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Originally published online as doi:10.1189/jlb.0604342 on November 29, 2004

Published online before print November 29, 2004
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(Journal of Leukocyte Biology. 2005;77:328-336.)
© 2005 by Society for Leukocyte Biology

Mannose-binding lectin enhances phagocytosis and killing of Neisseria meningitidis by human macrophages

Dominic L. Jack*,{dagger},1, Margaret E. Lee{dagger}, Malcolm W. Turner*, Nigel J. Klein{ddagger} and Robert C. Read{dagger}

* Immunobiology and
{ddagger} Infectious Diseases and Microbiology Units, Institute of Child Health, University College London, United Kingdom; and
{dagger} Academic Unit of Infection and Immunity, University of Sheffield Medical School, United Kingdom

1 Correspondence: Academic Unit of Infection and Immunity, University of Sheffield Medical School, Beech Hill Road, Sheffield, UK, S10 2RX. E-mail: D.L.Jack{at}sheffield.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Deficiency of mannose-binding lectin (MBL) is probably the most common human immunodeficiency and is associated with an increased risk of mucosally acquired infections including meningococcal disease. Tissue macrophages are an important component of mucosal defense, and so we determined the effect of MBL on uptake of meningococci by human monocyte-derived macrophages. Opsonization with MBL significantly increased the capture and doubled the amount of internalization of Neisseria meningitidis. Inhibition of f-actin polymerization indicated that MBL exerted this effect by a dose-dependent acceleration of uptake into phagosomes, which was maximal within the normal physiological concentration of MBL (1.5 µg/ml) and was independent of scavenger receptors. MBL accelerated the acquisition and subsequent loss of the early endosome marker, early endosomal antigen-1, and enhanced the acquisition of the late endosomal marker, lysosome-associated membrane protein-1. MBL reduced the survival of meningococci within macrophages by more than half, despite the increased uptake of organisms, and significantly reduced the number of viable extracellular bacteria by 80%. We conclude that MBL is a dependent opsonin able to accelerate microbial uptake and killing. These results suggest that MBL could modify disease susceptibility by modulating macrophage interactions with mucosal organisms at the site of initial acquisition.

Key Words: complement • opsonization • intracellular processing • monocyte-derived macrophages


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mannose-binding lectin (MBL) recognizes a large number of microbes by binding to mannose and N-acetylglucosamine (GlcNAc) sugars and their derivatives when presented in particular densities and orientations [1 ]. Human deficiency of MBL is now recognized as predisposing individuals to a variety of infectious diseases [2 ], including meningococcal disease [3 ].

On binding to its targets, MBL activates the complement system via a system of MBL-associated serine proteases (MASP-1, -2, and -3 and a truncated form of MASP-2, Map19), resulting in bacterial lysis or opsonization with C3 fragments [1 ]. Many reports of MBL function have concentrated on the consequences of complement activation in terms of direct killing of Gram-negative bacteria or increased opsonization of Gram-positive bacteria or other organisms and uptake by phagocytes [4 5 6 7 ].

However, a number of studies suggest that MBL has an opsonic role, which is independent of its ability to activate complement. MBL has been shown to increase the phagocytosis of bacteria such as Salmonella enterica serovar Montevideo [8 ] and Mycobacterium avium [9 ] and apoptotic cells [10 ]. The mechanism of uptake has been variously described as direct opsonization [not requiring the presence of other factors; ref. 8 ] or indirect opsonization [the enhancement of other phagocytic mechanisms, namely the immunoglobulin (Ig) and complement-phagocytic pathways; ref. 11 ].

