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The macrophage mannose receptor is an integral membrane protein expressed on the surface of tissue macrophages. After ligation of mannose-rich glycoconjugates or pathogens, the receptor mediates endocytosis and phagocytosis of the bound ligands by macrophages. The cDNA-derived primary structure of the mannose receptor predicts a cysteine-rich NH2-terminal domain, followed by a fibronectin type II region. The remainder of the ectodomain is comprised of eight carbohydrate recognition-like domains, followed by a transmembrane region, and a cytoplasmic tail. Transfection of the mannose receptor cDNA into Cos-I cells is necessary for receptor-mediated endocytosis of mannose-rich glycoconjugate as well as phagocytosis of yeasts. Deletion of the cytoplasmic tail results in a mutant receptor that is able to bind but not ingest the ligated pathogens, suggesting that the signal for phagocytosis is contained in the cytoplasmic tail.

Mycobacterium tuberculosis (M.tb) survives in macrophages in part by limiting phagosome–lysosome (P-L) fusion. M.tb mannose-capped lipoarabinomannan (ManLAM) blocks phagosome maturation. The pattern recognition mannose receptor (MR) binds to the ManLAM mannose caps and mediates phagocytosis of bacilli by human macrophages. Using quantitative electron and confocal microscopy, we report that engagement of the MR by ManLAM during the phagocytic process is a key step in limiting P-L fusion. P-L fusion of ManLAM microspheres was significantly reduced in human macrophages and an MR-expressing cell line but not in monocytes that lack the receptor. Moreover, reversal of P-L fusion inhibition occurred with MR blockade. Inhibition of P-L fusion did not occur with entry via Fcγ receptors or dendritic cell–specific intracellular adhesion molecule 3 grabbing nonintegrin, or with phosphatidylinositol-capped lipoarabinomannan. The ManLAM mannose cap structures were necessary in limiting P-L fusion, and the intact molecule was required to maintain this phenotype. Finally, MR blockade during phagocytosis of virulent M.tb led to a reversal of P-L fusion inhibition in human macrophages (84.0 ± 5.1% vs. 38.6 ± 0.6%). Thus, engagement of the MR by ManLAM during the phagocytic process directs M.tb to its initial phagosomal niche, thereby enhancing survival in human macrophages.

Radioiodinated recombinant human interferon-gamma (IFN gamma) bound to human monocytes, U937, and HL60 cells in a specific, saturable, and reversible manner. At 4 degrees C, the different cell types bound 3,000-7,000 molecules of IFN gamma, and binding was of comparable affinity (Ka = 4-12 X 10(8) M-1). No change in the receptor was observed after monocytes differentiated to macrophages or when the cell lines were pharmacologically induced to differentiate. The functional relevance of the receptor was validated by the demonstration that receptor occupancy correlated with induction of Fc receptors on U937. Binding studies using U937 permeabilized with digitonin showed that only 46% of the total receptor pool was expressed at the cell surface. The receptor appears to be a protein, since treatment of U937 with trypsin or pronase reduced 125I-IFN gamma binding by 87 and 95%, respectively. At 37 degrees C, ligand was internalized, since 32% of the cell-associated IFN gamma became resistant to trypsin stripping. Monocytes degraded 125I-IFN gamma into trichloroacetic acid-soluble counts at 37 degrees C but not at 4 degrees C, at an approximate rate of 5,000 molecules/cell per h. The receptor was partially characterized by SDS-polyacrylamide gel electrophoresis analysis of purified U937 membranes that had been incubated with 125I-IFN gamma. After cross-linking, the receptor-ligand complex migrated as a broad band that displayed an Mr of 104,000 +/- 18,000 at the top and 84,000 +/- 6,000 at the bottom. These results thereby define and partially characterize the IFN gamma receptor of human mononuclear phagocytes.

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Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 105 50% tissue culture infective doses/ml.

Background

Alveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60) are frequently used to model macrophage function.

Methods

The availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR) and ion channel expression in alveolar macrophages and their widely used surrogates.

Results

The complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates.

Conclusions

The data described in this report provides insight into the appropriate choice of cell models for investigating macrophage biology and highlights the importance of confirming experimental data in primary alveolar macrophages.

