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PubMed1993

Human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock.

Mannose-binding protein (MBP) is a plasma protein synthesized by hepatocytes. MBP, a structural analogue of the complement component C1q, can activate complement via the classical pathway and plays an important role in host defence. Expression of the human MBP gene was studied using the human hepatoma cell line HuH-7. RNA extracted from HuH-7 cells was reverse-transcribed to cDNA, amplified by the polymerase chain reaction and analysed by Southern blot hybridization. MBP mRNA expression in HuH-7 cells was increased by interleukin-6 (IL-6), dexamethasone and heat shock, decreased by interleukin 1 (IL-1), and unaffected by interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta). Gel shift assays demonstrated Sp-1 binding sites in the 5' region of the gene, and formation of specific complexes between DNA and nuclear protein extracted from HuH-7 cells treated with IL-1 or IL-6. Human MBP is an acute-phase protein, and transcription of its gene is enhanced by IL-6, dexamethasone and heat shock but inhibited by IL-1. The actions of the cytokines appear to be mediated by specific transcription factors.

Acute-Phase Reaction↗
PubMed1995

The Gly-54-->Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.

The human mannose-binding protein (MBP) is a pattern recognition molecule that appears to play a role in initial host defence. MBP activates the complement cascade and it may act as an opsonin both in the absence and in the presence of complement. A number of distinct MBP allelic forms exist in different population groups. An allele that occurs in 5-7% of Caucasians was identified by an inability to activate the complement system. A homozygous mutation at base pair 230 of the MBP gene results in a Gly-to-Asp substitution at the fifth collagen repeat. It appears that the resultant protein, MBPD, is able to form high-order multimers that bind bacteria but do not support complement activation. Recently a novel serine protease, the MBP-associated serine protease (MASP), has been described. MBP-MASP complexes circulate in serum and result in the direct activation of a novel complement pathway (lectin pathway) in the absence of the first complement components. In this study we demonstrate that MASP and its proenzyme proMASP are unable to bind to recombinant (r)MBPD. This lack of a MASP-rMBPD association corresponds to a failure of the Gly-54-->Asp form of MBP to activate complement. Our results provide a biochemical basis for the functional deficit in the Gly-54-->Asp allelic form of MBP and suggest that the proMASP/MASP binding site maps to the fifth collagen repeat of MBP.

Alleles↗
PubMed1989

Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella.

The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains, a cysteine-rich NH2-terminal domain that stabilizes the collagen alpha helix of the second domain and a third COOH-terminal carbohydrate recognition domain. Previous studies have shown that both native and recombinant human MBP bind to wild-type virulent Salmonella montevideo that expresses a mannose-rich lipopolysaccharide. Interaction with MBP results in opsonization and killing by phagocytes. In this report we show that low concentration of MBP (less than 10 micrograms/ml) markedly enhance complement deposition via the alternative complement pathway on S. montevideo. Despite structural similarities between MBP and the C1q subcomponent of the first complement component, MBP did not restore classical pathway activity to C1q-deficient serum, nor did it activate C1s when added to a mixture of C1r and C1s. In the presence of MBP the C3 bound to S. montevideo during incubation in serum was in the form of C3b and iC3b at a ratio of 1:2. Presensitization of S. montevideo with MBP rendered this normally serum resistant organism susceptible to complement-mediated killing. These results emphasize that MBP and complement cooperate in first line defense of the nonimmune host.

Amino Acid Sequence↗
PubMed1997

Ingestion of Candida albicans down-regulates mannose receptor expression on rat macrophages.

The frequency of infection and death due to various Candida species has increased steadily during the past decade, with mucocutaneous candidal infections as a common problem in the immunocompromised host. Mononuclear phagocytes are important in phagocytosis of this organism. In areas where there are low levels of opsonins, the macrophage-specific mannose receptor plays a dominant role in mediating Candida albicans ingestion. Following receptor-mediated infection, the host macrophage produces inflammatory cytokines and mediators that lead to ultimate killing of the invading Candida. Infection of macrophages by pathogens often leads to altered function that might effect their subsequent host defense properties. For example, function of both the complement receptor type 3 and the mannose receptor are down-regulated following exposure to pathogens or pathogen-derived products. In the current study, we have examined the down-regulation of mannose receptor expression following Candida infection and have investigated possible mechanisms that might be involved. Mannose receptor activity was decreased following 24 h postinfection with Candida. Both tumor necrosis factor and nitric oxide were produced during the infection, and inhibition of the these mediators partially blocked the effect on the receptor. Infection with Candida also inhibited the ability of dexamethasone to up-regulate mannose receptor expression. Finally, mannose receptor protein turnover was accelerated in Candida-infected macrophages. We conclude that Candida down-regulates one of the receptors involved in its internalization through a combination of production of modulatory molecules and enhanced receptor degradation. These results support the hypothesis that pathogens that infect macrophages have the ability to alter the phagocytic pathways available for subsequent host defense.

