunohistochemistry and in situ zymography (ISZ), respectively. We found that TDM-coated beads induced MMP-9 expression and activity in Matrigel granulomas. Macrophages were primarily responsible for MMP-9 expression, as granulomas from neutrophil-depleted mice showed staining patterns similar to that for wild-type mice. The relevance of these observations to human disease is supported by the similar induction of MMP-9 in human caseous tuberculosis (TB) granulomas. Given that MMPs likely play an important role in both the construction and breakdown of tuberculous granulomas, our results suggest that TDM may drive MMP expression during TB pathogenesis.

Topics: Animals; Cord Factors; Gene Expression Regulation; Granuloma; Humans; Macrophages; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Mycobacterium tuberculosis; Myeloid Differentiation Factor 88; Oligonucleotide Array Sequence Analysis; RNA; Transcriptome

The objective of this paper is to examine the role of NF-kappa B inhibitors A20 and ABIN-family proteins in the trehalose 6,6'-dimycolate (TDM)-induced model of tuberculous granulomatous lesions.. BALB/c mice were twice injected i.p. with w/o/w emulsions that contain TDM at a 1 week-interval. The mice were killed at days 0, 3, 7, 14, or 21 after the last injection. The mRNA and protein levels of A20 and ABIN-family proteins were measured by real-time PCR using mRNA or protein extract from the lesions. The activation status of NF-kappa B was analyzed by Western blotting and immunohistochemistry. Finally, the protein extracts were immunoprecipitated by anti-ABIN-3 antibody to identify the protein that potentially interacts with ABIN-3.. The activation of NF-kappa B pathway coincided with granuloma development, while A20 and ABIN-3 increased in accordance with granuloma regression. TAK-1 protein was co-precipitated with ABIN-3 by immunoprecipitation using anti-ABIN-3 antibody.. The results suggest that ABIN-3 contributed to granuloma regression by interacting with TAK-1 and, as a consequence, inhibiting activation of NF-kappa B pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cord Factors; Cysteine Endopeptidases; DNA-Binding Proteins; Female; Granuloma; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase Kinases; Mice; Mice, Inbred BALB C; NF-kappa B; Nuclear Proteins; Omentum; Peritoneal Diseases; RNA, Messenger; Tumor Necrosis Factor alpha-Induced Protein 3; Ubiquitin-Protein Ligases

The objective of this paper is to elucidate the factors contributing to the development and regression of trehalose 6,6'-dimycolate (TDM)-induced model of tuberculous granulomatous lesions.. BALB/c mice were twice injected i.p. with a 100 μl of w/o/w emulsion (100 μg of TDM, 3.2 μl of Freund's incomplete adjuvant, 3.2 μl of PBS, and 93.6 μl of saline containing 0.2% Tween 20) at a 1 week interval. The mice were killed at days 0, 3, 7, 14, or 21 after the last injection. Three mice were used per group.. We examined histopathological changes of the lesions and defined the expression levels of cytokines and suppressor of cytokine signaling (SOCS) family proteins by real-time PCR.. The levels of inflammatory cytokine, such as TNF-α and IL-1β, paralleled with the size of the lesions and the levels of TGF-β and SOCS-3 were high at regression phase.. Our results demonstrated that both the down-regulation of inflammatory cytokines and up-regulation of TGF-β and SOCS-3 are crucial for histopathological changes including alteration in the sizes of the lesions and changes in inflammatory cell populations.

Topics: Adjuvants, Immunologic; Animals; Chemokines; Cord Factors; Cytokines; Female; Granuloma; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; Suppressor of Cytokine Signaling Proteins; Tuberculosis

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6'-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was adsorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in an in vivo model of mycobacterial granuloma formation and in infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production in response to trehalose dimycolate, nor was there a difference in lung histopathology or overall bacterial burden in mice infected with Mycobacterium tuberculosis. In this model, fibrin(ogen) was not required for cell recruitment, cytokine response, or response to infection, but it promoted granulation tissue formation and suppressed leukocyte necrosis.

Topics: Animals; Cord Factors; Cytokines; Female; Fibrinogen; Granuloma; Inflammation; Leukocytes; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mycobacterium tuberculosis; Skin; Tuberculosis

The progression of human tuberculosis (TB) to active disease and transmission involves the development of a caseous granuloma that cavitates and releases infectious Mycobacterium tuberculosis bacilli. In the current study, we exploited genome-wide microarray analysis to determine that genes for lipid sequestration and metabolism were highly expressed in caseous TB granulomas. Immunohistological analysis of these granulomas confirmed the disproportionate abundance of the proteins involved in lipid metabolism in cells surrounding the caseum; namely, adipophilin, acyl-CoA synthetase long-chain family member 1 and saposin C. Biochemical analysis of the lipid species within the caseum identified cholesterol, cholesteryl esters, triacylglycerols and lactosylceramide, which implicated low-density lipoprotein-derived lipids as the most likely source. M. tuberculosis infection in vitro induced lipid droplet formation in murine and human macrophages. Furthermore, the M. tuberculosis cell wall lipid, trehalose dimycolate, induced a strong granulomatous response in mice, which was accompanied by foam cell formation. These results provide molecular and biochemical evidence that the development of the human TB granuloma to caseation correlates with pathogen-mediated dysregulation of host lipid metabolism.

