Characterization of the mannose/fucose receptor on human mononuclear phagocytes

Cells of the mononuclear phagocyte system contain a cell surface receptor which mediates the uptake of mannose- and fucose-terminated glycoproteins. We have extended the initial studies to human alveolar and monocyte-derived macrophages in culture using two radiolabelled ligands, the synthetic glycoconjugate mannose-bovine serum albumin and the lysosomal glycosidase, beta-glucuronidase. Uptake (37 degrees C) of 125I-mannose-BSA by freshly isolated alveolar macrophages is saturable with increasing concentrations of ligand. Kuptake values in macrophages of smokers and nonsmokers are similar, and resemble earlier reported values using rabbit alveolar macrophages (Kuptake = 40 nM). Uptake of 125I-mannose-BSA in cultured smoker macrophages is identical to that found in fresh cells, and uptake is stable for 5-10 days in culture. Fucose- and mannose-BSA are the most effective inhibitors of uptake, while N-acetylglucosamine-BSA is inhibitory at slightly higher concentrations. Binding (4 degrees C) of 125I-mannose-BSA is likewise ligand concentration dependent (KD = 30 nM). Freshly isolated human monocytes from healthy subjects and patients with cystic fibrosis do not have mannose-specific uptake. However, after monocytes are in culture for 3 days, mannose-specific uptake appears and Kuptake values and specificity of uptake are identical with the results from the alveolar macrophages. No uptake of mannose-BSA could be found in the human monocyte-like cell line, U937.     

Toll-like receptor 2 is required for control of pulmonary infection with Francisella tularensis

Toll-like receptor 2 (TLR2) deficiency enhances murine susceptibility to infection by Francisella tularensis as indicated by accelerated mortality, higher bacterial burden, and greater histopathology. Analysis of pulmonary cytokine levels revealed that TLR2 deficiency results in significantly lower levels of tumor necrosis factor alpha and interleukin-6 but increased amounts of gamma interferon and monocyte chemoattractant protein 1. This pattern of cytokine production may contribute to the exaggerated pathogenesis seen in TLR2-/- mice. Collectively, these findings suggest that TLR2 plays an important role in tempering the host response to pneumonic tularemia.     

Carbohydrate-independent recognition of collagens by the macrophage mannose receptor

Mannose receptor (MR) is the best characterised member of a family of four endocytic molecules that share a common domain structure; a cysteine-rich (CR) domain, a fibronectin-type II (FNII) domain and tandemly arranged C-type lectin-like domains (CTLD, eight in the case of MR). Two distinct lectin activities have been described for MR. The CR domain recognises sulphated carbohydrates while the CTLD mediate binding to mannose, fucose or N-acetylglucosamine. FNII domains are known to be important for collagen binding and this has been studied in the context of two members of the MR family, Endo180 and the phospholipase A2 receptor. Here, we have investigated whether the broad and effective lectin activity mediated by the CR domain and CTLD of MR is favoured to the detriment of FNII-mediated interaction(s). We show that MR is able to bind and internalise collagen in a carbohydrate-independent manner and that MR deficient macrophages have a marked defect in collagen IV and gelatin internalisation. These data have major implications at the molecular level as there are now three distinct ligand-binding sites described for MR. Furthermore our findings extend the range of endogenous ligands recognised by MR, a molecule firmly placed at the interface between homeostasis and immunity.     

Differential effects of Francisella tularensis lipopolysaccharide on B lymphocytes

Francisella tularensis, a designated Category A biological agent, can cause severe infection in humans. Previous studies have demonstrated a significant immunoprotective role for B lymphocytes in animal models, but the responses of human B lymphocytes to F. tularensis components are largely unknown. The LPS of F. tularensis is atypical and has been reported to lack biological activity on myeloid cells and mouse B cells. Our study characterized the immunological effects of highly purified LPS from different stains of F. tularensis on human B lymphocytes and compared these effects with those on mouse B cells and human monocyte-derived macrophages. Results indicate that marked differences exist between cell type and species in specific responses to this interesting bacterial component. In sharp contrast to responses of mouse splenic B cells or human macrophages, human peripheral B cells showed reproducibly elevated IL-6, TNF-alpha, and antibody production in response to F. tularensis LPS. Data also indicated that these activated human B lymphocytes may subsequently promote the activation of other immune cell types by direct cell-cell interaction. Further investigation into the potential usefulness of F. tularensis LPS as an adjuvant component of a more optimal subunit vaccine is warranted, as it is now clear that it is not biologically inactive, as assumed previously.     