Phagocytosis has traditionally been viewed as a specialized function of myeloid and monocytic cells. The mannose receptor (MR) is an opsonin- independent phagocytic receptor expressed on tissue macrophages. When human MR cDNA is transfected into Cos cells, these usually non- phagocytic cells express cell surface MR and bind and ingest MR ligands such as zymosan, yeast, and Pneumocystis carinii. Expression of cDNA for Fc gamma RI (CD64), the high-affinity Fc receptor, in Cos cells confers binding but barely detectable phagocytosis of antibody- opsonized erythrocytes (EA). We report here that chimeric receptors containing the ligand-binding ectodomain of the Fc receptor and the transmembrane and cytoplasmic domains of the MR ingest bound EA very efficiently, whereas chimeras with the Fc receptor ecto- and transmembrane domains and the MR tail, or the Fc receptor ecto- and cytoplasmic domains and the MR transmembrane region, are significantly less phagocytic. All of the chimeric receptors bind ligand with equal avidity, but gain of functional phagocytosis is only conferred by the MR transmembrane and cytoplasmic domains. Endocytosis of monomeric immunoglobulin G by chimeric receptors demonstrates a similar pattern, with optimal uptake by the chimera containing both tail and transmembrane regions from the MR. The chimeric receptors with only the transmembrane or the cytoplasmic domain contributed by the MR were less efficient. Site-directed mutagenesis of the single tyrosine residue in the cytoplasmic tail (which is present in a motif homologous to an endocytosis consensus motif in the LDL receptor cytoplasmic tail [Chen, W.-J., J. L. Goldstein, and M. S. Brown. 1990. J. Biol. Chem. 265:3116]) reduces the efficiency of phagocytosis and endocytosis to a similar extent.

Tumor-Associated Macrophages (TAMs) are abundantly present in the stroma of solid tumors and modulate several important biological processes, such as neoangiogenesis, cancer cell proliferation and invasion, and suppression of adaptive immune responses. Myeloid C-type lectin receptors (CLRs) constitute a large family of transmembrane carbohydrate-binding receptors that recognize pathogens as well as endogenous glycoproteins. Several lines of evidence demonstrate that some CLRs can inhibit the immune response. In this study we investigated TAM-associated molecules potentially involved in their immune suppressive activity. We found that TAMs isolated from human ovarian carcinoma samples predominantly express the CLRs Dectin-1, MDL-1, MGL, DCIR, and most abundantly the Mannose Receptor (MR). Components of carcinomatous ascites and purified tumoral mucins (CA125 and TAG-72) bound the MR and induced its internalization. MR engagement by tumoral mucins and by an agonist anti-MR antibody modulated cytokine production by TAM toward an immune-suppressive profile: increase of IL-10, absence of IL-12, and decrease of the Th1-attracting chemokine CCL3. This study highlights that tumoral mucin-mediated ligation of the MR on infiltrating TAM may contribute to their immune suppressive phenotype.

To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the persistently infected cells was more efficient in infecting and replicating in naive U937 cells than the virus which was used to establish the persistent infection. These results suggest that macrophages may serve as reservoirs of infection in vivo, sequestering virus which may subsequently be activated from its persistent state, resulting in clinical infection and death.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that can signal via several cell surface receptors. To investigate whether receptor biology could influence immune responses to gp120, we studied its interaction with human, monocyte-derived dendritic cells (MDDCs) in vitro. Gp120 from the HIV-1 strain JR-FL induced IL-10 expression in MDDCs from 62% of donors, via a mannose C-type lectin receptor(s) (MCLR). Gp120 from the strain LAI was also an IL-10 inducer, but gp120 from the strain KNH1144 was not. The mannose-binding protein cyanovirin-N, the 2G12 mAb to a mannose-dependent gp120 epitope, and MCLR-specific mAbs inhibited IL-10 expression, as did enzymatic removal of gp120 mannose moieties, whereas inhibitors of signaling via CD4, CCR5, or CXCR4 were ineffective. Gp120-stimulated IL-10 production correlated with DC-SIGN expression on the cells, and involved the ERK signaling pathway. Gp120-treated MDDCs also responded poorly to maturation stimuli by up-regulating activation markers inefficiently and stimulating allogeneic T cell proliferation only weakly. These adverse reactions to gp120 were MCLR-dependent but independent of IL-10 production. Since such mechanisms might suppress immune responses to Env-containing vaccines, demannosylation may be a way to improve the immunogenicity of gp120 or gp140 proteins.