Topics: Animals; Antigens, CD; Caseins; Cholesterol; Cholesterol Esters; Coenzyme A Ligases; Cord Factors; Gene Regulatory Networks; Granuloma; Humans; Lactosylceramides; Lipid Metabolism; Lung Diseases; Macrophages; Membrane Proteins; Mice; Necrosis; Peptides; Perilipin-2; Saposins; Triglycerides; Tuberculosis

The immune system responds to tuberculosis (TB) infection by forming granulomas. However, subsequent immune-mediated destruction of lung tissue is a cause of significant morbidity and contributes to disease transmission. Lactoferrin, an iron-binding glycoprotein, has demonstrated immunomodulatory properties that decrease tissue destruction and promote T(H)1 immune responses, both of which are essential for controlling TB infection. The cord factor trehalose 6,6'-dimycolate (TDM) model of granuloma formation mimics many aspects of TB infection with a similar histopathology accompanied by proinflammatory cytokine production. C57BL/6 mice were injected intravenously with TDM. A subset of mice was given 1 mg of bovine lactoferrin 24 h post-TDM challenge. Lung tissue was analyzed for histological response and for the production of proinflammatory mediators. C57BL/6 mice demonstrated a granuloma formation that correlated with an increased production of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α,) IL-12p40, interferon-gamma (IFN-γ), and IL-10 protein. Mice treated with lactoferrin postchallenge had significantly fewer and smaller granulomas compared with those given TDM alone. Proinflammatory and T(H)1 cytokines essential to the control of mycobacterial infections, such as TNF-α and IFN-γ, were not significantly different in mice treated with lactoferrin. Furthermore, the anti-inflammatory cytokines IL-10 and transforming growth factor-β were increased. A potential mechanism for decreased tissue damage observed in the lactoferrin-treated mice is proposed. Because of its influence to modulate immune responses, lactoferrin may be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection.

Topics: Animals; Cord Factors; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Granuloma; Interleukin-10; Lactoferrin; Lung; Lung Diseases; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Protein Biosynthesis; Transforming Growth Factor beta; Tuberculosis

Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6'-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM.

Topics: Animals; Cord Factors; Granuloma; Lectins, C-Type; Ligands; Lung Diseases; Macrophage Activation; Membrane Proteins; Mice; Mice, Inbred C57BL; Myeloid Differentiation Factor 88; Receptors, IgG

To establish the structure biological activity relationship of cord factor (trehalose 6,6'-dimycolate, TDM), we compared the molecular or supra-molecular structure of TDM micelles with toxicity, thymic atrophy and granulomatogenicity in lungs and spleen of BALB/c mice. According to the difference in the mycolyl subclass composition, TDM was divided into two groups, one possessing alpha-, methoxy- and keto-mycolates in M. tuberculosis H37Rv, M. bovis BCG and M. kansasii (group A) and the other having alpha-, keto- and wax ester-mycolates in M. avium serotype 4, M. phlei and M. flavescens (group B), although mycolic acid molecular species composition differed in each group considerably. Supra-molecular structure of TDM micelle differed species to species substantially and the micelle size of TDM from M. bovis BCG Connaught was the largest. The highest toxicity was shown with TDM from M. tuberculosis H37Rv which possessed the highest amount of alpha- (47.3%) and methoxy-mycolates (40.8%), while TDM from M. phlei having the low amount of alpha-mycolate (11.6%) showed almost no toxicity with the given doses. The thymic atrophy was observed with TDM from group A, but not with TDM from group B. On the other hand, TDM from group B showed massive lung granulomatogenic activity based on the histological observations and organ indices. Taken together, group A TDM showed a wide variety of micelle sizes and specific surface areas, high to low toxicity and marked to moderate granulomatogenicity, while group B TDM showed smaller sizes of micelles and larger specific surface areas, lower toxicity but higher granulomatogenicity in lungs. Existence of higher amount of longer chain alpha-mycolates in TDM appeared to be essential for high toxicity and thymic apoptotic activity, whereas TDM possessing wax ester-mycolate with smaller sized micelles seemed to be less toxic, but more granulomatogenic in lungs in mice. Thus, the mycolic acid subclass and molecular species composition of TDM affect critically the micelle forms, toxicity and granulomatogenicity in mice, while the relative abundances and carbon chain length of alpha-mycolate affected the toxicity in mice.

Topics: Animals; Apoptosis; Atrophy; Body Weight; Cord Factors; Female; Granuloma; Histocytochemistry; Lung; Lymphocytes; Mice; Mice, Inbred BALB C; Micelles; Models, Animal; Molecular Structure; Mycobacterium; Specific Pathogen-Free Organisms; Spleen; Structure-Activity Relationship; Thymus Gland

The granulomatous response is a characteristic histological feature of Mycobacterium tuberculosis infection responsible for organism containment. The development of cell-mediated immunity is essential for protection against disease, as well as being required for maintenance of the sequestering granulomatous response. Trehalose 6,6'-dimycolate (TDM; cord factor), a glycolipid associated with the cell wall of mycobacteria, is implicated as a key immunogenic component in M. tuberculosis infection. Models of TDM-induced hypersensitive granulomatous response have similar pathologies to that of active tuberculosis infection. Prior immunization (sensitization) of mice with TDM results in exacerbated histological damage, inflammation and lymphocytic infiltration upon subsequent TDM challenge. Adoptive transfer experiments were performed to ascertain the cell phenotype governing this response; CD4(+) cells were identified as critical for development of related pathology. Mice receiving CD4(+) cells from donor TDM-immunized mice demonstrated significantly increased production of Th1-type cytokines IFN-gamma and IL-12 within the lung upon subsequent TDM challenge. Control groups receiving naïve CD4(+) cells, or CD8(+) or CD19(+) cells isolated from TDM-immunized donors, did not exhibit an exacerbated response. The identified CD4(+) cells isolated from TDM-immunized mice produced significant amounts of IFN-gamma and IL-2 when exposed to TDM-pulsed macrophages in vitro. These experiments provide further evidence for involvement of a cell-mediated response in TDM-induced granuloma formation, which mimics pathological damage elicited during M. tuberculosis infection.

Topics: Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; Cord Factors; Granuloma; Interferon-gamma; Interleukin-12; Lung; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; T-Lymphocyte Subsets

The mechanisms by which pulmonary granuloma formation is caused by administration of mycobacterial glycolipids such as trehalose dimycolate (TDM), lipoarabinomannan (LAM) and phosphatidylinositol mannosides (PIM) were investigated. When peritoneal and alveolar macrophages were stimulated with TDM, LAM and PIM in vitro, TDM exhibited the strongest tumour necrosis factor (TNF)-inducing activity. Responsiveness of macrophages from mice defected Toll-like receptor 4 (TLR4) was much higher than that of the wild-type mice. Although PIM and LAM also had a significant activity, LAM rather than PIM stimulated higher TNF-alpha production by alveolar macrophage. When mycobacterial glycolipids were injected as water-in-oil-in-water emulsion into mice via the tail vein, development of pulmonary granuloma in response to glycolipids were related closely to their TNF-inducing activity and TDM exhibited the strongest activity. Granuloma formation was observed not only in mice lacking interleukin (IL)-12 signalling but also interferon (IFN)-gamma knock-out mice. Granuloma formation caused by glycolipids correlated with TNF-alpha levels in lungs. Administration of anti-TNF-alpha monoclonal antibody into TDM-injected IFN-gamma knock-out mice decreased in granuloma formation, suggesting that development of pulmonary granuloma by mycobacterial glycolipids such as TDM is due to IFN-gamma-independent and TNF-alpha-dependent pathway.

