Cellular, Molecular, and Immunological Characteristics of Langhans Multinucleated Giant Cells Programmed by IL-15.

Langhans multinucleated giant cells (LGCs) are a specific type of multinucleated giant cell containing a characteristic horseshoe-shaped ring of nuclei that are present within granulomas of infectious etiology. Although cytokines that trigger macrophage activation (such as IFN-γ) induce LGC formation, it is not clear whether cytokines that trigger macrophage differentiation contribute to LGC formation. Here, we found that IL-15, a cytokine that induces M1 macrophage differentiation, programs human peripheral blood adherent cells to form LGCs. Analysis of the IL-15‒treated adherent cell transcriptome identified gene networks for T cells, DNA damage and replication, and IFN-inducible genes that correlated with IL-15 treatment and LGC-type multinucleated giant cell formation. Gene networks enriched for myeloid cells were anticorrelated with IL-15 treatment and LGC formation. Functional studies revealed that T cells were required for IL-15‒induced LGC formation, involving a direct contact with myeloid cells through CD40L-CD40 interaction and IFN-γ release. These data indicate that IL-15 induces LGC formation through the direct interaction of activated T cells and myeloid cells.

Interleukin-22 receptor 1 is expressed in multinucleated giant cells: A study on intestinal tuberculosis and Crohn's disease.

BACKGROUND: It is challenging to distinguish intestinal tuberculosis from Crohn's disease due to dynamic changes in epidemiology and similar clinical characteristics. Recent studies have shown that polymorphisms in genes involved in the interleukin (IL)-23/IL-17 axis may affect intestinal mucosal immunity by affecting the differentiation of Th17 cells. AIM: To investigate the specific single-nucleotide polymorphisms (SNPs) in genes involved in the IL-23/IL-17 axis and possible pathways that affect susceptibility to intestinal tuberculosis and Crohn's disease. METHODS: We analysed 133 patients with intestinal tuberculosis, 128 with Crohn's disease, and 500 normal controls. DNA was extracted from paraffin-embedded specimens or whole blood. Four SNPs in the IL23/Th17 axis (IL22 rs2227473, IL1ß rs1143627, TGFß rs4803455, and IL17 rs8193036) were genotyped with TaqMan assays. The transcriptional activity levels of different genotypes of rs2227473 were detected by dual luciferase reporter gene assay. The expression of IL-22R1 in different intestinal diseases was detected by immunohistochemistry. RESULTS: The A allele frequency of rs2227473 (P = 0.030, odds ratio = 0.60, 95% confidence interval: 0.37-0.95) showed an abnormal distribution between intestinal tuberculosis and healthy controls. The presence of the A allele was associated with a higher IL-22 transcriptional activity (P < 0.05). In addition, IL-22R1 was expressed in intestinal lymphoid tissues, especially under conditions of intestinal tuberculosis, and highly expressed in macrophage-derived Langhans giant cells. The results of immunohistochemistry showed that the expression of IL-22R1 in patients with Crohn's disease and intestinal tuberculosis was significantly higher than that in patients with intestinal polyps and colon cancer (P < 0.01). CONCLUSION: High IL-22 expression seems to be a protective factor for intestinal tuberculosis. IL-22R1 is expressed in Langhans giant cells, suggesting that the IL-22/IL-22R1 system links adaptive and innate immunity.

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection.

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals.

Second-generation Langerhans cells originating from epidermal precursors are essential for CD8+ T cell priming.