Clearance of neutrophil-derived myeloperoxidase by the macrophage mannose receptor

Uptake of neutrophil-derived myeloperoxidase by the macrophage mannose receptor was studied. Rat bone marrow-derived macrophages internalized 75% of [125I]myeloperoxidase through a mannose-specific process. Uptake via the mannose receptor is highly sensitive to treatment with oxidants. Treatment of rat macrophages with 1 mM H2O2 for 30 min resulted in a 94% reduction in uptake of myeloperoxidase. By Percoll gradient fractionation studies, 38% of internalized myeloperoxidase was delivered to the lysosomal compartment during a 15-min chase period, similar to findings for delivery of other ligands for this receptor. Once in the lysosome, the myeloperoxidase remained enzymatically active for several hours, with 50% activity remaining at 8 h. Finally, myeloperoxidase-containing macrophages had an increased capacity to down-regulate their own mannose receptors or receptors on neighboring macrophages, possibly through the myeloperoxidase-mediated production of oxidized halogens. Thus, the macrophage mannose receptor plays a potentially physiologic role in regulating extracellular myeloperoxidase levels. The receptor-mediated uptake may either arm the macrophage to contribute to oxidant-mediated tissue damage or may function to clear extracellular myeloperoxidase during the resolution phase of the inflammatory process.     

Francisella gains a survival advantage within mononuclear phagocytes by suppressing the host IFNgamma response

Tularemia is a zoonotic disease caused by the Gram-negative intracellular pathogen Francisella tularensis. These bacteria evade phagolysosomal fusion, escape from the phagosome and replicate in the host cell cytoplasm. IFNgamma has been shown to suppress the intra-macrophage growth of Francisella through both nitric oxide-dependent and -independent pathways. Since Francisella is known to subvert host immune responses, we hypothesized that this pathogen could interfere with IFNgamma signaling. Here, we report that infection with Francisella suppresses IFNgamma-induced STAT1 expression and phosphorylation in both human and murine mononuclear phagocytes. This suppressive effect of Francisella is independent of phagosomal escape or replication and is mediated by a heat-stable and constitutively expressed bacterial factor. An analysis of the molecular mechanism of STAT1 inhibition indicated that expression of SOCS3, an established negative regulator of IFNgamma signaling, is highly up-regulated during infection and suppresses STAT1 phosphorylation. Functional analyses revealed that this interference with IFNgamma signaling is accompanied by the suppression of IP-10 production and iNOS induction resulting in increased intracellular bacterial survival. Importantly, the suppressive effect on IFNgamma-mediated host cell protection is most effective when IFNgamma is added post infection, suggesting that the bacteria establish a permissive environment within the host cell.     

Modulation of virulence factors in Francisella tularensis determines human macrophage responses

Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis live vaccine strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a sigma(54)-modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically defined medium. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses.     

Features of sepsis caused by pulmonary infection with Francisella tularensis Type A strain

The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F.?tularensis. The C57BL/6 mice infected intranasally with a lethal dose of SchuS4 exhibited high bacterial burden in systemic organs and blood indicative of bacteremia. In correlation, infected mice displayed severe tissue pathology and associated cell death in lungs, liver and spleen. Consistent with our studies with murine model strain Francisella novicida, infection with SchuS4 caused an initial delay in upregulation of inflammatory mediators followed by development of severe sepsis characterized by exaggerated cytokine release, upregulation of cardiovascular injury markers and sepsis mediator alarmins S100A9 and HMGB1. This study shows that pulmonary tularemia caused by the Type A strain of F.?tularensis results in a deregulated host response leading to severe sepsis and likely represents the major cause of mortality associated with this virulent pathogen.     

Francisella tularensis infection, or how to outsmart the certain death

Francisella tularensis is an intracellular bacterial pathogen and is the causative agent of tularemia. Human become infected when manipulating with infected animals (usually rodents) or via arthropod vectors. In spite of the high virulence of the microbe still very little is known about its overall interaction with host cells and virulence factors identified to date show properties different from other bacterial species.     

巨噬细胞甘露糖受体的结构与功能

巨噬细胞甘露糖受体属于哺乳动物类C型凝集素超家族成员,主要表达于巨噬细胞和未成熟树突状细胞表 面,不仅在抵抗病原体感染的天然免疫防御中起重要作用,还参与抗原提呈和获得性免疫应答。     

Toll-like receptor 2-mediated signaling requirements for Francisella tularensis live vaccine strain infection of murine macrophages

Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-kappaB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2(-/-) macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSDeltaguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-kappaB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.     