Animals↗
PubMed1985

Receptors and recognition mechanisms of Leishmania species.

This paper examines briefly receptors and recognition mechanisms involved in binding Leishmania parasites to the lumen of the sandfly gut and to cells of the vertebrate host's mononuclear phagocyte system. In particular, work carried out in our laboratory on the relative roles of the macrophage plasma membrane receptor (CR3) for the cleaved third complement component (iC3b) and the mannosyl/fucosyl receptor (MFR) in binding, ingestion and respiratory burst (RB) response elicited by promastigotes versus amastigotes of L. donovani, is discussed. In the absence of serum, soluble inhibitors (e.g. mannan) of the MFR cause a dose-dependent reduction in promastigote binding to murine resident peritoneal macrophages and in the proportion of bound parasites eliciting a RB response. For amastigotes, no consistent reduction in binding in the presence of mannan is observed but the proportion of parasites eliciting a RB is reduced. Serum-independent binding of promastigotes, which are good activators of the alternative complement pathway, is also inhibited by the anti-CR3 monoclonal antibody M1/70, by Fab anti-C3, and by an inhibitor of C3 fixation, sodium salicyl hydroxamate. With amastigotes, which are poor activators of the alternative pathway, a lesser effect is observed. These results provide strong evidence that cleaved macrophage-derived C3 (iC3b) mediates promastigote binding to CR3. Modulation experiments in which either CR3 or MFR are rendered inaccessible demonstrate that both receptors must be present on the segment of macrophage membrane with which the promastigote makes contact to mediate binding and ingestion. These receptor interactions may be important determinants of the infectivity and survival of Leishmania parasites in the vertebrate host.

Animals↗
PubMed1988

Receptor-mediated uptake of starch and mannan microparticles by macrophages: relative contribution of receptors for complement, immunoglobulins and carbohydrates.

The contribution of different macrophage receptors to the uptake of polyacryl starch and polyacryl mannan microparticles by macrophages was studied. Both types of microparticle were taken up to a larger and similar extent when they had been pre-incubated with serum, indicating the importance of serum factors in the uptake process. These results were supported by organ distribution studies in mice, showing that the two microparticles were rapidly targeted to the liver and spleen after i.v. injection. The carbohydrate-specific mannose/fucose receptor was found to contribute significantly to the uptake of non-opsonized but not opsonized mannan microparticles. The two different microparticles were found to activate the alternative complement pathway and to adsorb immunoglobulin G, human serum albumin and fibronectin to their surface. Inhibition experiments provided evidence for the involvement of a complement receptor (CR3) and an Fc-receptor (FCR) for IgG in the uptake of opsonized microparticles.

Animals↗
PubMed1985

Role of macrophage complement and lectin-like receptors in binding Leishmania parasites to host macrophages.

This paper reviews briefly work carried out in our laboratory on the relative roles of the macrophage plasma membrane receptor (CR3) for the cleaved third complement component (iC3b) and the mannosyl/fucosyl receptor (MFR) in binding, ingestion and respiratory burst (RB) response elicited by promastigotes versus amastigotes of Leishmania donovani. In the absence of serum soluble inhibitors (mannan, ribonuclease B) of the MFR cause a dose-dependent reduction in the numbers of promastigotes binding to murine resident peritoneal macrophages and in the proportion of bound parasites eliciting a RB response. For amastigotes no consistent reduction in binding in the presence of mannan is observed but the proportion of parasites eliciting a RB is reduced. Serum-independent binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, is also inhibited by the anti-CR3 monoclonal antibody M1/70, by Fab anti-C3, and by an inhibitor of C3 fixation, sodium salicyl hydroxamate. For amastigotes, which are poor activators of the alternative pathway, a lesser effect is observed with all three inhibitors of CR3-mediated binding. The results obtained with these three independent inhibitors provide strong evidence that cleaved macrophage-derived C3 (iC3b), which can be visualised on the parasite surface in electron microscope sections following addition of anti-C3 antibody and a protein A-gold conjugate, mediates binding to CR3. Modulation experiments in which either CR3 or MFR are rendered inaccessible demonstrate that both receptors must be present on the segment of the macrophage membrane with which the parasite makes contact to mediate binding and ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Circulating levels of mannose binding protein in human immunodeficiency virus infection.

