Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans

Lipoarabinomannans (LAM) and lipomannans (LM) are integral parts of the mycobacterial cell wall recognized by cells involved in the innate immune response and have been found to modulate the cytokine response. Typically, mannosylated LAM from pathogenic mycobacteria have been reported to be anti-inflammatory, whereas phosphoinositol-substituted LAM from nonpathogenic species are proinflammatory molecules. In this study, we show that LM from several mycobacterial species, including Mycobacterium chelonae, Mycobacterium kansasii, and Mycobacterium bovis bacillus Calmette-Guérin, display a dual function by stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages. LM, but none of the corresponding LAM, induce macrophage activation characterized by cell surface expression of CD40 and CD86 and by TNF and NO secretion. This activation is dependent on the presence of Toll-like receptor (TLR) 2 and mediated through the adaptor protein myeloid differentiation factor 88 (MyD88), but independent of either TLR4 or TLR6 recognition. Surprisingly, LM exerted also a potent inhibitory effect on TNF, IL-12p40, and NO production by LPS-activated macrophages. This TLR2-, TLR6-, and MyD88-independent inhibitory effect is also mediated by LAM from M. bovis bacillus Calmette-Guérin but not by LAM derived from M. chelonae and M. kansasii. This study provides evidence that mycobacterial LM bear structural motifs susceptible to interact with different pattern recognition receptors with pro- or anti-inflammatory effects. Thus, the ultimate response of the host may therefore depend on the prevailing LM or LAM in the mycobacterial envelope and the local host cell receptor availability.     

Adherent-invasive Escherichia coli isolated from Crohn's disease patients induce granulomas in vitro

Adherent-invasive Escherichia coli (AIEC) have been shown to be highly associated with ileal Crohn's disease (CD). AIEC survive within infected macrophages, residing within the phagolysosomal compartment where they take advantage of the low pH to replicate extensively. We investigated whether, like the tuberculous bacillus which also persists within macrophages, AIEC LF82 induces the formation of granulomas, which are a common histopathological feature of CD. For this purpose, we have taken advantage of an in vitro model of human granulomas that we recently developed, based on blood-derived mononuclear cells. We demonstrated that AIEC LF82 induces aggregation of infected macrophages, fusion of some of them to form multinucleated giant cells and subsequent recruitment of lymphocytes. Light microscopy and scanning electron microscopy analysis of the cell aggregates confirmed their granuloma features. This was further confirmed by histological analysis of granuloma sections. Noteworthy, this phenomenon can be reproduced by soluble protein extracts of AIEC LF82 coated onto beads. Although the cell aggregates not completely mimic natural CD-associated granulomas, they are very similar to early stages of epithelioid granulomas.     

Mycobacterium bovis strain bacillus Calmette-Guérin-induced liver granulomas contain a diverse TCR repertoire, but a monoclonal T cell population is sufficient for protective granuloma formation

Granuloma formation is a form of delayed-type hypersensitivity requiring CD4(+) T cells. Granulomas control the growth and dissemination of pathogens, preventing host inflammation from harming surrounding tissues. Using a murine model of Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) infection we studied the extent of T cell heterogeneity present in liver granulomas. We demonstrate that the TCR repertoire of granuloma-infiltrating T cells is very diverse even at the single-granuloma level, suggesting that before granuloma closure, a large number of different T cells are recruited to the lesion. At the same time, the TCR repertoire is selected, because AND TCR transgenic T cells (Valpha11/Vbeta3 anti-pigeon cytochrome c) are preferentially excluded from granulomas of BCG-infected AND mice, and cells expressing secondary endemic Vbeta-chains are enriched among AND cells homing to granulomas. Next, we addressed whether TCR heterogeneity is required for effective granuloma formation. We infected 5CC7/recombinase-activating gene 2(-/-) mice with recombinant BCG that express pigeon cytochrome c peptide in a mycobacterial 19-kDa bacterial surface lipoprotein. A CD4(+) T cell with a single specificity in the absence of CD8(+) T cells is sufficient to form granulomas and adequately control bacteria. Our study shows that expanded monoclonal T cell populations can be protective in mycobacterial infection.     

Mycobacteria exploit nitric oxide‐induced transformation of macrophages into permissive giant cells

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so‐called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double‐edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO‐induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria‐infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.     

A TNF-regulated recombinatorial macrophage immune receptor implicated in granuloma formation in tuberculosis

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis.     