We have previously shown that MBL binds to Neisseria meningitidis, the causative agent of meningococcal disease. MBL binds to the outer membrane proteins, opacity protein and porin B [12 ], but the degree of binding is largely determined by the structure of the bacterial lipo-oligosaccharide (LOS) and in particular, by the addition of sialic acid to the LOS [4 , 13 ]. We have also shown that MBL increases the phagocytosis of meningococci by human professional phagocytes in the absence of other serum factors such as Ig or complement. This could be taken as evidence of direct opsonization. In this study, we show that MBL enhances N. meningitidis phagocytosis by human macrophages and that this results in heightened efficiency of bacterial killing by the phagocyte. The relative enhancement of phagocytosis in the presence of MBL is a result of an active orchestration of cytoskeletal rearrangements rather than through the passive, high-affinity binding of a ligand to its receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of MBL
MBL, kindly provided by Clare Booth, Institute of Child Health (London, UK), was purified by a modification of the method of Kilpatrick [14 ] from human plasma paste, as described previously using a two-step, positive, mannan-affinity process, followed by removal of trace-contaminating Ig [15 , 16 ].

Growth and preparation of bacterial strains
A mutant derivative (cpsD–) of serogroup B strain 1940 lacking galE, donated by Dr. Matthias Frosch (University of Würzburg, Germany), was used throughout. This mutant has been described previously and has a truncation within the lacto-N-neotetraose of LOS and therefore lacks a normal sialylation site. The organism expresses pilin, opa, and opc with no detectable differences in expression compared with wild-type organisms [17 , 18 ]. We have described the binding characteristics between MBL and this organism previously [1 , 13 ].

Organisms were removed from liquid nitrogen storage 2 days before use and subcultured once on blood agar at 37°C in 5% CO2 and then grown in Mueller-Hinton broth at 37°C until in mid-log phase, as assessed by absorbance at 540 nm. Organisms were washed once in veronal-buffered saline supplemented with 5 mM CaCl2 and 10 mM glucose (VBS-Glc) before centrifugation at 2500 g for 2 min.

Organisms were incubated in VBS-Glc or VBS-Glc supplemented with purified MBL for 10 min. At the start of this incubation, two 2-mm glass beads were added to each tube, and the culture vortexed for 15 s to break up any clumps of bacteria. During optimization, it was found that MBL was able to stabilize preformed bacterial clumps effectively, as assessed by viable counts, although it did not cause aggregation if bacteria were separated at the start of incubation (data not shown). Bacteria were washed twice with RPMI containing 10% heat-inactivated fetal calf serum (RPMI/FCS) and vortexed for 30 s before addition to cells.

Macrophage culture
Monocyte-derived macrophages (MDMs) were prepared from mononuclear cells separated from the peripheral whole blood of healthy, anonymized human volunteers by Ficoll-paque density centrifugation as described elsewhere [19 ]. Macrophages were cultured at a cell density of 1 x 106 per well in 24-well tissue-culture plates on sterile glass coverslips in 1 ml RPMI with 2 mM L-glutamine and 10% newborn calf serum at 37°C in 5% CO2. After 24 h, the cells were washed to remove the nonadherent population and reincubated with 1 ml culture medium, which was exchanged at 3-day intervals until the cells were used after 12–14 days in culture. A different donor was used for each separate experiment.

The effect of MBL on macrophage phagocytosis
Macrophages were washed once with warm RPMI/FCS before organisms were added at a multiplicity of infection (MOI) of 250 or 500 to each cell in tissue-culture wells in triplicate. Control wells were incubated with RPMI/FCS alone. After 30 min incubation at 37°C in 5% CO2, the cell supernatants were aspirated, and the cells were fixed with 1% (w/v) paraformaldehyde for 30 min.

A number of experiments were performed using specific inhibitors of different phases of phagocytosis. To determine whether any effects observed on phagocytosis were MBL-specific, we added 25 mM GlcNAc, an inhibitor of MBL binding, or 25 mM mannitol (a 6-carbon molecule that does not inhibit MBL binding [6 ]) to the MBL preparation before addition to the organisms. Latrunculin B (25 µM) was added to macrophage cultures at the same time as organisms preincubated with MBL (0 and 1.5 µg/ml) at a MOI of 250:1. Toxin B from Clostridium difficile was added to macrophage cultures 1 h prior to washing and the addition of organisms preincubated with MBL (0 and 1.5 µg/ml) at a MOI of 250:1.