Topics: Adjuvants, Immunologic; Animals; Antigens, Bacterial; Cord Factors; Female; Glycolipids; Granuloma; Injections; Interferon-gamma; Lipopolysaccharides; Lung Diseases; Macrophages; Macrophages, Alveolar; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; Phosphatidylinositols; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Trehalose 6,6'-dimycolate (TDM) is the most abundant, most granulomagenic, and most toxic lipid extractable from the surface of virulent Mycobacterium tuberculosis (MTB). We further examined its toxicity, which requires activation by oily surfaces. Injections of MTB and/or TDM into sensitized mice induced caseating granulomas that centered on oil droplets. If large doses of MTB were injected in saline, caseating granulomas developed in adipose tissue, but MTB with surface TDM removed induced only acute inflammation that did not persist. Variations in protocols produced several variants of caseating granulomas, each with characteristics of human tuberculosis. In each instance, MTB were localized in fat cells or oil drops during initiation of caseating granulomas suggesting that necrosis was caused by activation of the toxicity of TDM toxicity. Evidence extending these findings to the lung was derived from the observation that in sensitized mice, as in humans, tuberculosis development stimulates accumulation of lipid selectively in alveoli. MTB preferentially associated with lipid droplets in developing necrotic foci in late-stage murine tuberculosis. This supports the hypothesis that pulmonary tuberculosis sequesters MTB in a protected environment that accumulates lipid until it is able to activate the toxicity of TDM and initiate necrosis that results in caseating granulomas.

Topics: Adipose Tissue; Animals; Cord Factors; Granuloma; Lipid Metabolism; Lung; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Necrosis; Peritoneum; Tuberculosis, Pulmonary

Virulence mechanism of infection with Mycobacterium tuberculosis is currently focused to be clarified in the context of cell surface lipid molecule. Comparing two mycobacterial glycolipids, we observed toxicity and prominent granulomatogenic activity of trehalose 6,6'-dimycolate (TDM) injection in mice, evident by delayed body weight gain and histological observations, whereas 2,3,6,6'-tetraacyl trehalose 2'-sulfate (SL) was non-toxic and non-granulomatogenic. Likewise, TDM but not SL caused temporarily, but marked increase of lung indices, indicative of massive granuloma formation. Interestingly, co-administration of TDM and SL prevented these symptoms distinctively and SL inhibited TDM-induced release of tumor necrosis factor alpha (TNF-alpha) in a dose-dependent manner. Histological findings and organ index changes also showed marked inhibition of TDM induced granuloma formation by co-administration of SL. Simultaneous injection of SL together with TDM was highly effective for this protection, as neither injection 1h before nor after TDM injection showed highly inhibitory. In parallel studies on a cellular level, TDM elicited strong TNF-alpha release from alveolar but not from peritoneal macrophages in vitro. This effect was blocked when alveolar macrophages were incubated in wells simultaneously coated with TDM and SL, indicating that SL suppresses TDM-induced TNF-alpha release from macrophages. Our results suggest a novel mechanism by which SL could contribute to virulence at early stage of mycobacterial infection or stimulation with the glycolipids by counteracting the immunopotentiating effect of TDM.

Topics: Animals; Cord Factors; Glycolipids; Granuloma; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred ICR; Mycobacterium tuberculosis; Species Specificity; Tumor Necrosis Factor-alpha; Virulence Factors

The granulomatous response is the characteristic histological feature of Mycobacterium tuberculosis infection that is essential for organism containment. Trehalose 6,6-dimycolate (TDM), a cell-wall glycolipid present on most mycobacterial species, has been implicated in the pathogenesis of M. tuberculosis infection. TDM has potent immunoregulatory and inflammatory properties, and can be used to model granulomatous reactions that mimic, in part, pathology caused during active infection. This study examined the hypersensitive granulomatous response, focusing on cellular responses specific to TDM. Lungs from mice immunized with TDM emulsion demonstrated exacerbated histological damage, inflammation, and lymphocytic infiltration upon subsequent challenge with TDM. Splenocytes recovered from these mice demonstrated significant interferon (IFN)-gamma production during recall response to TDM, as well as increased production of proinflammatory mediators (tumour necrosis factor-alpha, interleukin-6 and macrophage inflammatory protein-1alpha). The exacerbated response could be adoptively transferred to naïve mice. Administration of non-adherent lymphocytes or purified CD3(+) cells from TDM-immunized mice led to increased inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon challenge with TDM. Recipient mice that received immunized CD3(+) lymphocytes demonstrated significant increases in Th1-type cytokines and proinflammatory mediators in lung tissue following TDM challenge. When CD1d(-/-) mice were immunized with TDM, they failed to generate a specific IFN-gamma response, suggesting a role for this molecule in the generation of hypersensitivity. These experiments provide further evidence for the involvement of TDM-specific CD3(+) T cells in pathological damage elicited during M. tuberculosis infection.