Author Summary

Dendritic cells (DCs) initiate immune responses to pathogens or vaccine antigens. The HIV-1 gp120 envelope glycoprotein is an antigen that is a focus of vaccine design strategies. We have studied how gp120 proteins interact with DCs in cell culture. Certain gp120s stimulate DCs from some, but not all, human donors to produce IL-10, a cytokine that is generally immunosuppressive. In addition, whether or not the DCs produce IL-10, their ability to mature properly when activated is impaired by gp120—the gp120-treated DCs have a reduced ability to stimulate T cell growth when the two cell types are cultured together. These various effects of gp120 are caused by its binding to cell surface receptors of the mannose C-type lectin receptor family, including (but probably not exclusively) one called DC-SIGN. Gp120 binds to these receptors via mannose residues that are present on some of the glycan structures that overlay much of its protein surface. Removing the mannoses by digesting gp120 with a suitable enzyme prevents IL-10 induction and impairment of DC maturation, as does the use of inhibitors of the binding of gp120 to DC-SIGN and similar receptors. This work could help with the design of better HIV-1 vaccines.

Mannose-terminated glucocerebrosidase (alglucerase; Ceredase) was designed for enzyme replacement therapy in Gaucher disease to take advantage of mannose receptor-mediated endocytosis by macrophages. To provide a rational basis for designing enzyme replacement therapy protocols, we examined the in vitro binding, uptake, and degradation of alglucerase by murine and human macrophages. Both were found to have approximately 500,000 mannose-dependent receptors for alglucerase per cell with a Kd of 10(-7) M at 0 degrees C. In contrast, the number of binding sites for mannose-bovine serum albumin (mannose-BSA), the classical ligand for the mannose receptor, was only approximately 20,000 with a Kd of 10(-8) M. Alglucerase was also bound in a mannose-dependent manner by cells that lack the capacity to bind mannose-BSA, such as Cos-1 cells, endothelial cells, and peripheral blood monocytes. The fact that the binding of alglucerase by macrophages was mediated principally by a receptor distinct from the classical mannose receptor that binds mannose-BSA was confirmed by differential inhibitors, viz., alpha-methyl-glucoside, fucose, and mannose-BSA, and by its independence on Ca2+. Uptake of alglucerase by macrophages at 37 degrees C was concentration dependent and half maximal at 10(-6) M. However, at a concentration of 10(-7) M, only 0.5% of the added alglucerase was incorporated into macrophages and approximately 50% of the alglucerase taken up was quickly released into the medium. Endothelial cells also manifest mannose-dependent binding and uptake of alglucerase and may therefore account for a large proportion of the infused alglucerase. Our data suggest that only a small amount of the alglucerase administered is effectively delivered to macrophages and that a more efficiently targeted enzyme might have a marked therapeutic advantage over mannose-terminated glucocerebrosidase.

We generated human dendritic cell (DC) hybridoma cell lines by fusing HGPRT-deficient promonocytic U937 cells with immature DCs obtained by culturing peripheral blood monocytes with interleukin-4 (IL-4; 1,000 U/ml) and granulocyte-macrophage colony-stimulating factor (100 U/ml) for 7 days and mature DCs by treatment with tumor necrosis factor alpha (12.5 μg/ml) for 3 days. Only one fusion with immature DCs was successful and yielded four cell lines—HB-1, HB-2, HB-3, and HB-9—with an overall fusion efficiency of 0.0015%. The cell lines were stable in long-term culture, displayed morphological features typical of DCs, and expressed distinct class I and class II molecules not present on U937 (A*031012, B*51011, Cw*0701, DRB3*01011 52, and DR5*01011). A representative cell line, HB-2, that expressed DC markers including CD83, CD80 and CD86 could be induced to produce IL-12 through CD40 stimulation. After human immunodeficiency virus (HIV) infection, there was impairment of antigen-presenting cell (APC) function, which was manifested by an inability to stimulate allogeneic T-cell responses. There was no change in expression of major histocompatibility complex class I and class II antigens, CD83, CD40, CD4, CD11c, CD80, CD86, CD54, and CD58, or IL-12 production in the HIV-infected HB-2 cells. The HIV-infected HB-2 cells induced T-cell apoptosis in the cocultures. T-cell proliferation could be partially restored by using ddI, indinivir, and blocking anti-gp120 and anti-IL-10 antibodies. Our data suggest that there are multiple mechanisms that DCs use to inhibit T-cell responses in HIV-infected patients. The HB-2 cell line could be a useful model system to study APC function in HIV-infected DCs.