Scavenger receptors (SRs) were inhibited by incubation of MDMs with the polyanion, polyinosinic acid [poly(I); 50 µg/ml], for 30 min before the addition of organisms preincubated in buffer alone or 1.5 µg/ml MBL at a MOI of 250:1. As a control, MDMs were incubated with 50 µg/ml polyadenylic acid [poly(A)], a similar molecule to poly(I) but is not a ligand for SRs before the addition of organisms. Treatment of macrophages with poly(I) reduced the uptake of fluorescently labeled (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate), acetylated low-density lipoprotein by MDMs (data not shown). The cells were incubated for 30 min at 37°C before fixation and staining for binding and internalization as described above.

Fluorescence microscopy
Cells were washed twice with phosphate-buffered saline (PBS) and stained for external and internalized organisms as described [20 ]. Briefly, cells were incubated with rabbit N. meningitidis serogroup B-specific antiserum (Difco, Cowley, UK). The cells were washed and incubated with fluorescein-conjugated goat anti-rabbit Ig (Sigma, Poole, UK) and then blocked with heat-inactivated goat serum. To study the presence of intracellular components, cells were incubated with H4A3 hybridoma supernatant or 1/150 dilution of monoclonal anti-early endosomal antigen (EEA)-1 antibody (BD Transduction Laboratories, Cowley, UK), anti-MBL (clone 131-1, Statens Serum Institut, Copenhagen, Denmark), or anti-C3d (Quidel, San Diego, CA) in RPMI containing 0.1% (w/v) saponin for 30 min. H4A3 has specificity for lysosome-associated membrane protein (LAMP)-1 [21 ]. After washing, cells were incubated with 0.01% (v/v) Triton X-100, 0.002% (w/v) sodium dodecylsulfate, in PBS containing 85 ng/ml 4',6-aminido-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Eugene, OR) and a Cy3-conjugated goat anti-mouse Ig to detect bound mouse antibody (Sigma). The cells were washed once with PBS and then with distilled water before the coverslips were inverted onto mounting medium (Vectashield, Vector Laboratories, Burlingame, CA). Slides were randomized and examined blind to sample identity using a Leica DMRB fluorescence microscope at 100x objective magnification. A minimum of 100 cells was counted, and the following was recorded: number of cells positive; total number of organisms associated with cells, including internalized organisms and those organisms bound but not yet internalized; the total number of internalized organisms. In preliminary experiments, we found that at lower MOIs (e.g., 100:1), there was insufficient phagocytosis to measure intracellular processing accurately (data not shown).

The effect of MBL on binding and internalization of N. meningitidis
To determine whether any effects of MBL on internalization or processing were a result of changes in the association of organisms with cells in the presence of MBL, MDMs were incubated with organisms preincubated with or without MBL at a MOI of 500:1 for 1 h at 4°C. At this temperature, binding of bacteria occurs, but internalization is inhibited [20 ]. After 1 h, cells were fixed to determine binding or allowed to warm to 37°C to study internalization. For internalization studies, the cell supernatants were gently aspirated, and the cells were washed three times with cold RPMI/FCS to remove nonadherent organisms before cold RPMI/FCS (250 µl) was added and the tissue culture trays placed in a 37°C/5% CO2 incubator. Cells in triplicate wells were fixed at 30 min and 60 min for fluorescent staining. The intention was to adjust the MOI until equal numbers of organisms were adherent to macrophages after 1 h incubation at 4°C such that changes in organism internalization would be independent of changes in binding as described previously [19 , 20 ].

Killing of meningococci by macrophages
We used a penicillin and gentamicin-exclusion assay to determine the killing of meningococci by macrophages. Organisms prepared with or without MBL as described above were added to MDMs and incubated for 1 h on ice. Nonadherent organisms were removed by washing with cold medium and incubated for a further 1.5 h at 37°C. The cells were then pulsed with penicillin (10 µg/ml) and gentamicin (40 µg/ml) for 30 min, washed, and then lysed with 1% saponin (w/v) in PBS for 10 min, and viable counts of organisms were determined. In some samples, no antibiotics were added, and the viable count of the supernatant was determined. We also fixed macrophage wells with paraformaldehyde before incubation with organisms to ensure that viable counts did not change with the addition of MBL. The use of a cold incubation step allowed us to load an equivalent number of organisms ± MBL onto the macrophage surface at time 0.