Topics: Adoptive Transfer; Animals; Bacterial Proteins; CD3 Complex; Chemokine CCL4; Cord Factors; Granuloma; Hypersensitivity, Delayed; Interferon-gamma; Interleukin-6; Lung; Lymphocyte Subsets; Macrophage Inflammatory Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Mycobacterium tuberculosis; Tuberculosis; Tumor Necrosis Factor-alpha

The hallmark of Mycobacterium-induced pathology is granulomatous inflammation at the site of infection. Mycobacterial lipids are potent immunomodulators that contribute to the granulomatous response and are released in appreciable quantities by intracellular bacilli. Previously we investigated the granulomagenic nature of the peripheral cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin (BCG) by coating the lipids onto 90-microm diameter microspheres that were mixed into Matrigel matrix with syngeneic bone marrow-derived macrophages and injected i.p. into mice. These studies demonstrated that BCG lipids elicit proinflammatory cytokines and recruit leukocytes. In the current study we determined the lipids responsible for this proinflammatory effect. BCG-derived cell wall lipids were fractionated and purified by liquid chromatography and preparative TLC. The isolated fractions including phosphatidylinositol dimannosides, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, trehalose monomycolate, trehalose dimycolate, and mycoside B. Trehalose dimycolate, when delivered to bone marrow-derived murine macrophages, induced the greatest secretion of IL-1beta, IL-6, and TNF-alpha in vitro. Trehalose dimycolate similarly induced the greatest secretion of these proinflammatory cytokines in ex vivo matrices over the course of 12 days. Trehalose monomycolate and dimycolate also induced profound neutrophil recruitment in vivo. Experiments with TLR2 or TLR4 gene-deficient mice revealed no defects in responses to trehalose mycolates, although MyD88-deficient mice manifested significantly reduced cell recruitment and cytokine production. These results demonstrate that the trehalose mycolates, particularly trehalose dimycolate, are the most bioactive lipids in the BCG extract, inducing a proinflammatory cascade that influences granuloma formation.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, Differentiation; Cord Factors; Cytokines; Female; Granuloma; In Vitro Techniques; Inflammation Mediators; Macrophages; Male; Membrane Lipids; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Microspheres; Mycobacterium bovis; Myeloid Differentiation Factor 88; Neutrophils; Receptors, Cell Surface; Receptors, Immunologic; Toll-Like Receptor 2; Toll-Like Receptor 4

The development of pulmonary granulomatous lesions during mycobacterial infection is a complex phenomenon, in part caused by responses elicited towards the surface glycolipid trehalose 6,6'-dimycolate (TDM; cord factor). The molecular mechanisms underlying granuloma formation following challenge with TDM are not yet completely understood. The present study defines pathologic differences in acute response to Mycobacterium tuberculosis TDM in C57BL/6 mice and mice lacking the C5a receptor (C5aR-/-). Mice were intravenously injected with TDM prepared in water-in-oil-in-water emulsion and examined for histologic response and changes in proinflammatory cytokines and chemokines in lung tissue. Control C5a receptor-sufficient mice demonstrated a granulomatous response that peaked between days 4 and 7. Increased production of macrophage inflammatory protein-1 alpha (MIP-1alpha), interleukin-1beta (IL-1beta) and CXC chemokine KC (CXCL1) correlated with development of granulomas, along with modest change in tumor necrosis factor-alpha (TNF-alpha). In contrast, the C5aR-/- mice revealed markedly exacerbated inflammatory response. The receptor-deficient mice also demonstrated a lack of coherent granulomatous response, with severe oedema present and instances of lymphocytic cuffing around pulmonary vessels. Lung weight index was increased in the C5aR-/- mice, correlating with increased MIP-1alpha, KC, IL-1beta and TNF-alpha over that identified in the congenic C5aR-sufficient controls. Correlate experiments performed in C5-deficient (B10.D2-H2d H2-T18c Hco/oSnJ) mice revealed similar results, leading to the conclusion that C5 plays a significant role in mediation of chemotactic and activation events that are the basis for maturation of granulomatous responses to TDM.

Topics: Animals; Chemokine CCL3; Chemokine CCL4; Chemokine CXCL1; Chemokines, CXC; Complement C5; Cord Factors; Granuloma; Histocytochemistry; Intercellular Signaling Peptides and Proteins; Interleukin-1; Macrophage Inflammatory Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mycobacterium tuberculosis; Receptor, Anaphylatoxin C5a; Tuberculosis; Tumor Necrosis Factor-alpha

The present study defines pathologic differences in acute and hypersensitive responses to Mycobacterium tuberculosis glycolipid trehalose-6,6'-dimycolate (TDM, cord factor) in normal BALB/c mice and those deficient in group II CD1 molecule CD1d1. Mice immunized against TDM demonstrate hypersensitive responses, yet the mechanisms for TDM presentation remain elusive. Mice lacking CD1d (CD1D(-/-)) demonstrate dysregulated granulomatous response to TDM, compared with CD1D(+/-) heterozygous controls. Because CD1d-restricted T cells can regulate macrophage immune functions at mucosal surfaces, we hypothesized that CD1D(-/-) mice would show deficient TDM-induced hypersensitive pulmonary granulomatous response in which T cells play a central role. Control CD1D(+/+) mice sensitized and subsequently challenged with TDM demonstrated aggressive inflammation defined by monocytic lesions contained by CD3(+) lymphocytic cuffing. CD1D(-/-) mice demonstrated distinctly different pathologies, with edema present concurrent with extended, nonfocal mononuclear cell-based granulomatous reactions. Furthermore, CD1D(-/-) mice did not demonstrate destructive lesions, and CD3(+) lymphocytes were only loosely organized in proximity to reactive pathology. The CD1d-deficient mice demonstrated rapid increases in proinflammatory mRNAs, with significant differences in interferon-gamma (IFN-gamma) compared to the wild-type group. IFN-gamma, interleukin-6 (IL-6), and IL-12 proteins were significantly elevated in the CD1D(-/-) group compared with wild-type mice (p < 0.05) 2 days after TDM challenge. However, by 7 days postadministration, similar production for all cytokines and proinflammatory molecules examined was present in both groups of mice. These experiments provide evidence for a role for CD1d in mediation of pathology during hypersensitive responses to the mycobacterial glycolipid TDM.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD1; Antigens, CD1d; Cord Factors; Cytokines; Granuloma; Humans; Hypersensitivity; Lung; Mice; Mice, Inbred BALB C; Mice, Knockout; Mycobacterium tuberculosis; RNA, Messenger

This study was designed to determine the identity of granulomatogenic substances in Mycobacterium bovis BCG Pasteur. When heat-treated BCG Pasteur bacilli were introduced into the lungs of guinea-pigs by an inhalation exposure apparatus, pulmonary granulomas without necrosis developed. Furthermore, when four kinds of mycolates derived from M. tuberculosis Aoyama B strain were introduced into the lungs by the same method, only trehalose 6,6'-dimycolate (TDM) and methyl ketomycolate induced pulmonary granulomas without central necrosis. The pulmonary granulomas consisted of epithelioid macrophages and lymphocytes. When a mixture of TDM and anti-TDM antibody was introduced into the lungs, development of granulomatous lesions was reduced. These data indicate that TDM and methyl ketomycolate are potent granulomatogenic reagents.