Mannose binding protein (MBP) is a serum lectin which, upon binding to a carbohydrate extremity, acquires the ability to activate the classical complement pathway. MBP binds human immunodeficiency virus (HIV) in vitro via glycans on gp120 and thus, it may play a defensive role in HIV infection and contribute to virus clearance through the activation of complement associated with this condition. We measured serum MBP and activation indices of the classical complement pathway (plasma C4d and C3d) in HIV-seropositive patients at different stages of disease severity, and in normal subjects. MBP was higher in HIV patients as a whole and in each Centers for Disease Control (CDC) group than controls (P<0.01). MBP was not significantly different between CDC groups and and did not significantly correlate either with CD4-positive lymphocytes, neopterin or beta2-microglobulin or with C4d and C3d. The possibility that MBP plays a defensive role in HIV infection cannot be excluded, but, it it is, it does not appear to act by recruiting complement for vital elimination.

Adult↗
PubMed1992

Distinct and overlapping functions of allelic forms of human mannose binding protein.

Human mannose binding protein (MBP) is a C-type serum lectin involved in first-line host defense against a variety of bacterial, fungal and viral pathogens. Recently an association was found between low levels of serum MBP and an increased frequency of recurrent infections in infants. A particular genotype, in which glycine is substituted by aspartic acid at codon 54 of MBP in the fifth collagen repeat, shows apparent concordance with the clinical phenotype. We report, however, that this genotype occurs in 5% of the population and encodes a functional protein. Our results indicate that the Gly54Asp allele does not account for a deficiency state, but instead suggest that MBP may have two predominant allelic forms that have overlapping function and differ only in their ability to activate the classical pathway of complement.

Alleles↗
PubMed1997

The role of collectins in host defense.

Mannose-binding protein (MBP) belongs to a group of Ca(2+)-dependent lectins called collectins that play a role in first-line host defense. It recognizes specific carbohydrate residues (mannose and N-acetylglucosamine) on the surface of microorganisms and promotes the killing of microbes either by acting directly as an opsonin or by activating the lectin complement pathway. The collagenlike domain of MBP is important for the binding of MBP to the collectin receptors expressed on different phagocytes, and for activation of complement. The binding of MBP to bacteria, viruses, and parasites has been demonstrated in vitro. Three major mutations have been found in exon 1 of the MBP gene, which encodes the collagenous domain of the protein. These mutations cause low levels of serum MBP and have been linked with lifelong risk of infection. The homozygotes for these mutations are especially susceptible to severe infections.

Animals↗
PubMed1984

Local opsonization by secreted macrophage complement components. Role of receptors for complement in uptake of zymosan.

We have examined the role of macrophage (M phi plasma membrane receptors for the cleaved third complement component (iC3b; CR3) and mannosyl, fucosyl terminated glycoproteins (MFR) in uptake of unopsonized zymosan. Monoclonal antibodies against CR3, M1/70 (Mac-1) and MO1, each inhibited approximately 50% of uptake of 125I-zymosan by murine and human M phi, respectively. Yeast mannan inhibited 0-50% of zymosan uptake in various M phi, in parallel with their expression of MFR. We demonstrated that M phi were the source of C3 in our assay and that the activity of other components of the complement system, namely a C3 convertase, factor I, and a factor I cofactor were also present in serum-free cultures of human monocytes. Macrophage C3 was deposited rapidly, within 10 min, on the zymosan particles and mediated binding, ingestion, and stimulation of superoxide release in BCG-activated and thioglycollate-elicited peritoneal M phi via CR3. Local secretion of complement proteins by M phi themselves can therefore opsonize pathogens and cells able to activate the alternative pathway and effect their destruction.