In vivo studies questioned the ability of Langerhans cells (LCs) to mediate CD8(+) T cell priming. To address this issue, we used intradermal immunization with plasmid DNA, a system in which activation of CD8(+) T cells depends on delayed kinetics of Ag presentation. We found that dendritic cells (DCs) located in the skin at the time of immunization have limited ability to activate CD8(+) T cells. This activity was mediated by a second generation of DCs that differentiated in the skin several days after immunization, as well as by lymph node-resident DCs. Intriguingly, CD8(+) T cell responses were not affected following treatment with clodronate liposomes, immunization of CCR2(-/-) mice, or local neutralization of CCL20. This suggests that local, rather than blood-derived, DC precursors mediate CD8(+) T cell priming. Analysis of DC differentiation in the immunized skin revealed a gradual increase in the number of CD11c(+) cells, which reached their maximum 2 wk after immunization. A similar differentiation kinetics was observed for LCs, with the majority of differentiating LCs proliferating in situ from epidermal precursors. By using B6/Langerin-diphtheria toxin receptor chimeric mice and LC ablation, we demonstrated that epidermal LCs were crucial for the elicitation of CD8(+) T cell responses in vivo. Furthermore, LCs isolated from lymph nodes 2 wk after immunization contained the immunization plasmid and directly activated Ag-specific CD8(+) T cells ex vivo. Thus, these results indicate that second-generation Ag-expressing LCs differentiating from epidermal precursors directly prime CD8(+) T cells and are essential for optimal cellular immune responses following immunization with plasmid DNA.

Cutting edge: microRNA regulation of macrophage fusion into multinucleated giant cells.

Cellular fusion of macrophages into multinucleated giant cells is a distinguishing feature of the granulomatous response to inflammation, infection, and foreign bodies (Kawai and Akira. 2011. Immunity 34: 637-650). We observed a marked increase in fusion of macrophages genetically deficient in Dicer, an enzyme required for canonical microRNA (miRNA) biogenesis. Gene expression profiling of miRNA-deficient macrophages revealed an upregulation of the IL-4-responsive fusion protein Tm7sf4, and analyses identified miR-7a-1 as a negative regulator of macrophage fusion, functioning by directly targeting Tm7sf4 mRNA. miR-7a-1 is itself an IL-4-responsive gene in macrophages, suggesting feedback control of cellular fusion. Collectively, these data indicate that miR-7a-1 functions to regulate IL-4-directed multinucleated giant cell formation.

The CD40-CD40L axis and IFN-γ play critical roles in Langhans giant cell formation.

The presence of Langhans giant cells (LGCs) is one of the signatures of systemic granulomatous disorders such as tuberculosis and sarcoidosis. However, the pathophysiological mechanism leading to LGC formation, especially the contribution of the T cells abundantly found in granulomas, has not been fully elucidated. To examine the role of T cells in LGC formation, a new in vitro method for the induction of LGCs was developed by co-culturing human monocytes with autologous T cells in the presence of concanavalin A (ConA). This system required close contact between monocytes and T cells, and CD4+ T cells were more potent than CD8+ T cells in inducing LGC formation. Antibody inhibition revealed that a CD40-CD40 ligand (CD40L) interaction and IFN-γ were essential for LGC formation, and the combination of exogenous soluble CD40L (sCD40L) and IFN-γ efficiently replaced the role of T cells. Dendritic cell-specific transmembrane protein (DC-STAMP), a known fusion-related molecule in monocytes, was up-regulated during LGC formation. Moreover, knock-down of DC-STAMP by siRNA inhibited LGC formation, revealing that DC-STAMP was directly involved in LGC formation. Taken together, these results demonstrate that T cells played a pivotal role in a new in vitro LGC formation system, in which DC-STAMP was involved, and occurred via a molecular mechanism that involved CD40-CD40L interaction and IFN-γ secretion.

Tumor necrosis factor-α can induce Langhans-type multinucleated giant cell formation derived from myeloid dendritic cells.