Topics: Administration, Inhalation; Animals; Cord Factors; DNA, Bacterial; Female; Granuloma; Guinea Pigs; Lung; Lung Diseases; Mycobacterium bovis; Mycolic Acids

Granulomatous inflammation is characterized morphologically by a compact organized collection of macrophages and their derivatives. It is classified as either a hypersensitivity type or a foreign-body type. Lipid components of the Mycobacterium tuberculosis cell wall participate in the pathogenesis of infection. Strains of M. tuberculosis have cord factor (trehalose 6,6'-dimycolate [TDM]) on their surface. To clarify host responses to TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reaction, histopathology, and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mice challenged with TDM. In the present study, we have demonstrated for the first time that TDM can induce both foreign-body-type (nonimmune) and hypersensitivity-type (immune) granulomas by acting as a nonspecific irritant and T-cell-dependent antigen. Immunized mice challenged with TDM developed more severe lesions than unimmunized mice. At the active lesion, we found monocyte chemotactic, proinflammatory, and immunoregulatory cytokines. The level was enhanced in immunized mice challenged with TDM. This result implies that both nonimmune and immune mechanisms participate in granulomatous inflammation induced by mycobacterial infection. Taken together with a previous report, this study shows that TDM is a pleiotropic molecule against the host and plays an important role in the pathogenesis of tuberculosis.

Topics: Animals; Chemokines; Cord Factors; Cytokines; Female; Foreign Bodies; Granuloma; Hypersensitivity, Delayed; Lung; Mice; Mice, Inbred BALB C

Novel mycoloyl glycolipids with short carbon chains were isolated and purified from Rhodococcus sp. 4306, a soil origin of Actinomycetales. Their chemical structures were identified as trehalose 6,6'-dimycolate (TDM), trehalose 6-monomycolate, glucose 6-monomycolate, mannose 6-monomycolate and fructose 6-monomycolate. The length of carbon chains and number of double bonds of mycolic acids were C(34), C(36)and C(38)saturated, monoenoic and dienoic molecular species, which were much shorter than those of Mycobacterium tuberculosis (C(78-88)monoenoic and dienoic). Among them, only TDM could induce prominent granulomatous inflammation of the lung and spleen in mice. By contrast, other mycoloyl glycolipids induced mild lesions. The small-sized TDM of Rhodococcus possessed granulomatogenic activity, however, the toxicity was much lower than that of M. tuberculosis. Rhodococcal TDM was composed of mycolic acid with the shortest carbon chains, when compared to granulomatogenic TDM of Mycobacterium, Nocardia and Rhodococcus reported previously. Our results imply that rhodococcal TDM is a pathogenetic factor similar to that of M. tuberculosis, although rhodococcal TDM exhibits low toxicity.

Topics: Actinomycetales Infections; Animals; Chromatography, Thin Layer; Cord Factors; Granuloma; Male; Mice; Mice, Inbred ICR; Mycobacterium tuberculosis; Rhodococcus; Structure-Activity Relationship; Trehalose

Trehalose 6,6'-dimycolate (TDM) plays important roles in the development of granulomatous inflammation during infection with Mycobacterium spp., Rhodococcus spp., etc. To reveal the augmenting effect of TDM on vascular endothelial growth factor (VEGF) production and neovascularization, we investigated murine granulomatous tissue air pouches induced by Rhodococcus sp. strain 4306 TDM dissolved in Freund's incomplete adjuvant (FIA), comparing them to pouches treated with FIA alone. Histologically, granulomatous tissue and new vessel formation, which reached a maximum at day 7, was greatly enhanced by treatment with TDM. At day 1, VEGF-positive neutrophils accumulated in the pouch wall with frequency of 95% of total infiltrating cells, adhering to TDM-containing micelles. By day 3, granulomatous tissue and new vessels started to develop, and VEGF-positive macrophages appeared in a small number and gradually increased in number thereafter. The pouch contents of VEGF, interleukin-1beta, tumor necrosis factor alpha, and transforming growth factor beta were significantly elevated in TDM-treated pouches, with peaks at days 1, 0.5, 1, and 3, respectively, compared to those of control pouches, while that of basic fibroblast growth factor showed no significant increase. Treatment with anti-VEGF antibody inhibited TDM-induced granulomatous tissue formation and neovascularization, and administration of recombinant VEGF into pouches treated with FIA alone induced neovascularization comparable to that in the TDM-treated pouches. Incubation of neutrophils and macrophages on TDM-coated plastic dishes increased the VEGF release. The present results indicate that TDM augments VEGF production by neutrophils and macrophages and induces neovascularization in the granulomatous tissue.

Topics: Animals; Antibodies; Cord Factors; Dose-Response Relationship, Drug; Endothelial Growth Factors; Fibroblast Growth Factor 2; Freund's Adjuvant; Granuloma; Immunoglobulin G; Immunohistochemistry; Interleukin-1; Lipids; Lymphokines; Macrophages; Male; Mice; Mice, Inbred ICR; Microscopy, Electron; Neovascularization, Physiologic; Neutrophils; Rhodococcus; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Trehalose 6,6'-dimycolate (TDM) is a cell surface molecule of Mycobacterium tuberculosis. TDM induced a loss of body weight and prominent granulomas in the liver and lungs by the intravenous injection of TDM into rabbits. TDM also induced atrophy of the thymus and spleen due to apoptosis. By contrast, sulfolipid (2,3,6, 6'-tetraacyl trehalose 2'-sulfate) induced neither toxicity, nor granuloma formation, nor atrophy of the thymus and spleen. In rabbits the histopathological changes were more dramatic than in mice. The rabbit model may be more sensitive and may provide more information on the beneficial or pathological effects of TDM.