Statistical analysis
In all cases, data were examined for normality using SPSS version 10 software (SPSS Inc., Chicago, IL). Each parameter after preincubation of organisms with MBL was calculated as a percentage of the parameter in the absence of MBL (set at 100%), as described by Tino and Wright [22 ] for lung surfactant protein-A-mediated phagocytosis. The percentage differences were normally distributed, and experimental data were compared against a test value of 100 using a one-sample t-test. Similarly, the influence of monosaccharides, actin inhibitors, and poly(I)/(A) was determined as a percentage of each parameter in the absence of MBL (100%). Inhibition was tested by one-sample t-tests against a test value of 100% with pairwise t-tests between groups where required using the Bonferroni correction for multiple comparisons. To analyze kinetic data from cells incubated first at 4°C and then over a time-course at 37°C, the area under the curve (at 37°C) was calculated by the trapezoid rule using the "Area under Curve" macro function of Sigmaplot 2001 software (SPSS Inc). Area under curve analysis was chosen, as macrophages from different donors apparently recover from +4°C incubation at different rates. The curve areas with and without MBL were compared using the Wilcoxon nonparametric test for paired samples. Data for organism killing were log-transformed to normality and expressed as geometric means. Differences ± MBL were examined by paired t-test. Statistical tests were performed using SPSS or Sigmastat 2 (SPSS Inc.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MBL mediates phagocytosis
The influence of MBL on the association of meningococci with human MDMs is summarized in Figure 1A . At a MOI of 250:1, there was a significant increase in the number of organisms associated with macrophages in the presence of MBL (P=0.039, one-sample t-test). This was, in part, a result of a 1.5-fold increase in the percentage of macrophages associated with bacteria (P=0.042).



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  		Figure 1. Association and internalization of meningococci by macrophages. N. meningitidis B1940cpsD– were preincubated with MBL (5 µg/ml; +MBL) or buffer alone (–MBL). Organisms were then incubated with MDMs at a MOI of 250:1 or 500:1 for 30 min at 37°C. MDMs were fluorescently stained to discriminate between associated and internalized organisms. Samples with MBL were calculated as a percentage of samples without MBL, which was set at 100% and shown as the control bars. (A) The Cell associated organisms bars represent the total number of organisms associated with 100 MDMs. The Cells with associated organisms bars indicate the percentage of MDMs associated with one or more organisms. (B) The Organisms internalized bars represent the total number of organisms internalized by 100 MDMs. The Internalization (%) bars indicate the percentage of organisms associated with cells that are internalized rather than being merely bound. Each bar is the mean of ≥7 separate experiments + SEM. *, P <0.05, one-sample t-test.

 
In addition, MBL caused a twofold increase in the number of bacteria internalized by cells (P=0.003) and an increase in the fraction of the cell-associated bacteria taken up by the macrophages (P=0.017; Fig. 1B ).

At the higher MOI of 500:1, there was also a significant increase in the number of organisms associated with macrophages in the presence of MBL (P=0.008). There were significant increases (P=0.033) in the percentage of cells positive for organisms, in the number of organisms associated with each cell (P=0.002), and in the number of organisms that had been internalized by the macrophages (P=0.017). However, at this higher MOI, the ratio of associated-to-internalized bacteria did not alter significantly in the presence of MBL (P=0.065).

To test the specificity of the effect of MBL on phagocytosis, we investigated the effect of pretreatment of bacteria with a monosaccharide inhibitor of MBL binding, GlcNAc, and a noninhibitor of binding, mannitol, and observed their effect on internalization (Fig. 2 ). GlcNAc reversed the enhanced internalization seen in the presence of MBL (P>0.05, no significant difference above background in the presence of MBL and GlcNAc). In the presence of mannitol, however, the significant increase in internalization persisted when MBL was present (P=0.032). This indicates that the effect could be inhibited in a manner consistent with the sugar-binding activity of MBL [23 ].