Animals↗
PubMed1985

Macrophage complement and lectin-like receptors bind Leishmania in the absence of serum.

We have examined the relative roles of the macrophage (M phi) plasma membrane receptor for the cleaved third complement component (iC3b, CR3) and of the mannosyl/fucosyl receptor (MFR) in binding and ingestion of Leishmania donovani. In the absence of exogenous complement, the binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, were inhibited by the anti-CR3 monoclonal antibody M1/70, by the Fab portion of an anti-C3 antibody, or by the nucleophile, sodium salicyl hydroxamate, an inhibitor of C3 fixation. This provides strong evidence that M phi-derived, cleaved C3 (iC3b) present on the promastigote surface mediates binding to CR3. Equivalent inhibition of promastigote binding and ingestion was also observed using the soluble inhibitors of MFR activity, mannan or ribonuclease B. No additive effect for blocking the two M phi receptors simultaneously was observed. For amastigotes, which are poor activators of the alternative pathway, a lesser but nevertheless equivalent effect was observed for the three soluble inhibitors of CR3-mediated binding vs. the two soluble inhibitors of MFR-mediated binding. Modulation experiments in which either CR3 or MFR had been rendered inaccessible demonstrated that both receptors must be present on the segment of M phi membrane to which the parasite binds. The combined function of these two distinct M phi receptors may provide a general mechanism for recognition and ingestion of other pathogenic protozoa known to activate the alternative pathway.

Animals↗
PubMed1992

High frequencies in African and non-African populations of independent mutations in the mannose binding protein gene.

We have previously identified, in three British families having an index child with frequent infections, a point mutation (GGC-->GAC) in codon 54 of exon 1 of the gene for the human lectin mannose binding protein (MBP). This was associated with low serum levels of this complement activating protein and would be anticipated to impair opsonization of mannose rich microorganisms. We now report a second point mutation (GGA-->GAA) in Gambians from West Africa, involving codon 57 of exon 1. By substituting carboxylic acids for axial glycines in the translated proteins both mutations would be expected to disrupt the secondary structure of the collagenous triple helix of the 96 kDa MBP subunits. In the Gambians the codon 57 mutation was studied by PCR, sequence analysis and restriction analysis and found to be remarkably common (frequency of the mutant gene 0.29 in adults and 0.23 in newborns) whereas the codon 54 mutation was very rare (frequency 0.003). However, the codon 54 mutation was frequent in both a British Caucasian and a Hong Kong Chinese population (frequency of the mutant gene 0.17 and 0.11 respectively). It was predicted that both homozygous and heterozygous individuals would have profoundly reduced serum levels of the protein and this was confirmed by immunoassay as was the reduced capacity of such sera to activate complement through the MBP initiated classical pathway. Our data indicate that the two mutations have arisen independently since the divergence of African and non African populations and both have attained high frequencies.

Adult↗
PubMed1996

Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis survives and replicates within human macrophages, but the mechanisms whereby tubercle bacilli resist killing are incompletely understood. We tested the general model in which M. tuberculosis evades killing by entering naive macrophages through receptors that are unable to activate cellular microbicidal activities. Complement receptor types 1 (CR1), 3 (CR3), and 4 (CR4) were blocked with monoclonal antibodies, and mannose receptors were blocked with a competitive ligand, mannosylated bovine serum albumin (MBSA). Survival and replication of M. tuberculosis (Erdman) were evaluated after the bacteria were phagocytosed in the presence of blocking agents (directing binding to the unblocked receptors). Although there was significant variation in the growth rate of virulent M. tuberculosis in monocyte-derived macrophages from different donors, the intracellular survival and replication of mycobacteria were equivalent regardless of the receptor(s) used for binding and phagocytosis. We conclude that the mechanisms whereby M. tuberculosis evades killing by human macrophages are independent of the receptor-mediated route of entry, and operate at one or more steps common to all entry pathways. Blocking complement and mannose receptors in combination did not completely abrogate binding of M. tuberculosis to macrophages. However, we found that two polyanionic scavenger-receptor ligands exhibited a concentration-dependent ability to block binding of M. tuberculosis to macrophages. Moreover, blocking class A scavenger receptors abrogated nearly all binding that persisted after blocking complement and mannose receptors. This indicates that class A scavenger receptors are quantitatively important mediators of M. tuberculosis-macrophage interactions. M. tuberculosis has evolved multiple mechanisms to promote its efficient entry into macrophages. This suggests that passage of the organism through macrophages may be an essential early step in the pathogenesis of tuberculosis.