Organ-dependent differences in composition and function observed in hepatic and intestinal granulomas isolated from mice with Schistosomiasis mansoni

In murine schistosomiasis mansoni, eggs deposited in the liver and intestines induce a cell-mediated granulomatous reaction. Previous studies have shown that maximal granuloma size differs in the liver and various segments of the gastrointestinal tract. The objective of this investigation was to isolate intestinal granulomas and to determine whether organ-dependent differences in cell composition and granuloma function exist. Intestinal granulomas representative of those in tissue were isolated by a combination of chemical and mechanical techniques. When dissociated by collagenase, these lesions yielded a viable heterogeneous population of inflammatory cells. Granulomas from the liver, colon, and ileum showed differences in cellular composition. Liver lesions contained the largest number of T and B lymphocytes, eosinophils, and mast cells whereas ileal granulomas comprised mostly macrophages. Immunofluorescence studies on frozen tissue sections revealed that T and B lymphocytes also displayed different patterns of distribution within granulomas from different tissues. In contrast to isolated cultured liver granulomas that produced MIF-like substances, isolated colonic and ileal granulomas had weak or no MIF activity. It thus appears that granuloma formation in various organs is influenced by local factors that could affect the ultimate resolution of the lesions.     

Exacerbation of granuloma formation in IL-1 receptor antagonist-deficient mice with impaired dendritic cell maturation associated with Th2 cytokine production

Dendritic cell (DC) maturation at the site of inflammation and migration into draining lymph nodes is fundamental to initiate Ag-specific immune responses. Although several proinflammatory cytokines, including IL-1, are known to promote DC maturation in vitro, their contributions to DC activation and migration within peripheral inflamed tissue compartments are not yet fully understood. We show here that endogenous IL-1 receptor antagonist (IL-1ra) controls the activation state of liver-recruited DCs and their migration in a Propionibacterium acnes-induced murine granulomatous liver disease model. After P. acnes treatment, formation of portal tract-associated lymphoid tissue was conversely impaired in IL-1ra-deficient mice. IL-1ra-deficient mice developed hepatic granulomas within 3 days after P. acnes administration and showed a more pronounced granuloma formation than wild-type mice. Although sinusoidal granulomas contained numerous CD11c+ DCs at day 7, expressions of CCR7, IL-12p40 by these DCs were dramatically decreased in IL-1ra-deficient mice, suggesting aberrant DC maturation and sinusoid portal migration in the absence of endogenous IL-1ra. This was accompanied with enhanced intrahepatic Th2 cytokine production and severe hepatocellular damage. Thus, hepatocyte-derived IL-1ra may control optimal activation and migration of inflammatory DCs within the liver and thereby determine the local immune responses in granulomatous liver disease.     

MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis

The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.     

Culture at high density improves the ability of human macrophages to control mycobacterial growth

The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 x 10(4) cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.     

Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.     

Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response

The cell wall component lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis is involved in the inhibition of phagosome maturation, apoptosis and interferon (IFN)-gamma signalling in macrophages and interleukin (IL)-12 cytokine secretion of dendritic cells (DC). All these processes are important for the host to mount an efficient immune response. Conversely, LAM isolated from non-pathogenic mycobacteria (PILAM) have the opposite effect, by inducing a potent proinflammatory response in macrophages and DCs. LAMs from diverse mycobacterial species differ in the modification of their terminal arabinose residues. The strong proinflammatory response induced by PILAM correlates with the presence of phospho-myo-inositol on the terminal arabinose. Interestingly, recent work indicates that the biosynthetic precursor of LAM, lipomannan (LM), which is also present in the cell wall, displays strong proinflammatory effects, independently of which mycobacterial species it is isolated from. Results from in vitro assays and knock-out mice suggest that LM, like PILAM, mediates its biological activity via Toll-like receptor 2. We hypothesize that the LAM/LM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. Recent progress in the identification of genes involved in the biosynthesis of LAM is discussed, in particular with respect to the fact that enzymes controlling the LAM/LM balance might represent targets for new antitubercular drugs. In addition, inactivation of these genes may lead to attenuated strains of M. tuberculosis for the development of new vaccine candidates.     

Ultrastructure of Mycobacterium marinum granuloma in striped bass Morone saxatilis