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  		Figure 2. GlcNAc inhibits MBL-mediated internalization of meningococci. MDMs were incubated with N. meningitidis B1940cpsD– at a MOI of 250:1. Organisms had been incubated with buffer alone, MBL (5 µg/ml), 100 mM GlcNAc, 100 mM GlcNAc + MBL (5 µg/ml), 100 mM mannitol, or 100 mM mannitol + MBL (5 µg/ml). The internalization of meningococci by MDMs was determined by an epifluorescence technique. Each bar represents the mean + SEM of four separate experiments, calculated as a percentage of the relevant buffer control. *, Parameters significantly increased in the presence of MBL (P<0.05, one-sample t-test).

 
We were able to observe MBL on internalized organisms that had been preincubated with the lectin, suggesting that MBL was localized to the phagosome (Fig. 3 ). No MBL was present on the surface of organisms that had not been preincubated with MBL, and no C3 was detectable on any organism in the presence or absence of MBL (data not shown).



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  		Figure 3. Fluorescence micrographs showing the phagocytosis of meningococci by human MDMs, which at 12–14 days, were incubated with N. meningitidis B1940cpsD– at a MOI of 500:1 for 30 min at 4°C. MDMs were fixed with 1% paraformaldehyde and then stained to discriminate between organisms that were internalized and those that were merely associated. (A–D) The intracellular expression of LAMP-1 (A and B) and EEA-1 (C and D) was demonstrated using saponin and appropriate antibodies. (A) Cells and organisms examined under blue fluorescence, illustrating DAPI staining of the DNA of cell nuclei and bacteria. (B) The same field with three-color fluorescence. Green fluorescence indicates meningococcal capsule staining for organisms not internalized by macrophages. Red fluorescence represents LAMP-1 staining, a marker of late phagolysosomes. Arrow 1 indicates an organism showing green and blue fluorescence, suggesting that it is associated with a macrophage, but has not been internalized. Arrow 2 designates an organism showing blue fluorescence only, indicating that it has been internalized. Arrow 3 identifies an organism showing blue and red fluorescence, indicating an organism that has been internalized and is within a LAMP-1 compartment. (C) Cells and organisms examined under blue and green fluorescence and (D) the same field with the addition of red fluorescence. The organisms identified by Arrow 1 are external to the macrophage, whereas those indicated by Arrows 2 and 3 are internalized. In addition, the organism indicated by Arrow 3 is colocalized with anti-EEA-1 staining. (E and F) Intra- and extracellular staining for MBL was achieved using saponin and an anti-MBL antibody. Green and blue fluorescence are the same as in A and B, but red fluorescence indicates MBL staining. (E) A macrophage nucleus and four associated organisms examined under blue and red fluorescence. (F) The same field with three-color fluorescence. The organism labeled 1 is positive for MBL staining (red) but negative for green fluorescence, indicating that it has been internalized. The organisms labeled 2 are positive for MBL but also positive for anticapsular antibody, indicating that they have not yet been internalized by the macrophage.

 
MBL is not a "direct" opsonin
To determine whether MBL increased phagocytosis by an effect on binding to cells or by an effect on the rate of internalization, we incubated bacteria with macrophages at 4°C to inhibit uptake of bound bacteria [20 ]. Nonadherent organisms were removed by washing, and the cells were warmed to allow synchronous internalization.

After 60 min cold incubation, there was no significant effect of MBL on the percentage of cells with bound organisms or the number of organisms per cell and therefore, no effect on the number of organisms associated with 100 cells (P>0.05, one-sample t-test). This suggested that MBL did not influence the binding of meningococci to the macrophage plasma membrane and therefore was not operating as a direct opsonin.

The cells were then washed to remove nonadherent organisms and warmed to 37°C for up to 1 h. MBL significantly increased the percentage of organisms internalized over 60 min (P=0.021, Wilcoxon signed ranks test of the area under the curve±MBL; Fig. 4A ) with an increase at the 60-min time-point from 28% to 44%.