Antibodies, Monoclonal↗
PubMed1985

Interaction of human monocytes, macrophages, and polymorphonuclear leukocytes with zymosan in vitro. Role of type 3 complement receptors and macrophage-derived complement.

Macrophages take up zymosan in the absence of exogenous complement via receptors for iC3b (type 3 complement receptors) acting with or without lectin-like receptors for mannosyl-fucosyl-terminated glycoconjugates. We previously provided evidence that macrophages themselves secrete complement-alternative pathway components able to opsonize zymosan locally (Ezekowitz et al., J. Exp. Med. 1984. 159:244-260). We show here that covalently bound C3 cleavage products C3b and iC3b can be eluted from zymosan particles cultivated with 36-h adherent human monocytes in the absence of serum. The ligand binding site of type 3 complement receptors is involved in macrophage-zymosan interactions as shown by inhibition studies of zymosan binding and uptake with Fab fragments of anti-C3 antibodies and monoclonal antireceptor antibodies M01 and OKM10. In contrast, antibody IB4, which binds to a receptor epitope distinct from the binding site, does not inhibit zymosan uptake. Selective modulation of macrophage receptors onto anticomplement receptor antibody and mannose-rich yeast mannan, respectively, confirms that the complement and lectin-like receptors are distinct. Human polymorphonuclear leukocytes, which express receptors for complement, but are not known to secrete complement proteins, bind and ingest only exogenously opsonized zymosan. Unopsonized zymosan is a poor trigger of respiratory burst activity in neutrophils or 7-d adherent human macrophages, but induces cell aggregation and secretion of large amounts of superoxide anion when these cells are co-cultivated in serum-free medium and challenged with zymosan. Our studies indicate that complement and/or other products synthesized by macrophages at extravascular sites could play an important role in opsonization and lysis of pathogens able to activate the alternative pathway and mediate macrophage-neutrophil collaboration in first-line host defence.

Animals↗
PubMed1995

Mannose binding protein is involved in first-line host defence: evidence from transgenic mice.

Mannose binding protein (MBP) is a calcium-dependent C-type lectin secreted by the liver which seems to be an important component of innate or natural immunity. We have investigated the effects of Candida albicans and thioglycolate injection into transgenic mice bearing the human MBP gene. The transgenes contained a 15 kb fragment of the MBP gene which included the complete coding sequence. Northern blot hybridization showed human MBP mRNA transcripts in the liver of two transgenic lines with low and high copy number respectively. Western blot analysis showed the presence in serum of human MBP which associated into the higher multimeric forms which are capable of activating complement. Enzyme-linked immunosorbent assays (ELISA) showed that serum human MBP concentrations in the transgenes (1.90 +/- 0.16 mg/l, mean +/- SEM) were about twice as high as the levels in man. The serum concentration of MBP A, which is the mouse homologue of MBP, (13.9 +/- 0.45 mg/l) was about seven times that of human MBP. Intravenous injection of Candida albicans caused the serum human MBP level to fall by more than 50% in the first hour and then slowly recover, but it did not return the initial value by 72 hr. Candida injection caused a 25% fall in serum mouse MBP A in the first hour which then rose to supranormal levels by 72 hr. Following Candida injection mouse MBP A mRNA concentrations increased over 72 hr in contrast to human MBP mRNA which remained constant in both transgenic lines. These data indicate that the human MBP gene fragment in the transgene did not include the regulatory elements of the gene. Total haemolytic complement activity and C3 concentrations also fell immediately after Candida and thioglycolate injection while the concentrations of mannose specific immunoglobulin G (IgG) and immunoglobulin M (IgM) did not fall. The data indicate that mannose binding protein plays an important role in the initial stages of defence against infection which, in this model, is quantitatively greater than that of mannose-specific IgG and IgM antibodies. Mannose binding protein is probably most important in defense of previously unexposed and non-immune hosts.

Animals↗