The formation of the rich cellular features of MGCs, where the nuclei are arranged circularly at the periphery of the cell (morphologically epithelioid; Langhans-type), is assumed to be associated with any granulomatous disease. The mechanism by which TNF controls the formation of human MGCs in vitro was investigated, focusing on the effect of the TNF-neutralizing antibody. Peripheral blood monocytes were isolated with mAb-coated immunologic magnetic beads and cultured for 10 days in the presence of 20 ng/mL GM-CSF and 10 ng/mL IL-4. These cells were further incubated in the presence of TNF-α with/without its blockade antibodies for 14 days. Myeloid DCs can be generated from peripheral blood monocytes, and both IL-4 and GM-CSF can provide sufficient stimulus for their differentiation. The formation of MGC can be induced in the presence of TNF-α. This reaction was prohibited by the presence of the TNF-neutralizing antibody but not by the presence of anti-TNF receptor II antibody. The activation of Rho and focal adhesion kinases induced by TNF-α stimulation might be linked to cell assembling and the formation of Langhans-type MGCs. MGCs can produce only small amounts of superoxide anions compared to isolated macrophages such as myeloid DCs.

[TUBERCULOSIS GRANULOMA: THE CURRENT VIEW OF ITS IMMUNOGENESIS AND CELLULAR COMPOSITION].

The review presents the available basic scientific data characterizing the current views of the immunological homeostasis of intact lung tissue, the population and subpopulation composition of the cells forming a tuberculosis granuloma, as well as their phenotypic characteristics, the factors contributing to macrophageal transformation into epitheliod cells and Pirogov-Langhans giant cells, and the effect of mycobacterial antigens on this process.

Thalidomide prevents formation of multinucleated giant cells (Langhans-type cells) from cultured monocytes: possible pharmaceutical applications for granulomatous disorders.

Thalidomide is an effective drug for chronic inflammatory diseases, but the mechanism underlying its immunomodulatory action remains uncertain. Thalidomide has been reported to clinically improve chronic inflammatory granulomatous disorders. In such disorders, the granulomas consist of epithelioid cells, scattered lymphocytes and multinucleated giant cells (MNGC; Langhans-type cells). The present experimental approach permitted the reproduction of MNGC formation from peripheral blood monocytes and examination of thalidomides effect on it. MNGC can be effectively generated from monocytes cultured in the presence of interleukin-4 (IL-4) and macrophage colony-stimulating factor(M-CSF) for 14 days. Thalidomide can inhibit the formation of MNGC in a dose-dependent manner. MNGC formation was partly inhibited by the presence of neutralizing TNF-alpha antibody in the responses induced by IL-4 and M-CSF. Autocrinal TNF-alpha production and modulation of cadhelin expression to regulate cell adhesion might be involved in this inhibitory action of thalidomide. Our results support thalidomides clinical efficacy in the treatment of chronic granulomatous disorders (granulomatosis).

Why does tuberculosis lead to specific inflammation?

When Mycobacterium tuberculosis infects humans, about 20% of those infected actually develop tuberculosis (TB). In Japan, the incidence of TB in 2008 was 24,760 cases (19.4/100,000 persons) and the rate has been decreasing gradually, but is still higher than in the USA, Holland, and Belgium, for example. Histologically, tuberculosis displays exudative inflammation, proliferative inflammation and productive inflammation depending on the time course. In productive inflammation, granulomatous lesions with necrotic centers are formed. The typical granulomas consist of epithelioid macrophages, Langhans' multinucleated giant cells, lymphocytes and fibroblasts, and the process of their formation involves many cytokines, chemokines and transcription factors. These findings have been derived primarily from animal experiments utilizing an airborne infection apparatus. The conditions for airborne infection have been described in detail elsewhere. This mini-review focuses on what has been found through animal experiments, and also indicates areas for which data are not currently available.

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice.

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.

Increased susceptibility of mice lacking T-bet to infection with Mycobacterium tuberculosis correlates with increased IL-10 and decreased IFN-gamma production.

A sustained CD4+ Th1-dominated type 1 immune response is required to successfully control Mycobacterium tuberculosis infection. Considerable work has demonstrated that the transcription factor, T-bet, is required for IFN-gamma expression and fundamental to the generation of type 1 immunity in multiple cell types. Mice lacking T-bet are susceptible to virulent M. tuberculosis infection. Susceptibility of T-bet-deficient mice is associated with increased systemic bacterial burden, diminished IFN-gamma production, and the striking accumulation of eosinophilic macrophages and multinucleated giant cells in the lung. Interestingly, T-bet(-/-) mice did not develop a fully polarized Th2 response toward M. tuberculosis, but exhibited selective elevation of IL-10 production. These results indicate that T-bet plays a central role in controlling M. tuberculosis disease progression, in part through the regulation of both IFN-gamma and IL-10.