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  		Figure 4. Internalization of meningococci after binding at 4°C. (A) N. meningitidis B1940cpsD– preincubated or not with MBL (5 µg/ml) were incubated with MDMs at a MOI of 250:1 for 60 min at 4°C. Cells were washed with cold buffer and then warmed to 37°C and fixed at 30 min or 60 min, and internalization (the percentage of associated bacteria that was internalized) was determined by fluorescence microscopy. The first time-point (0 min) represents internalization immediately after 60 min of cold incubation. The graph shows the median ± the interquartile range for internalization (n=8). The triangles show samples in the presence of MBL; the circles show samples in the absence of MBL, which significantly increased internalization of meningococci (P<0.05, Wilcoxon test of the area under the curve). (B) Dose response of MBL-mediated internalization. MDMs were incubated with meningococci preincubated with different concentrations of MBL at a MOI of 500:1 for 60 min at 4°C; cells were then washed and incubated for a further 60 min at 37°C. Each point represents the median ± the interquartile range of five separate experiments.

 
The effect of MBL on internalization was dose-dependent and maximal at 1.5 µg/ml (Fig. 4B) , a concentration within the range for Caucasoid individuals lacking structural gene mutations [24 ]. We found that preincubation of cells with MBL, rather than preincubation of organisms with MBL, had no effect on the subsequent internalization of MBL-coated or non-MBL-coated organisms (data not shown).

As an alternative to cold incubation to prevent actin reorganization, we used two soluble inhibitors of the actin cytoskeleton, latrunculin B (lat B), which actively depolymerizes actin [25 ], and toxin B, which inhibits the function of the Rho family of small GTPases involved in directing actin polymerization in phagocytic interactions [26 ].

The addition of lat B caused a profound inhibition (mean 86%, n=4) in the number of organisms internalized in the absence of MBL (P<0.001, one-sample t-test) and a significant reduction in the overall number of organisms associated with macrophages (data not shown). The addition of MBL did not reverse this effect (P>0.05, t-tests of association/internalization±MBL with Bonferroni correction). The addition of toxin B reduced the number of organisms internalized by almost 50% (P=0.007, one-sample t-test, n=4). As with the experiments containing lat B, the addition of MBL did not reverse this effect (P>0.05, t-test of internalization±MBL with Bonferroni correction).

MBL bypasses SR-mediated internalization of N. meningitidis
To explore further the effect of MBL on intrinsic interactions between phagocytes and the meningococcus, we examined the effect of inhibiting SRs. SR-A has recently been described as a molecule that mediates the uptake of meningococci by macrophages [27 , 28 ].

The addition of the SR inhibitor, poly(I), in the absence of MBL decreased the number of organisms internalized by 30% (P<0.001, one-sample t-test; Fig. 5 ) with no inhibition with poly(A), a noninhibitor of SRs (P>0.05). These results are similar to those we have observed previously [28 ]. However, neither poly(I) nor poly(A) affected the number of organisms internalized when MBL was present (P>0.05, Bonferroni t-tests). There was still a significant difference between the number of organisms internalized in the presence of MBL compared with its absence in samples in which poly(I) was also present (P<0.001, Bonferroni t-test), indicating that MBL was able to overcome the inhibition of internalization by poly(I).



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  		Figure 5. The effect of SR-mediated internalization inhibition. The addition of poly(I) to MDMs 30 min prior to the addition of organisms significantly decreased the number of organisms internalized in the absence of MBL but had no effect on the number of organisms internalized in the presence of MBL. Poly(A) does not inhibit SRs and did not affect the internalization of organisms in the presence or absence of MBL. Bars are labeled as in the presence (+) or absence (–) of MBL (1.5 µg/ml). *, P < 0.05, one-sample t-test. Bonferroni t-test; n = 6.

 
MBL enhances maturation of phagosomes
The observation that MBL did not directly bind organisms to macrophages but rather changed the efficiency with which they were internalized raised the possibility that the protein might alter the eventual intracellular fate of phagocytosed organisms.