Delayed presence of alternatively activated macrophages during a Francisella tularensis infection

Francisella tularensis is an intracellular bacterium that has the ability to multiply within the macrophage. The phenotype of a macrophage can determine whether the infection is cleared or the host succumbs to disease. Previously published data has suggested that F. tularensis LVS actively induces the alternative phenotype as a survival mechanism. In these studies we demonstrate that this is not the case for the more virulent strain of F. tularensis SCHU-S4. During an intranasal mouse model of infection, immuno-histochemistry identified that iNOS positive (“classical”) macrophages are present at 72 h post-infection and remain high (supported by CCL-5 release) in numbers. In contrast, arginase/FIZZ-1 positive (“alternative”) cells appear later and in low numbers during the development of the disease tularemia.     

Live vaccine strain of Francisella tularensis: infection and immunity in mice.

The live vaccine strain (LVS) of Francisella tularensis caused lethal disease in several mouse strains. Lethality depended upon the dose and route of inoculation. The lethal dose for 50% of the mice (LD50) in four of six mouse strains (A/J, BALB/cHSD, C3H/HeNHSD, and SWR/J) given an intraperitoneal (i.p.) inoculation was less than 10 CFU. For the other two strains tested, C3H/HeJ and C57BL/6J, the i.p. log LD50 was 1.5 and 2.7, respectively. Similar susceptibility was observed in mice inoculated by intravenous (i.v.) and intranasal (i.n.) routes: in all cases the LD50 was less than 1,000 CFU. Regardless of the inoculation route (i.p., i.v., or i.n.), bacteria were isolated from spleen, liver, and lungs within 3 days of introduction of bacteria; numbers of bacteria increased in these infected organs over 5 days. In contrast to the other routes of inoculation, mice injected with LVS intradermally (i.d.) survived infection: the LD50 of LVS by this route was much greater than 10(5) CFU. This difference in susceptibility was not due solely to local effects at the dermal site of inoculation, since bacteria were isolated from the spleen, liver, and lungs within 3 days by this route as well. The i.d.-infected mice were immune to an otherwise lethal i.p. challenge with as many as 10(4) CFU, and immunity could be transferred with either serum, whole spleen cells, or nonadherent spleen cells (but not Ig+ cells). A variety of infectious agents induce different disease syndromes depending on the route of entry. Francisella LVS infection in mice provides a model system for analysis of locally induced protective effector mechanisms.     

Activation of cytotoxic lymphocytes by interferon-alpha: role of oxygen radical-producing mononuclear phagocytes

A significant part of the therapeutic benefit of interferon-alpha (IFN-alpha) therapy in malignant diseases and in chronic viral infections is assumed to result from activation of lymphocytes with natural killer (NK) and T cell phenotype. In tumor tissue and in chronically infected tissue, the function and viability of these lymphocytes are frequently impaired. Mononuclear phagocyte (MP)-derived reactive oxygen species (ROS) have been proposed to contribute to the lymphocyte suppression in these tissues. Here, we report that three types of human cytotoxic lymphocytes of relevance to immunoactivation by IFN-alpha, CD3epsilon+/8+/56- T cells, CD3epsilon-/56+ NK cells, and CD3epsilon+/56+ NK/T cells became anergic to IFN-alpha induction of the cell-surface activation marker CD69 after exposure to autologous MPs in vitro. In addition to their incapacity to express CD69, cytotoxic lymphocytes acquired features characteristic of apoptosis after incubation with MPs. The lymphocyte apoptosis and nonresponsiveness to IFN-alpha were prevented by two inhibitors of reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent formation of ROS in MPs, histamine dihydrochloride and diphenylene ionodonium, as well as by catalase, a scavenger of ROS. We conclude that MP-derived ROS may negatively affect IFN-alpha-induced immunostimulation and propose that ROS inhibitors or scavengers may be useful to improve lymphocyte activation during treatment with IFN-alpha.     

Toll-like receptor 2 controls the gamma interferon response to Francisella tularensis by mouse liver lymphocytes

The production of gamma interferon (IFN-gamma) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-gamma expression. The present study was undertaken to determine whether or not macrophages (Mphi) or dendritic cells (DC) provide coactivating signals for the liver IFN-gamma response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mphi and DC significantly augmented the IFN-gamma response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-gamma production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-gamma at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-gamma responses to F. tularensis by regulating DC IL-12 production. While Mphi also coinduced hepatic IFN-gamma production in response to F. tularensis, they did so in a fashion less dependent on TLR2.     