Muramyl dipeptide and mononuclear cell supernatant induce Langhans-type cells from human monocytes.

Muramyl dipeptide (MDP) in bacterial cell walls reportedly evokes epithelioid cell granulomas. We examined its effects on multinucleated-giant-cell (MGC) formation from monocytes. Supernatant of concanavalin A-stimulated peripheral blood mononuclear cells (conditioned medium) generated MGCs from monocytes. MDP significantly increased the fusion index of Langhans-type MGCs (LGCs) but did not affect total MGCs. N-Acetylmuramyl-L-alanyl-L-isoglutamine, an MDP analogue, had no effect on MGC formation. MGCs were produced by conditioned medium from CD14(++)/CD16(-) monocytes. MDP enhanced the LGC fusion index from CD14(++)/CD16(-) monocytes. MGCs were not produced from CD14(+)/CD16(+) monocytes or immature dendritic cells induced by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL) 4 and only weakly produced from macrophage (M)-CSF- or GM-CSF-induced macrophages. Added MDP did not generate MGCs from CD14(+)/CD16(+) monocytes or dendritic cells but enhanced LGC formation from macrophages. Because IFN-gamma, IL-3, and GM-CSF reportedly are important in LGC induction, we added anti-IFN-gamma, anti-IL-3, or anti-GM-CSF monoclonal antibody (mAb) concomitantly to the monocyte culture treated with conditioned medium alone or plus MDP. Anti-IFN-gamma mAb completely abrogated MGC generation, whereas anti-GM-CSF and anti-IL-3 mAbs significantly inhibited LGCs. These findings suggest that CD14(++)/CD16(-) monocytes are fused to form LGCs by MDP derived from granulomatous-disease-causing pathogens with inflammatory mediators such as IFN-gamma, IL-3, and GM-CSF.

Sistema monocítico-macrofágico y mycobacterium leprae: su importancia en la interpretación patogénica de las distintas formas clínico-patológicas de la Lepra y de las reacciones leprosas / Monocytic-macrophagic system and mycobacterium leprae

Se realiza una revisión de la patogenia de la Lepra (Hanseniasis), en sus vinculaciones con el "sistema monocítico-macrofágico y se efectúa el estudio histopatológico, histoquimico (lipídico) y con microscopía electrónica de cuatro enfermos de Lepra, dos lepromatosos ( sin y con eritema nudoso) y otros dos dimorfos ("Borderline"). Se concluye que: 1) La Lepra presenta sus manifestaciones clínico-patológicas más ostensibles, en vinculación con lesiones del "sistema monocítico-macrofágico". 2)Existen distintos comportamientos de los macrófagos frente al mycobacterium leprae" a)en personas Mitsuda positivos se puede demostrar el predominio de "macrófagos lisadores del M.leprae" con muerte y desaparición de los bacilos; b)en individuos Mitsuda-negativos predominan los "magrófagos no lisadores del M.leprae",que también producen la muerte de los bacilos, pero con persistencia de la "envoltura lipídica bacilar", que se deposita en las vacuolas de los virchowcitos; y c)en enfermos Mitsuda-negativos, luego de formado el "Granuloma virchowiano", se desarrollan los "macrófagos lisadores de las células de Virchow" que permitirían el recambio celular de los virchowitos. 3)Los "macrófagos lisadores del M.leprae" obtienen información antigénica del "bacilo aislado" que pueden transmitir al sistema de inmunidad celular, cuyos linfocitos T activados generan los "granulomas epitelioides con células de Langhans" (granulomas tuberculoides), de la Lepra tuberculoide. 4) Los "macrófagos no lisadores del M.leprae" no obtendrían información antigénica adecuada y no tendrían capacidad para estimular ningún sistema inmunológico (celular, o humoral), originando los "granulomas virchowianos" de la Lepra lepromatosa 5)Luego del envejecimiento y muerte de los vinchowcitos, los "macrófagos lisadores de las celulas de Virchow" obtendrían información antigénica de los "bacilos degenerados asociados a lípidos y glucoproteinas citoplasmáticas", con capacidad de estimulación del sistema inmunológico humoral cuyos linfocitos B activados generarían anticuerpos complejos: a)contra bacilos ubicados en los virchowcitos en la Reacción leprosa tipo 2 (eritema nudoso leprótico); b) contra la pared de vasos en la reacción leprosa tipo 3(fenómeno de Lucio); y c)contra tejidos del huésped (enfermedad autoagresiva hanseniana-Azulay). 6)En la lepra no existiría "polaridad inmunológica" en los períodos iniciales de la instalación de la enfermedad, pero parecería secundariamente a la formación del "granulo