We determined the effect of MBL on the incorporation of EEA-1 and LAMP-1 into the phagosome membrane, the latter a marker suggestive of maturing phagosomes (Fig. 3) . MDMs were infected at a MOI of 500:1 and then incubated for up to 45 min at 37°C. At 15 min, there was substantial colocalization of meningococci with EEA-1, but there was rarely appreciable staining for LAMP-1. An analysis of LAMP-1 colocalization was therefore not conducted for this time-point. As shown in Figure 6 , MBL increased the proportion of internalized meningococci, which colocalized with EEA-1 at 15 min by 64% (P=0.032, one-sample t-test). At 45 min, in the presence of MBL, there was a decrease of 38% in the proportion of internalized organisms colocalized with EEA-1 (P=0.008). At 30 min and 45 min, LAMP-1 colocalization was 148% and 278%, respectively, in the presence of MBL (P=0.011 and 0.015, one-sample t-test), more than doubling the total number of organisms that was associated with LAMP-1-positive phagolysosomes from 2.8 to 6.6 at 30 min and from 1.85 to 4.3 at 45 min (P=0.012 and 0.015, one-sample t-test).



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  		Figure 6. Effect of MBL on intracellular processing of meningococci. MDMs at 12–14 days were incubated with N. meningitidis B1940cpsD–, preincubated or not with MBL (5 µg/ml) at a MOI of 500:1 for up to 45 min at 37°C. MDMs were fixed with 1% paraformaldehyde and then stained to identify internalized organisms and EEA-1- or LAMP-1-positive endosomes. Samples were randomized and scored blind to sample identity. Each point represents the proportion of internalized organisms, which colocalized with LAMP-1 (late endosomal marker, •) or EEA-1 (early endosome marker, {blacksquare}) and is the mean of up to nine separate experiments + SEM. Each point is significantly different from processing in the absence of MBL (P<0.05, one-sample t-test).

 
Effect of MBL on meningococcal killing by macrophages
We next examined the effect of the lectin on the killing of meningococci by macrophages. We found that if macrophages were incubated on ice for 1 h, washed, and then incubated at 37°C for a further 2 h, the total number of organisms in macrophage tissue-culture wells was reduced by 80% in the presence of MBL (P=0.003, paired samples t-test; Fig. 7 ). If the cells were washed and then lysed with saponin and viable counts determined, there was a trend toward reduced survival with MBL, but this was not significant (data not shown). If the number of surviving organisms from the supernatant and the lysed cells were added together, the net effect of MBL was a reduction in survival of 80% (P=0.015, paired samples t-test). There was no significant effect of MBL on the number of viable organisms associated with cells immediately after incubation on ice.



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  		Figure 7. The effect of MBL on macrophage-mediated killing of meningococci. Cells were incubated with organisms for 1 h at 4°C, washed, and then incubated for a further 2 h at 37°C with or without antibiotics for the last 30 min. Data are the geometric mean + SE of seven separate experiments. (A) Survival of N. meningitidis in supernatants derived from macrophages not treated with antibiotics. MBL reduced survival by 80% (P<0.003, paired t-test). (B) Survival of intracellular N. meningitidis in macrophages treated with antibiotics. The presence of MBL reduced meningococcal survival by 61% (P<0.015, paired t-test) despite the increased uptake noted earlier. cfu, Colony-forming units.

 
To determine the effect of MBL on intracellular survival, we used an antibiotic exclusion assay. At 2 h, the number of viable, intracellular organisms was reduced by 61% in the presence of MBL (P<0.015; Fig. 7 ). MBL did not enhance killing if the cells were fixed with paraformaldehyde prior to the addition of organisms (P>0.05), indicating the requirement for viable macrophages.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previously, we have shown that MBL in serum-free conditions is able to increase the uptake of meningococci by neutrophils and monocytes. Using a series of mutants that differ in MBL binding, we were able to demonstrate that the amount of MBL bound did not correlate with the amount of phagocytosis observed. One suggestion arising from these observations is that MBL was not acting as a direct opsonin [16 ]. In the present study, we aimed to characterize the enhancement of phagocytosis of N. meningitidis by MBL and explore the consequences of this method of uptake. Our experiments suggest that this enhancement occurs mainly through an effect on the process of internalization with more rapid processing of meningococci to lethal compartments with consequent accelerated bacterial killing. MBL does not appear to be involved in the initial attachment of the organism to the macrophage, which likely occurs via a separate pathogen-host interaction. The current data suggest that the opsonic effect of MBL is different than that of complement or Ig, which are capable of mediating the attachment to phagocytes of the particles with which they are associated as well as enhancing internalization [29 ]. We therefore propose that MBL is a "dependent" opsonin, not a direct opsonin.