C-type lectins such as DC-SIGN and L-SIGN, which bind mannose-enriched carbohydrate modifications of host and pathogen proteins, have been shown to bind glycoproteins of several viruses and facilitate either cis or trans infection. DC-SIGN and L-SIGN are expressed in several early targets of arbovirus infection, including dendritic cells (DCs) and cells of the reticuloendothelial system. In the present study, we show that DC-SIGN and L-SIGN can function as attachment receptors for Sindbis (SB) virus, an arbovirus of the Alphavirus genus. Human monocytic THP-1 cells stably transfected with DC-SIGN or L-SIGN were permissive for SB virus replication, while untransfected controls were essentially nonpermissive. The majority of control THP-1 cells were permissive when attachment and entry steps were eliminated through electroporation of virus transcripts. Infectivity for the DC-SIGN/L-SIGN-expressing cells was largely blocked by yeast mannan, EDTA, or a DC-SIGN/L-SIGN-specific monoclonal antibody. Infection of primary human DCs by SB virus was also dependent upon SIGN expression by similar criteria. Furthermore, production of virus particles in either C6/36 mosquito cells or CHO mammalian cells under conditions that limited complex carbohydrate content greatly increased SB virus binding to and infection of THP-1 cells expressing these lectins. C6/36-derived virus also was much more infectious for primary human DCs than CHO-derived virus. These results suggest that (i) lectin molecules such as DC-SIGN and L-SIGN may represent common attachment receptor molecules for arthropod-borne viruses, (ii) arbovirus particles produced in and delivered by arthropod vectors may preferentially target vertebrate host cells bearing these or similar lectin molecules, and (iii) a cell line has been identified that can productively replicate alphaviruses but is deficient in attachment receptors.

Background

Glucocorticoids (GCs) are commonly used in the treatment of (chronic) inflammatory diseases and cancer, but inherent or acquired resistance to these drugs limits their optimal efficacy. The availability of drugs that could modulate GC resistance is therefore of potential clinical interest.

Objective

To explore the molecular basis of GC sensitisation of GC resistant monocytic/macrophage cells after chronic exposure to sulfasalazine.

Methods

Human monocytic/macrophage THP1 and U937 cells represent a cell line model system characterised by inherent resistance to the GCs dexamethasone and prednisolone. Both cell lines were chronically exposed in vitro to 0.3–0.6 mM sulfasalazine (SSZ) for approximately 3 months, after which they were characterised for GC sensitivity, expression levels of GC receptor and components of the nuclear factor kappa B (NFκB) signalling pathway, and their ability to undergo GC induced apoptosis.

Results

Chronic exposure to SSZ markedly sensitised both U937 and THP1 cells to dexamethasone (781‐fold and 1389‐fold, respectively) and prednisolone (562‐fold and 1220‐fold, respectively). Restoration of GC sensitivity in cells exposed to SSZ was provoked via GC induced apoptosis, coinciding with inhibition of NFκB activation. Moreover, western blot analysis revealed a markedly increased expression of glucocorticoid receptor α (GRα) in cells exposed to SSZ. Since GRα mRNA levels were only marginally increased, these results suggest that an altered post‐transcriptional mechanism was operable which conferred a stable GRα protein on SSZ exposed cells.

Conclusion

These results suggest that chronic targeting of the NFκB signalling pathway by SSZ may be exploited as a novel strategy to stabilise GRα expression and thereby sensitise primary resistant cells to GCs.

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the β2-microglobulin (β2m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical FcγRs: FcγRI, FcγRII, and FcγRIII.

