Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 μg/ml and 10 μg/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.     

Characteristics of the beta-glucan receptor of murine macrophages

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.     

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.     

Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.     

IL-13 attenuates gastrointestinal candidiasis in normal and immunodeficient RAG-2(-/-) mice via peroxisome proliferator-activated receptor-gamma activation

We recently demonstrated that in vitro peroxisome proliferator-activated receptor-gamma (PPARgamma) activation of mouse peritoneal macrophages by IL-13 or PPARgamma ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2(-/-)) mice with natural and synthetic PPARgamma-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPARgamma antagonist, and are reduced in PPARgamma(+/-) mice. Overall, these data demonstrate that IL-13 or PPARgamma ligands attenuate C. albicans infection of the GI tract through PPARgamma activation and hence suggest that PPARgamma ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.     

Studies towards the potential of poliovirus as a vector for the expression of HPV 16 virus-like-particles

In this study we tested the hypothesis that after administration of a single intraperitoneal dose of concanavalin A (Con-A) to mice, the proportion of neutrophils and macrophages in the peritoneal exudate and their phagocytic and candidacidal activities should change with time. The number of neutrophils in the peritoneal exudate was greatly increased 6 h after administration of Con-A, and those cells were able to kill both intracellular and extracellular yeast and germ tube forms of Candida albicans. Addition of catalase to the culture medium reduced the killing of C. albicans, suggesting that the candidacidal activity depended on the myeloperoxidase system. The survival of mice pretreated with Con-A and submitted to an inoculum of C. albicans 6 h afterwards was twice higher than that of controls, which suggests that neutrophils were able to clear the experimental infection. One day after the treatment, the population of neutrophils in the exudate was about 45%, but after 2 days it was reduced to only 5% and the candidacidal activity was also reduced. After 4 days the exudate contained over 95% of macrophages, the candidacidal activity reached a maximum, and the phagocytosis mediated by both complement receptors and mannose receptors was increased. Uptake of FITC-mannose-BSA by macrophages was maximal on about the 4th day and was inhibited by mannan, suggesting that treatment with Con-A increased the activity of mannose receptors. These results support the hypothesis that activation of cellular immunity by Con-A occurred in two phases, one dominated by neutrophils, and the other by macrophages expressing increased activity of mannose receptors.     

Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro

The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man-liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man-liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man-liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.     

Serum mannan binding protein inhibits mannosylated liposome-mediated transfection to macrophages

The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man–liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man–liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man–liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.     

Effector function of leucocytes from susceptible and resistant mice against distinct isolates of Candida albicans

Neutrophils and macrophages were generated in vitro from mice that display either high or low tissue susceptibilities to Candida albicans infection and their ability to phagocytose and kill three isolates of the yeast with different virulence characteristics was evaluated. In the absence of opsonization, phagocytosis by BALB/c and CBA/CaH neutrophils was comparable, but the killing was very poor. Opsonization with normal serum slightly decreased phagocytosis, but it had markedly different effects on killing, either enhancing or inhibiting candidacidal activity, depending on the combination of yeast isolate and mouse strain. In contrast, BALB/c macrophages showed high levels of phagocytosis and killing of both unopsonized yeasts and opsonized yeasts; whereas killing of unopsonized yeasts by CBA/CaH macrophages was poor, it was markedly enhanced by opsonization.     

Phagocytic receptors on macrophages distinguish between different Sporothrix schenckii morphotypes

Sporothrix schenckii is a human pathogen that causes sporotrichosis, a cutaneous subacute or chronic mycosis. Little is known about the innate immune response and the receptors involved in host recognition and phagocytosis of S. schenckii. Here, we demonstrate that optimal phagocytosis of conidia and yeast is dependent on preimmune human serum opsonisation. THP-1 macrophages efficiently ingested opsonised conidia. Competition with d-mannose, methyl α-d-mannopyranoside, d-fucose, and N-acetyl glucosamine blocked this process, suggesting the involvement of the mannose receptor in binding and phagocytosis of opsonised conidia. Release of TNF-α was not stimulated by opsonised or non-opsonised conidia, although reactive oxygen species (ROS) were produced, resulting in the killing of conidia by THP-1 macrophages. Heat inactivation of the serum did not affect conidia internalization, which was markedly decreased for yeast cells, suggesting the role of complement components in yeast uptake. Conversely, release of TNF-α and production of ROS were induced by opsonised and non-opsonised yeast. These data demonstrate that THP-1 macrophages respond to opsonised conidia and yeast through different phagocytic receptors, inducing a differential cellular response. Conidia induces a poor pro-inflammatory response and lower rate of ROS-induced cell death, thereby enhancing the pathogen's survival.     