Topics: Animals; Apoptosis; Atrophy; Body Weight; Cord Factors; Female; Glycolipids; Granuloma; Granuloma, Respiratory Tract; Lipids; Liver; Lung; Mycolic Acids; Rabbits; Spleen; Thymus Gland; Trehalose

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Cytokines; Disease Models, Animal; Female; Gene Expression; Granuloma; Inflammation; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium; RNA, Messenger

Antibody production against the trehalose 6,6'-dimycolate (TDM, cord factor) of Rhodococcus ruber, a non-pathogenic species of the Actinomycetales group, was investigated in mice by repeated intraperitoneal injection of TDM in water-in-oil-in-water micelles without carrier protein. The antigenic TDM was isolated and purified chromatographically from the chloroform-methanol extractable lipids of R. ruber. The hydrophobic moiety of this TDM was composed of two molecules of monoenoic or dienoic alpha-mycolic acids with a carbon chain length ranging from C44 to C48 centering at C46. To detect the antibody, an enzyme-linked immunosorbent assay (ELISA) system was employed using plastic plates coated with TDM. The antibody reacted against the TDM of R. ruber. The antibody was reactive in similar fashion against glycosyl monomycolates differing in the carbohydrate moiety, such as that of glucose mycolate (GM) and mannose mycolate (MM), obtained from R. ruber. Moreover, the antibody reacted against mycolic acid methyl ester itself when it was used as the antigen in ELISA, and trehalose did not absorb the antibody to TDM or inhibit the reaction. These results indicate that the epitope of TDM recognized by the antibody is mycolic acid, an extremely hydrophobic part of the molecule. Next, we prepared monoclonal anti-TDM antibody (moAb) in mice myeloma cells to examine its biological activities and the role of humoral immunity in mycobacterial infection. MoAb reacted against the TDM, glycosyl mycolate, and mycolic acid methyl ester in ELISA in the same manner as our polyclonal antibody did. The administration of moAb suppressed granuloma formation in the lungs, spleen, and liver induced by TDM and inhibited the production of interleukin-1 (IL-1) and chemotactic factor, which is reported to precede granuloma formation.

Topics: Absorption; Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Carbohydrates; Chemotactic Factors; Cord Factors; Glycolipids; Granuloma; Interleukin-1; Male; Mice; Mice, Inbred ICR; Mycolic Acids; Rhodococcus

Cord factor (trehalose 6,6'-dimycolate: TDM) is a well-known toxic glycolipid in Mycobacterium tuberculosis. We isolated various mycoloyl glycolipids from Nocardia asteroides 23,167 and related species which are closely related taxonomically to Mycobacterium. Since Nocardia is also an opportunistic pathogen co-infected with HIV, we examined in vivo granuloma formation and the in vitro macrophage activation in mice. We found that cord factor (TDM) and glucose monomycolate (GM) from Nocardia asteroides and Rhodococcus species with shorter chain mycolic acids also exhibited distinctive granuloma-forming activity in lungs, spleen and liver in mice and in vitro induction of prostaglandin E2 (PGE2) and interleukin 1 (IL-1) synthesis. We also found that mycoloyl glycolipids possessing trehalose or glucose as a carbohydrate moiety exhibited immunomodulatory activity, and that mycoloyl glycolipids with longer chain mycolic acids exhibited stronger activity in mice than did those with shorter chain mycolic acids. The mycoloyl glycolipids from Nocardia asteroides directly activated macrophages. Stimulation of above concentration with 0.16 microgram/ml of TDM or 0.8 microgram/ml of GM markedly enhanced production of PGE2 by mouse peritoneal macrophages. At higher concentrations above 100 micrograms/ml of TDM or 500 micrograms/ml of GM, IL-1 release was also enhanced.

Topics: Animals; Cord Factors; Glycolipids; Granuloma; Macrophages; Male; Mice; Mice, Inbred ICR; Nocardia asteroides; Rhodococcus

We analyzed the mycolic acid composition of trehalose 6,6'-dimycolate (TDM) obtained from Mycobacterium, using thin layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Utilizing TDM, whose structure was confirmed, granuloma formation in mice was investigated. All TDM used exhibited considerable granuloma formation activity in the lung and spleen. In particular, TDM from M. bovis showed the greatest activity and toxicity among mycobacterial TDM. We therefore discussed the relationship between the chemical structure and granuloma-forming activity of TDM, especially in relation to the structure of mycolic acid in TDM.

Topics: Animals; Cord Factors; Granuloma; Mice; Mycobacterium; Mycolic Acids

It is reported that some bacteria or bacterial components cause thymic atrophy via the apoptotic process. The present study demonstrated for the first time in vivo induction of apoptosis in the mouse thymus by mycobacterial cord factor (CF) (trehalose 6,6'-dimycolate). When 300 microg of purified CF from Mycobacterium tuberculosis was intravenously administered to BALB/c mice in the form of water-in-oil-in-water (w/o/w) emulsion, thymic atrophy and pulmonary granulomas were induced with a peak on day 7, whereas, in the form of liposomes, CF induced thymic atrophy on days 14 to 21 in parallel with the development of hepatic granulomas. Thymic atrophy resulted from the depletion of cortical lymphocytes via apoptosis as revealed by DNA fragmentation and karyorrhectic changes. In contrast, mycobacterial sulfatide (2,3,6,6'-tetraacyl trehalose 2'-sulfate) caused neither thymic atrophy nor granuloma formation. Compared to lipopolysaccharide-induced thymocyte apoptosis, CF (w/o/w)-induced thymocyte apoptosis developed more slowly, reached a maximum later, and lasted longer but was less intense. Although serum tumor necrosis factor alpha (TNF-alpha) levels in CF-treated mice were not significantly elevated, administration of anti-TNF-alpha antibody almost completely inhibited thymic atrophy and granuloma formation. Serum corticosterone levels were only slightly elevated by CF administration. The present results indicate that mycobacterial CF induces thymic atrophy via apoptosis, which is closely linked with granuloma formation.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Cord Factors; Corticosterone; DNA; DNA Fragmentation; Drug Carriers; Electrophoresis, Agar Gel; Granuloma; Lipopolysaccharides; Liposomes; Liver; Lung; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron; Specific Pathogen-Free Organisms; Spleen; Sulfoglycosphingolipids; T-Lymphocytes; Thymus Gland; Tumor Necrosis Factor-alpha