We have developed a novel system to study monocytic function after human immunodeficiency virus type 1 (HIV-1) infection by infecting a series of human macrophage hybridoma cell lines with HIV-1. Since ethanol has detrimental effects on immune function, we investigated the effect of ethanol and its metabolites acetaldehyde and acetate on monocytic function by utilizing one human macrophage hybridoma cell line, clone 43, as well as primary monocytes. Pretreatment of clone 43 and primary monocytes with ethanol and its metabolites resulted in diminished accessory cell function for mitogen-, anti-CD3-, and antigen-induced T-cell proliferation. The decreased accessory cell function was associated with reduced interleukin 1α (IL-1α), IL-1β, and tumor necrosis factor alpha production with loss of intracellular cytokine and mRNA production and the induction of transforming growth factor β. In ethanol-, acetaldehyde-, and acetate-treated HIV-1-infected clone 43 cells (43HIV), there was a more rapid loss (3 days after infection) of accessory cell function at a lower infecting dose of HIV-1 than that in untreated 43HIV cells. We also observed a more rapid loss of surface class II antigen expression in the ethanol-, acetaldehyde-, and acetate-treated 43HIV cells, but no change in surface expression of CD80 or CD86. Ethanol-induced impairment of monocytic function may compound the immunologic defects of AIDS, making the infected individual more susceptible to the complications of the disease.

Expression of the macrophage mannose receptor is inhibited by interferon gamma (IFN-gamma), a T helper type 1 (Th-1)-derived lymphokine. Interleukin 4 (IL-4), a Th-2 lymphocyte product, upregulates major histocompatibility class II antigen expression but inhibits inflammatory cytokine production by macrophages. We have studied the effect of IL-4 on expression of the macrophage mannose receptor (MMR) by elicited peritoneal macrophages. We found that recombinant murine IL-4 enhances MMR surface expression (10-fold) and activity (15-fold), as measured by the respective binding and degradation of 125I-mannose-bovine serum albumin. Polymerase chain reaction analysis of cDNAs from purified primary macrophage populations revealed that MMR, but not lysozyme or tumor necrosis factor alpha, mRNA levels were markedly increased by IL-4. The above effects were associated with morphologic changes. These data establish IL-4 as a potent and selective enhancer of murine MMR activity in vitro. IL-4 induces inflammatory macrophages to adopt an alternative activation phenotype, distinct from that induced by IFN-gamma, characterized by a high capacity for endocytic clearance of mannosylated ligands, enhanced (albeit restricted) MHC class II antigen expression, and reduced proinflammatory cytokine secretion.

Objectives

To examine selective macrophage differentiation occurring in areas of intraplaque hemorrhage in human atherosclerosis.

Background

Macrophage subsets are recognized in atherosclerosis but the stimulus for and importance of differentiation programs remains unknown.

Methods

We used freshly isolated human monocytes, a rabbit model, and human atherosclerotic plaques to analyze macrophage differentiation in response to hemorrhage.

Results

Macrophages characterized by high expression of both mannose and CD163 receptors preferentially exist in atherosclerotic lesions at sites of intraplaque hemorrhage. These hemoglobin (Hb)-stimulated macrophages, M(Hb), are devoid of neutral lipids typical of foam cells. In vivo modeling of hemorrhage in the rabbit model demonstrated that sponges exposed to red cells showed an increase in mannose receptor positive macrophages only when these cells contained hemoglobin (Hb). Cultured human monocytes exposed to hemoglobin:haptoglobin complexes (Hb:Hp), but not IL-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and upregulation of ABC transporters. M(Hb) demonstrated increased ferroportin (FPN) expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of FPN using hepcidin increased ROS, inhibited ABCA1 expression, and cholesterol efflux to ApoAI, suggesting reduced ROS triggers these effects. Knockdown of liver x receptor alpha (LXRα) inhibited ABC transporter expression in M(Hb) and macrophages differentiated in the anti-oxidant superoxide dismutase. Lastly, liver X receptor α (LXR) luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport (RCT).

Conclusions

Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A reduction of macrophage intracellular iron plays an important role in this non- foam cell phenotype by reducing ROS, which drives transcription of ABC transporters through activation of LXRα. Reduction of macrophage intracellular iron may be a promising avenue to increase macrophage RCT.

As the role of monocytes and macrophages in a range of diseases is better understood, strategies to target these cell types are of growing importance both scientifically and therapeutically. As particulate carriers, liposomes naturally target cells of the mononuclear phagocytic system (MPS), particularly macrophages. Loading drugs into liposomes can therefore offer an efficient means of drug targeting to MPS cells. Physicochemical properties including size, charge and lipid composition can have a very significant effect on the efficiency with which liposomes target MPS cells. MPS cells express a range of receptors including scavenger receptors, integrins, mannose receptors and Fc-receptors that can be targeted by the addition of ligands to liposome surfaces. These ligands include peptides, antibodies and lectins and have the advantages of increasing target specificity and avoiding the need for cationic lipids to trigger intracellular delivery. The goal for targeting monocytes/macrophages using liposomes includes not only drug delivery but also potentially a role in cell ablation and cell activation for the treatment of conditions including cancer, atherosclerosis, HIV, and chronic inflammation.