An emerging epizootic of mycobacteriosis currently threatens striped bass Morone saxatilis populations in Chesapeake Bay, USA. Several species of mycobacteria, including Mycobacterium marinum, species resembling M. avium, M. gordonae, M. peregrinum, M. scrofulaceum and M. terrae, and the new species M. shottsii have been isolated from diseased and healthy bass. In this study, we describe the ultrastructure of developing M. marinum granulomas in experimentally infected bass over a period of 45 wk. The primary host response to injected mycobacteria was formation of large macrophage aggregations containing phagocytosed bacilli. M. marinum were always contained within phagosomes. Close association of lysosomes with mycobacterial phagosomes, as well as the presence of electron-opaque material within phagosomes, suggested phagolysosomal fusion. Development of granulomas involved epithelioid transformation of macrophages, followed by appearance of central necrosis. Desmosomes were present between mature epithelioid cells. The necrotic core region of M. marinum granulomas was separated from overlying epithelioid cells by several layers of flattened, electron-opaque spindle-shaped cells. These cells appeared to be formed by compression of epithelioid cells and, aside from a flattened nucleus, did not possess recognizable organelles. Following the development of well-defined, paucibacillary granulomas, secondary disease was observed. Recrudescence was marked by bacterial replication followed by disruption of granuloma architecture, including loss of epithelioid and spindle cell layers. In advanced recrudescent lesions, normal tissue was replaced by macrophages, fibroblasts, and other inflammatory leukocytes. Large numbers of mycobacteria were observed, both intracellular and suspended in cellular debris.     

IL-10 Modulates In Vitro Multinucleate Giant Cell Formation in Human Tuberculosis

Background

Multinucleated giant cells (MGC) are the histologic hallmark of granuloma which is known to limit tuberculosis infection. Both Th1 and Th2 type of cytokines regulate the immune response occurring within the granulomas. The objective of the study was to determine whether tuberculosis patient monocytes differed in their MGC forming ability as compared to healthy controls.

Methods

In vitro MGC formation was carried out by treatment of monocytes with cytokine containing culture supernatant of ConA or PPD stimulated peripheral mononuclear cells. IL-2, TNF-α, IL-4, IL-10 and TGF-β cytokine levels were analysed in culture supernatants using ELISA. IL-4 and IL-10 were added to culture supernatant separately and simultaneously along with their respective neutralizing antibodies and their consequent effect on MGC formation was evaluated.

Results

MGC formation was significantly low in patient monocytes incubated with autologous culture supernatant as compared to control culture supernatant. Cytokine analysis of the culture supernatants revealed that while IL-4 levels were similar in patients and controls, increased IL-10 levels were found in patients. Exogenous addition of IL-10 resulted in reduced MGC formation. Contrastingly, when IL-4 was added exogenously, it led to increased MGC formation. The effects of both IL-10 and IL-4 were reversed upon addition of their respective antibodies.

Conclusion

The findings suggest that one of the factors contributing to the disease could be the effect of cytokines on the functionality of monocytes, which are crucial in the fight against the organism. Significantly reduced MGC formation was observed on addition of IL-10. The findings imply an overriding role of IL-10 in MGC formation. The suppressive effect of IL-10 on MGC formation was further confirmed by addition of IL-10 neutralizing antibody.     

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.     

Dynamic Assembly of Brambleberry Mediates Nuclear Envelope Fusion during Early Development

To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres.     

Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.     

Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant

Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.     

Tuberculous Granuloma Formation Is Enhanced by a Mycobacterium Virulence Determinant

Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.     

Lipomannans,but not lipoarabinomannans,purified from Mycobacterium chelonae and Mycobacterium kansasii induce TNF-alpha and IL-8 secretion by a CD14-toll-like receptor 2-dependent mechanism

Lipoarabinomannans (LAMs) are glycolipids from the mycobacterial cell wall that exhibit various biological activities, including proinflammatory and anti-inflammatory responses. However, little is known about the properties of lipomannans (LMs), considered to be precursors of LAMs. In this study, we provide evidence that LMs purified from Mycobacterium chelonae and a clinical strain of Mycobacterium kansasii stimulated mRNA expression and secretion of TNF-alpha and IL-8 from human macrophage-like differentiated THP-1 cells. In contrast to LMs, LAMs were not able to induce a significant cytokine-inducing effect. The mechanism of activation by LMs was investigated using various Abs raised against surface receptors for multiple bacterial products. The presence of anti-CD14 or anti-Toll-like receptor 2 (TLR2) Abs profoundly affected production of TNF-alpha and IL-8, suggesting that both CD14 and TLR2 participate in the LM-mediated activation process. Furthermore, stimulation of cells was dependent on the presence of the LPS-binding protein, a plasma protein that transfers glycolipids to CD14. Chemical degradation of the arabinan domain of mannose-capped LAM from M. kansasii, which presented no cytokine-eliciting effect, restored the cytokine-inducing activity at a level similar to those of LMs. These results support the hypothesis that the presence of an arabinan in LAMs prevents the interaction of these glycolipids with TLR2/CD14 receptors. In addition, we found that phosphatidylinositol dimannosides isolated from M. kansasii did not induce cytokine secretion. This study suggests that LMs isolated from different mycobacterial species participate in the immunomodulation of the infected host and that the D-mannan core of this glycolipid is essential for this function.