Mouse model of oral infection with virulent type A Francisella tularensis

Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Little is known about the immunopathogenesis of oral infection with this pathogen. Here, for the first time, we examined the susceptibility of mice to intragastric inoculation with virulent type A F. tularensis and characterized the course of infection and the associated host responses. Both immunocompetent and immunodeficient mice were relatively susceptible to intragastric inoculation of type A F. tularensis with a 50% lethal dose (LD(50)) of 10(6) organisms, which was 100,000-fold higher than the LD(100) for intradermal or respiratory routes of infection. Mice deficient in gamma interferon or tumor necrosis factor receptors 1 and 2 were more susceptible than wild-type controls to oral infection with a high dose of the pathogen. After oral inoculation, F. tularensis appeared first in the mesenteric lymph nodes (MLN) and then rapidly spread to the livers and spleens, where the organism multiplied to high numbers and induced marked neutrophilic infiltration and severe tissue necrosis. Infected mice showed rapid increases in tissue cytokine mRNA expression, which peaked in the MLN at 2 days postinfection (dpi) and in the liver and spleen at 3 dpi. The levels of gamma interferon, interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha, KC, interferon-inducible protein 10, and monocyte chemotactic protein 1 were elevated from day 2 postinoculation onward. Moreover, mice intradermally immunized with the live vaccine strain of F. tularensis showed little survival advantage over naive mice after oral challenge with type A F. tularensis. These results suggest that type A F. tularensis is an effective oral pathogen that can cause fatal systemic infection and could pose a public health concern, particularly to immunocompromised individuals, if ingested in contaminated water and food.     

From pattern recognition receptor to regulator of homeostasis: the double-faced macrophage mannose receptor

The mannose receptor (MR) is an endocytic and phagocytic receptor belonging to the C-type lectin superfamily. A number of functions have been ascribed to this receptor, which is involved in innate and adaptive immune responses. The MR binds carbohydrate moieties on several pathogens, such as bacteria, fungi, parasites, and viruses, and, therefore, is considered a pattern recognition receptor (PRR). In addition, MR binds endogenous molecules and was originally described as a membrane-associated component binding lysosomal glycosidases in alveolar macrophages. Since its identification more than 25 years ago, many other endogenous ligands were described, including hormones, enzymes, cell membranes, extracellular matrix components, and normal as well as tumoral mucins. The MR is preferentially expressed on immune cells of myeloid lineage, especially subsets of dendritic cells (DC) and tissue macrophages. In addition to immune cells, specialized endothelial cells are also MR-positive. Increasing evidence shows that the MR is involved in the silent clearance of inflammatory molecules. In this review, we discuss current knowledge about the receptor and show that endogenous ligands activate an anti-inflammatory and tolerogenic program in DC and macrophages, thus reinforcing the hypothesis that the MR has an important role in the maintenance of homeostasis.     

Leishmania infection impairs beta 1-integrin function and chemokine receptor expression in mononuclear phagocytes

Leishmania spp. are intracellular parasites that cause lesions in the skin, mucosa, and viscera. We have previously shown that Leishmania infection reduces mononuclear phagocyte adhesion to inflamed connective tissue. In this study, we examined the role of adhesion molecules and chemokines in this process. Infection rate (r = -0.826, P = 0.003) and parasite burden (r = -0.917, P = 0.028) negatively correlated to mouse phagocyte adhesion. The decrease (58.7 to 75.0% inhibition, P = 0.005) in phagocyte adhesion to connective tissue, induced by Leishmania, occurred as early as 2 h after infection and was maintained for at least 24 h. Interestingly, impairment of cell adhesion was sustained by phagocyte infection, since it was not observed following phagocytosis of killed parasites (cell adhesion varied from 15.2% below to 24.0% above control levels, P > 0.05). In addition, Leishmania infection diminished cell adhesion to fibronectin (54.1 to 96.2%, P < 0.01), collagen (15.7 to 83.7%, P < 0.05), and laminin (59.1 to 82.2%, P < 0.05). The CD11b(hi) subpopulation was highly infected (49.6 to 97.3%). Calcium and Mg(2+) replacement by Mn(2+), a treatment that is known to induce integrins to a high state of affinity for their receptors, reverted the inhibition in adhesion caused by Leishmania. This reversion was completely blocked by anti-VLA4 antibodies. Furthermore, expression of CCR4 and CCR5, two chemokine receptors implicated in cell adhesion, was found to be downregulated 16 h after infection (2.8 to 4.1 times and 1.9 to 2.8 times, respectively). Together, these results suggest that mechanisms regulating integrin function are implicated in the change of macrophage adhesion in leishmaniasis.