Background

Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested.

Results

We show here that cell-surface expressed nucleolin is a receptor for Francisella tularensis Live Vaccine Strain (LVS) and promotes LVS binding and infection of human monocyte-like THP-1 cells. The HB-19 pseudopeptide that binds specifically carboxy-terminal RGG domain of nucleolin inhibits LVS binding and infection of monocyte-like THP-1 cells. In a pull-down assay, elongation factor Tu (EF-Tu), a GTP-binding protein involved in protein translation, usually found in cytoplasm, was recovered among LVS bacterial membrane proteins bound on RGG domain of nucleolin. A specific polyclonal murine antibody was raised against recombinant LVS EF-Tu. By fluorescence and electron microscopy experiments, we found that a fraction of EF-Tu could be detected at the bacterial surface. Anti-EF-Tu antibodies reduced LVS binding to monocyte-like THP-1 cells and impaired infection, even in absence of complement and complement receptors. Interaction between EF-Tu and nucleolin was illustrated by two different pull-down assays using recombinant EF-Tu proteins and either RGG domain of nucleolin or cell solubilized nucleolin.

Discussion

Altogether, our results demonstrate that the interaction between surface nucleolin and its bacterial ligand EF-Tu plays an important role in Francisella tularensis adhesion and entry process and may therefore facilitate invasion of host tissues. Since phagosomal escape and intra-cytosolic multiplication of LVS in infected monocytes are very similar to those of human pathogenic F. tularensis ssp tularensis, the mechanism of entry into monocyte-like THP-1 cells, involving interaction between EF-Tu and nucleolin, might be similar in the two subspecies. Thus, the use of either nucleolin-specific pseudopeptide HB-19 or recombinant EF-Tu could provide attractive therapeutic approaches for modulating F. tularensis infection.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary markedly in the ability to infect cells of the monocyte/macrophage (M/M) lineage. M/M are generally resistant to infection with T-cell-tropic (T-tropic) strains of HIV-1. Recently, the chemokine receptors CCR5 and CXCR4 were identified as cofactors for fusion/entry of macrophage- and T-tropic strains of HIV-1, respectively. To investigate the mechanisms of resistance of M/M to T-tropic HIV-1 infection, we examined a number of subclones of the U937 promonocytic cell line. We found that certain subclones of U937 (plus clones) could, while others (minus clones) could not, support replication of T-tropic strains of HIV-1. We demonstrate that (i) both minus and plus clones support HIV-1 replication when transfected with an infectious molecular cDNA clone of a T-tropic HIV-1; (ii) minus clones do not, but plus clones do, efficiently support fusion with cells expressing HIV-1 IIIB Env; (iii) both plus and minus clones (with the exception of one clone) express physiologically functional CXCR4 protein as well as CD4 on the cell surface; (iv) introduction of CXCR4 into the CXCR4-negative clone does not restore fusogenicity with or susceptibility to T-tropic HIV-1; and (v) a ligand (stromal cell-derived factor 1) for or a monoclonal antibody (12G5) to CXCR4 does not effectively inhibit HIV-mediated cell-to-cell fusion of U937 cells. These data indicate that resistance to T-tropic HIV-1 infection of U937 minus clones occurs at fusion/ entry events and that expression of functional CXCR4 and CD4 is not a sole determinant for susceptibility to T-tropic HIV-1 infection; furthermore, they suggest that other factors are positively or negatively involved in HIV-mediated cell-to-cell fusion in U937 promonocytic cells.