We investigated here the kinetics of natural killer (NK) cells and extrathymic T cells, which include intermediate CD3 cells and gamma delta T cells, in the cord factor-induced granulomatous inflammation of the lungs and liver. In Balb/c mice, pulmonary inflammation elevated the proportion of NK cells and that of extrathymic T cells to mononuclear cells in the lungs. C3H/He mice exhibited shorter-term inflammation of the lungs than Balb/c mice and accordingly showed a smaller increase in the proportions of pulmonary NK cells and intermediate CD3 cells. In the liver of Balb/c mice, hepatic NK cells increased as well with the granulomatous changes, while intermediate CD3 cells exhibited a transient decrease before they increased. The present study has demonstrated that granulomatous inflammation is accompanied by the increase of lung-associated NK cells and extrathymic T cells and that there exists a difference between these two mouse strains in the induction of these lymphocyte subsets by cord factor.

Topics: Animals; CD3 Complex; Cord Factors; Flow Cytometry; Fluorescent Antibody Technique, Direct; Granuloma; Killer Cells, Natural; Kinetics; Liver; Liver Diseases; Lung; Lung Diseases; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Receptors, Antigen, T-Cell, gamma-delta; Specific Pathogen-Free Organisms; T-Lymphocytes

Diffuse pulmonary inflammation in interstitial lung diseases is associated with increased coagulation in the extravascular spaces of the lung. We hypothesized that conditions favoring coagulation over fibrinolysis in the lung are related to inflammation. Pulmonary coagulation and fibrinolysis were studied in two strains of mice susceptible or resistant to the development of lung inflammation in response to the mycobacterial cell wall glycolipid trehalose-6,6'-dimycolate (TDM). Susceptible animals treated with TDM intravenously develop well-organized collections of mononuclear cells in the lung parenchyma referred to as granulomas in this report. More granulomas were found in the susceptible ICR mice than in the resistant A/J mice after intravenous administration of TDM (7 +/- 1 granulomas/mm2 versus 1 +/- 0.3 granulomas/mm2, p = 0.005). Granuloma formation was associated with increased lung procoagulant activity (PCA) measured in bronchoalveolar lavage (BAL) cell lysates from susceptible mice. In contrast, TDM-resistant A/J mice challenged with TDM did not have a significant BAL cell PCA response, but expressed several-fold greater levels of lung BAL fluid plasminogen activator activity (PAA) than ICR mice. To examine the role of coagulation in the TDM pulmonary inflammatory response, susceptible C57Bl/10SnJ mice were anticoagulated by oral administration of warfarin prior to challenge of TDM; these mice developed fewer pulmonary granulomas than TDM-treated mice without warfarin treatment (2.6 +/- 0.5 granulomas/mm2 versus 6.5 +/- 0.8 granulomas/mm2, p < 0.001) but had similar BAL cell PCA and lung inflammatory changes as measured by lung weights and BAL cellularity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Coagulation; Bronchoalveolar Lavage Fluid; Cord Factors; Female; Fibrinolysis; Granuloma; Lung; Lung Diseases, Interstitial; Mice; Mice, Inbred ICR; Mice, Inbred Strains; Plasminogen Activators; Warfarin

A newly isolated mycoloyl glycolipid (Rt. GM-2) from Rhodococcus terrae 70012 was identified and the granulomagenic and antitumor activities were studied as compared with trehalose-6,6'-dimycolate (cord factor) also from R. terrae (Rt. TDM). The alkaline hydrolysis products of Rt. GM-2 contained trehalose, methyl-alpha-mycolate and a less-polar ester than the usual methyl-alpha-mycolate, possibly beta-keto mycolate (1:1:1, by mol. ratios). On the other hand, analysis of alditol acetate obtained after the mild permethylation, NaBH4 reduction, and acetylation showed the occurrence of 2,3,4-tri-O-methyl-6-O-acetylglucitol. Therefore, the original glycolipid (Rt. GM-2) was identified tentatively as 6-O-alpha-mycoloyl 6'-O-beta-ketomycoloyl trehalose. Intravenous injection of Rt. GM-2 in the form of water-in-oil-in-water emulsion caused prominent granulomas in lungs and spleen of ICR and BALB/c mice. The granulomagenic effects were as strong as those caused by Rt. TDM. The lung and spleen weights reached peaks one week after an injection of Rt. GM-2 in mice and then gradually decreased. Multiple intravenous injections of Rt. GM-2 and Rt. TDM showed antitumor activity against subcutaneously implanted Sarcoma-180, and caused prominent granulomatous changes and growth suppression of mice.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Cord Factors; Glycolipids; Granuloma; Male; Mice; Mice, Inbred BALB C; Mycolic Acids; Organ Specificity; Rhodococcus; Sarcoma; Tumor Cells, Cultured

Topics: Animals; Cord Factors; Glycolipids; Granuloma; Mice; Sarcoma 180; Structure-Activity Relationship; Tumor Necrosis Factor-alpha

Trehalose-6,6'-dimycolate (TDM) and its monosaccharide-type analogues were synthesized, and their lethal and adjuvant activities were examined in mice. All the monosaccharide-type analogues with a glucose or N-acetylglucosamine moiety were devoid of lethal toxicity to mice; in particular, D-GlcNAcM(1-deoxy) and D-GlcNM did not cause any loss of body weight at an early stage after intravenous administration as a 9% oil-in-water emulsion. Intraperitoneal administration of D-GlcNAcM(1-deoxy) in aqueous suspension, as well as TDM, could activate macrophages to become tumoricidal against tumour cells, whereas D-GlcNAcM(1-deoxy) in oil emulsion, unlike TDM, caused no granulomatous formation in the lung after intravenous injection. Squalane-treated D-GlcNAcM(1-deoxy) showed significant inhibition of spontaneous lung metastases by B16-BL6 melanoma cells when it was administered twice intratumorally. The non-toxic monosaccharide-type analogue of TDM [D-GlcNAcM(1-deoxy)] was a beneficial adjuvant for the activation of macrophages and the prevention of cancer metastasis.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Female; Glycolipids; Granuloma; Lung Neoplasms; Macrophage Activation; Mice; Mice, Inbred C57BL