Isolation of soluble yeast beta-glucans that inhibit human monocyte phagocytosis mediated by beta-glucan receptors

The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE-cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis-inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of soluble beta-glucan with immune-enhancing activity from the cell wall of yeast

Beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae, has been found to enhance immune functions, especially by activating macrophages. However, a major obstacle to the clinical application of beta-(1-->3)-glucan is its low solubility in aqueous media. In this study, soluble beta-glucan, free of mannoprotein, was prepared, and its effects on TNF-alpha secretion and phagocytosis by macrophages were evaluated. Beta-glucan was first rendered soluble from the yeast cell wall by alkaline extraction (glucan-p1). The extract contained 2.8% of protein which was subsequently removed by successive DEAE-cellulose and ConA chromatography. Beta-glucan thus prepared was completely free of mannoprotein and was soluble at neutral pH (glucan-p3). The effects of beta-glucan on phagocytosis and TNF-alpha release activity were investigated. While glucan-p1 moderately induced TNF-alpha secretion at 200 microg/ml (550 pg of TNF-alpha/5 x 10(5) cells), glucan-p3 markedly stimulated macrophages at 200 microg/ml (2,860 pg of TNF-alpha/5 x 10(5) cells). Furthermore, glucan-p3 stimulated phagocytosis about 20% more than glucan-p1 did. In conclusion, we purified water-soluble beta-glucan which was completely devoid of mannoprotein and effectively stimulated the macrophage function, enabling it to be used as an intravenous injection for sepsis.     

Fluid phase and mannose receptor-mediated uptake of horseradish peroxidase in mouse bone marrow-derived macrophages. Biochemical and ultrastructural study

A detailed study of horseradish peroxidase (HRP) uptake by in vitro cultured bone marrow-derived macrophages was undertaken. Biochemical quantitations performed over a wide range of HRP concentrations, in the absence or presence of yeast mannan, showed that these macrophages pinocytose HRP by both fluid phase and mannose receptor-mediated uptake. The relative contribution of these two types of endocytosis varied with the concentration of enzyme in the extracellular medium. A morphological study at the light and electron microscope levels conducted in parallel confirmed the biochemical data.     

Leukotrienes target F-actin/cofilin-1 to enhance alveolar macrophage anti-fungal activity

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.     

Peptides that mimic Candida albicans-derived beta-1,2-linked mannosides

Beta-1,2-linked mannosides from Candida albicans phosphopeptidomannan (PPM) bind to macrophages through a receptor independent from the macrophage alpha-linked mannose receptor and stimulate these cells to secrete immune mediators. Anti-beta-1,2-linked mannoside but not anti-alpha-linked mannoside antibodies produced after immunization with neoglycoproteins protect animals from disseminated candidiasis. In this study, peptides that mimic beta-1,2-linked mannosides were isolated using phage display methodology. A phage library expressing random peptides was panned with an anti-beta-1,2-linked mannoside monoclonal antibody (mAb). After three rounds of biopanning, the isolated phages were able to inhibit recognition of C. albicans by the mAb. Sixty percent of the phages had an identical DNA insert corresponding to the peptide sequence FHENWPS that was recognized specifically by the mAb. Injection of KLH-coupled peptide into mice generated high titers of polyclonal antibodies against C. albicans yeast cell walls. The anti-FHENWPS antibodies bound to C. albicans PPM and were inhibited by soluble beta-1,2-mannotetraose. Together, these data provide evidence for mimotopic activity of the peptide selected by biopanning with the anti-beta-1,2-oligomannoside mAb.     

The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.     

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.     

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.     

Quantitative evaluation of macrophage phagocytosing capacity by a fluorometric assay

This paper reviews sensitive and simple quantitative evaluation of macrophage phagocytosing capacity by applying fluoresecin-labeled Sacharomyces cerevisiae cells. Yeast cells were conjugated with fluoresceinisothiocyanate (FITC) and used as fluorescent particles. A time course analysis within this method showed that phagocytosis of yeast cells was temperature dependent and that the number of that ones ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C. Free fluorescent cells can be effectively removed by aspiration from the well. Furthermore, yeast cells required preopsonization with serum to achieve optimal uptake of the cells. The uptake of nonopsonized yeast cells by macrophages was significantly lower than that of opsonized cells (P < 0.05). We propose that about 50% of mouse macrophages can carry functionally active FcR responsible for phagocytosis.     

The non-phagocytic route of collagen uptake: a distinct degradation pathway

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.