The macrophage mannose receptor (MMR) facilitates the binding and internalization of microorganisms and glycoproteins with terminal mannose residues. The receptor is progressively upregulated as bone marrow precursor cells mature into macrophages and thus may serve as a marker of differentiation. Prostaglandins of the E series (PGE) are known inhibitors of monocyte and macrophage precursor proliferation, an effect often associated with cellular maturation. MMR expression was therefore assessed after exposure of bone marrow macrophage precursor (BMMP) cells to these prostanoids. Receptor expression was determined by ligand binding and via immunoprecipitation of newly synthesized receptor molecules. PGE1 and PGE2 at 10(-9)-10(-6) M upregulated MMR surface expression and biosynthesis four- to sixfold in a dose-dependent manner. BMMPs responsive to prostaglandins were characterized by plastic adherence, F4/80 antigen expression, and nonspecific esterase activity. Prostaglandins accelerated the expression of the MMR in cells by 48-72h, with maximal levels of receptor expression being identical in control or treated cells. Thus, prostaglandins enhanced mannose receptor expression in adherent but not fully differentiated macrophage precursors. This effect is specific for PGE and is mimicked by dibutyrl cyclic AMP. These results indicate that prostaglandins accelerate MMR expression and hence the differentiation of macrophage precursor cells. Cells resident in the bone marrow secrete abundant prostaglandins, suggesting that a paracrine mechanism may exist to regulate MMR expression and function.

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Although studies have been performed to characterize responses of macrophages from individual anatomical sites (e.g., alveolar macrophages) or of murine-derived macrophage cell lines to microbial ligands, few studies compare these cell types in terms of phenotype and function. We directly compared the expression of cell surface markers and functional responses of primary cultures of three commonly used cells of monocyte-macrophage lineage (splenic macrophages, bone-marrow derived macrophages, and bone-marrow derived dendritic cells) with those of the murine-leukemic monocyte-macrophage cell line, RAW 264.7. We hypothesized that RAW 264.7 cells and primary bone marrow-derived macrophages would be similar in phenotype and would respond similarly to microbial ligands that bind to either Toll-like receptors 2, 3, and 4. Results indicate that RAW 264.7 cells most closely mimic bone marrow-derived macrophages in terms of cell surface receptors and response to microbial ligands that initiate cellular activation via Toll-like receptors 3 and 4. However, caution must be applied when extrapolating findings obtained with RAW 264.7 cells to those of other primary macrophage-lineage cells, primarily because phenotype and function of the former cells may change with continuous culture.

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.

Background

An experimental approach for improving vaccine efficacy involves targeting antigens to mannose receptors (MRs) on dendritic cells (DCs) and other professional antigen presenting cells. Previously, we demonstrated that mannosylated Pichia pastoris-derived recombinant proteins exhibited increased immunogenicity compared to proteins lacking mannosylation. In order to gain insight into the mechanisms responsible for this observation, the present study examined the cellular uptake of the mannosylated and deglycosylated recombinant proteins.

Methodology/Principal Findings

Utilizing transfected cell lines, roles for the macrophage mannose receptor (MMR, CD206) and DC-SIGN (CD209) in the recognition of the mannosylated, but not deglycosylated, antigens were demonstrated. The uptake of mannosylated antigens into murine bone marrow-derived DCs (BMDCs) was inhibited by yeast mannans (YMs), suggesting a mannose-specific C-type lectin receptor-dependent process, while the uptake of deglycosylated antigens remained unaffected. In particular, antigens with both N-linked and extensive O-linked mannosylation showed the highest binding and uptake by BMDCs. Finally, confocal microscopy studies revealed that both mannosylated and deglycosylated P. pastoris-derived recombinant proteins localized in MHC class II+ compartments within BMDCs.

Conclusions/Significance

Taken together with our previous results, these data suggest that increased uptake by mannose-specific C-type lectin receptors is the major mechanism responsible for the enhanced antigenicity seen with mannosylated proteins. These findings have important implications for vaccine design and contribute to our understanding of how glycosylation affects the immune response to eukaryotic pathogens.

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1-long terminal repeat (HIV-1-LTR) CAT constructs transfected into monocyte/macrophage-like cell lines but not a T cell line. This effect appears to be mediated through the induction of nuclear factor kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrate that LPS induces a DNA binding activity indistinguishable from NF-kappa B in U937 and THP-1 cells. LPS is also shown to dramatically increase HIV-1 production from a chronically infected monocyte/macrophage-like cloned cell line, U1, which produces very low levels of HIV-1 at baseline. The stimulation of viral production from this cell line occurs only if these cells are treated with granulocyte/macrophage colony-stimulating factor (GM-CSF) before treatment with LPS. This stimulation of HIV-1 production is correlated with an increase in the level of HIV-1 RNA and and activation of NF- kappa B. LPS is not able to induce HIV-1 production in a cloned T cell line. The effect of LPS on HIV-1 replication occurs at picogram per milliliter concentrations and may be clinically significant in understanding the variability of the natural history of HIV-1 infection.