When spread as a monolayer on the surface of hydrophobic beads and injected into mice, the mycobacterial glycolipid, trehalose 6,6'-dimycolate, reproduces the biologic effects traditionally associated with virulent mycobacteria, including acute inflammation, granuloma formation, and immune adjuvancy. Repeated intraperitoneal injection of glycolipid-coated beads into young C57Bl/6 mice elicits a granulomatous peritonitis, with concomitant dissemination of beads from the peritoneum to distant organs. Glycolipid-coated beads which disseminate from the peritoneum to other sites elicit neither acute inflammation nor granulomata. The coagulation system may be involved in the dissemination of glycolipid-coated beads as evidenced by the following: fibrinogen is a necessary cofactor of the trehalose dimycolate monolayer; diffuse peritoneal and pulmonary hemorrhage accompanies bead dissemination; peritoneal exudate collected shortly after intraperitoneal injection of glycolipid-coated beads is enriched in coagulant activity; coagulability of blood from trehalose dimycolate-treated animals is reduced; and anticoagulation inhibits the inflammatory response to glycolipid-coated beads. In this report, the dissemination of trehalose dimycolate-coated beads is characterized, and a role for the coagulation system in this process is proposed.

Topics: Animals; Blood Coagulation; Cord Factors; Glycolipids; Granuloma; Inflammation; Leukocytes; Lymphatic Diseases; Mice; Microspheres; Peritoneal Diseases; Polystyrenes; Time Factors; Tissue Adhesions; Tissue Distribution

The distribution of an aqueous suspension of cord factor (CF) from Mycobacterium bovis BCG in several mouse organs was examined after intravenous injection, and the correlation between evolution of the inflammatory granulomatous reaction and the presence of CF in these organs was determined. CF was preferentially deposited in the lungs and liver, and the kinetics of the pulmonary and hepatic inflammatory reaction, evaluated by determining the indices for these organs, showed a gradual increase on day 2 after injection, reached a peak around the fifth day, and declined thereafter. Histological analysis showed that on day 5 both the lungs and the liver were diffusely damaged by a mononuclear inflammatory infiltrate arranged in a granulomatous manner and consisting predominantly of histiocytes. CF elimination was more marked in the liver than in the lungs: 2 d after injection 76% of the material deposited in the liver had been eliminated. Little or no CF was detected in the liver and lungs by day 16, when the inflammatory reaction was also substantially decreased. A second CF dose administered 8 d after the first exacerbated the inflammatory process in both the lungs and the liver, indicating that the intensity of this process depends on CF concentration in the lesion site.

Topics: Animals; Cord Factors; Glycolipids; Granuloma; Inflammation; Liver Diseases; Lung Diseases; Mice; Mycobacterium bovis

Factors contributing to protection against experimental tuberculosis have been studied with refined and well characterized fractions from mycobacteria and with certain unrelated antigens. Mice were vaccinated intravenously with various combinations of materials presented on minute oil droplets in saline emulsions and were later challenged by aerosol. The minimal composition of an effective vaccine was P3 (a trehalose mycolate similar to cord factor) plus an antigen, which could be tuberculoprotein, or a low-molecular-weight tuberculin-active peptide, or unrelated antigen such as bovine serum albumin or bacterial endotoxin. Development of a hypersensitivity granuloma in the lungs appeared to be essential to protection in this laboratory model.

Topics: Animals; Bacteria; Bacterial Proteins; BCG Vaccine; Cell Migration Inhibition; Cord Factors; Granuloma; Hypersensitivity, Delayed; Lung Diseases; Macrophages; Male; Mice; Mycobacterium tuberculosis; Peptides; Serum Albumin, Bovine; Tuberculin; Tuberculosis, Pulmonary

Topics: Acute Disease; Adjuvants, Immunologic; Animals; Blood Coagulation; Cattle; Cord Factors; Female; Fibrinogen; Glycolipids; Granuloma; Inflammation; Kinetics; Lung Diseases; Mice; Mice, Inbred C57BL; Micelles; Microspheres; Mycobacterium tuberculosis; Phagocytosis; Polystyrenes; Serum Albumin, Bovine

Intratumoral injection of ultrasonically prepared emulsions of the synthetic glycolipid methly 6-O-(2-tetradecyl-3-hydroxyoctadecanoyl)-alpha-D-glucopyranoside (designated C39) induced complete regression of transplants of a syngeneic murine fibrosarcoma in most of the treated animals as did 6,6'-di-O(2-tetradecyl-3-hydroxyoctadecanoyl)-alpha,alpha,-trehalose (designated C76) in a previous study. The C76 compound, about twice the molecular weight of C39, was more effective therapeutically than the smaller molecule. Ultrasonically prepared emulsions of C39 and C76 were not toxic when given intravenously. Intravenously administered emulsions of C39 prepared by mechanical grinding were more toxic, but less granulomagenic, than those containing C76. Squalane and squalene, but not peanut oil, were effective substitutes for mineral oil as carriers of C39 in the treatment of the tumor.

Topics: Animals; Chemical Phenomena; Chemistry; Cord Factors; Emulsions; Fibrosarcoma; Glycolipids; Granuloma; Male; Methylcholanthrene; Mice; Neoplasm Transplantation; Sarcoma, Experimental

The intensity of the granulomatous reaction evoked in lungs of mice by trehalose-6,6'-dimycolate administered intravenously in oil-water emulsion depended on the size distribution of the oil droplets. Emulsions containing the greatest number of the largest oil droplets were the most granulomagenic.

Topics: Animals; Cord Factors; Emulsions; Glycolipids; Granuloma; Injections, Intravenous; Lung Diseases; Male; Mice; Oils; Particle Size; Polysorbates