Sistema monocítico-macrofágico y mycobacterium leprae: su importancia en la interpretación patogénica de las distintas formas clínico-patológicas de la Lepra y de las reacciones leprosas / Monocytic-macrophagic system and mycobacterium leprae

Se realiza una revisión de la patogenia de la Lepra (Hanseniasis), en sus vinculaciones con el "sistema monocítico-macrofágico y se efectúa el estudio histopatológico, histoquimico (lipídico) y con microscopía electrónica de cuatro enfermos de Lepra, dos lepromatosos ( sin y con eritema nudoso) y otros dos dimorfos ("Borderline"). Se concluye que: 1) La Lepra presenta sus manifestaciones clínico-patológicas más ostensibles, en vinculación con lesiones del "sistema monocítico-macrofágico". 2)Existen distintos comportamientos de los macrófagos frente al mycobacterium leprae" a)en personas Mitsuda positivos se puede demostrar el predominio de "macrófagos lisadores del M.leprae" con muerte y desaparición de los bacilos; b)en individuos Mitsuda-negativos predominan los "magrófagos no lisadores del M.leprae",que también producen la muerte de los bacilos, pero con persistencia de la "envoltura lipídica bacilar", que se deposita en las vacuolas de los virchowcitos; y c)en enfermos Mitsuda-negativos, luego de formado el "Granuloma virchowiano", se desarrollan los "macrófagos lisadores de las células de Virchow" que permitirían el recambio celular de los virchowitos. 3)Los "macrófagos lisadores del M.leprae" obtienen información antigénica del "bacilo aislado" que pueden transmitir al sistema de inmunidad celular, cuyos linfocitos T activados generan los "granulomas epitelioides con células de Langhans" (granulomas tuberculoides), de la Lepra tuberculoide. 4) Los "macrófagos no lisadores del M.leprae" no obtendrían información antigénica adecuada y no tendrían capacidad para estimular ningún sistema inmunológico (celular, o humoral), originando los "granulomas virchowianos" de la Lepra lepromatosa 5)Luego del envejecimiento y muerte de los vinchowcitos, los "macrófagos lisadores de las celulas de Virchow" obtendrían información antigénica de los "bacilos degenerados asociados a lípidos y glucoproteinas citoplasmáticas", con capacidad de estimulación del sistema inmunológico humoral cuyos linfocitos B activados generarían anticuerpos complejos: a)contra bacilos ubicados en los virchowcitos en la Reacción leprosa tipo 2 (eritema nudoso leprótico); b) contra la pared de vasos en la reacción leprosa tipo 3(fenómeno de Lucio); y c)contra tejidos del huésped (enfermedad autoagresiva hanseniana-Azulay). 6)En la lepra no existiría "polaridad inmunológica" en los períodos iniciales de la instalación de la enfermedad, pero parecería secundariamente a la formación del "granulo