The first stage of phagocytosis is the initial binding of the target to the phagocyte. We observed that MBL increased the phagocytosis of meningococci when cells were incubated at 37°C. However, when macrophages were cooled to 4°C, which inhibits the rearrangement of actin and the internalization of bound bacteria [20 ], there was no effect of MBL on the association of meningococci with macrophages as determined by microscopy and viable counting, indicating that MBL was not involved in the initial attachment of the organism to the phagocyte. A similar effect was observed when actin rearrangement was inhibited by lat B or toxin B.

It is known that pathogenic Neisseria have a number of mechanisms for adhering or modifying binding to host cells in the absence of "opsonization" [30 , 31 ] including Rho GTPase-dependent and -independent pathways mediated by gonococcal opa [32 ]. Our results would be consistent with a Rho-independent mechanism being present for Neisseria, as a low level of bacterial uptake still occurred in the presence of toxin B, which inhibits Rho GTPase activity. We observed no effect of MBL on toxin B-treated macrophages, which suggests that MBL-mediated phagocytosis is dependent on Rho GTPases. Two of the outer membrane proteins of Neisseria, which modify pathogen-cell interactions opa and porB, are MBL ligands. The attachment does not appear to occur via the normal carbohydrate-recognition mode of binding [12 ]. However, the truncated lipopolysaccharide of the organism used here may also be a target of the lectin through exposure of glucose, and binding is very sensitive to inhibition with GlcNAc [13 ]. We found that phagocytosis was inhibited by GlcNAc, which may suggest that the effect on phagocytosis is bacterial ligand-independent, but further studies are required.

We proceeded to investigate whether there was an effect of MBL on the internalization of bacteria by incubating organisms with macrophages at 4°C, washing to remove nonadherent organisms, and then warming the cells to allow internalization. Under these conditions, MBL caused a 60% increase in microbial uptake. From this, we conclude that MBL primarily influences phagocytosis by enhancing internalization, a process that might be termed "phagosomal transduction".

Although the primary effect of MBL appeared to be on internalization, our data at 37°C showed an increase, not only in the number of organisms internalized by macrophages but also in the number of organisms associated with cells. This suggests that the cytoskeleton is involved in MBL-enhanced association of bacteria with macrophages. This would be consistent with our observation that the two cytoskeletal inhibitors reduced the association of bacteria with macrophages as well as internalization. We suggest that rearrangement of the cell surface is necessary for adequate stabilization of the bound organism for effective capture, perhaps by improving the functional affinity of organism binding. MBL might enhance stabilization of organisms on the surface of the phagocyte by accelerating the internalization process.

We showed that at 15 min, more meningococci colocalized with an early marker of endosomes, EEA-1, but at 45 min, there was a decrease in colocalization with EEA-1 in the presence of MBL. At 30 and 45 min, there was an increase in colocalization with a later marker of endosomes, LAMP-1, in the presence of MBL. This suggests that MBL can accelerate the incorporation of endosomal markers after an organism has been internalized. We did not find evidence for the incorporation of different endosomal markers in the presence of MBL, suggesting that MBL does not alter the intracellular fate of organisms but has an augmenting role in endosomal maturation. A wider range of markers could be examined in the future.

MBL more than halved the number of viable organisms within macrophages and in the supernatant of these cells. This stimulation of macrophage killing of organisms by MBL suggests that this is one way that the protein could increase protection against bacterial infection. Increased intracellular killing is consistent with the acceleration of intracellular processing that we observed, but as with our enumeration of colocalization with EEA-1 and LAMP-1, this only gives a snapshot of the processes occurring within the cells. The effect of MBL on the numbers of extracellular organisms is a realistic measure of the role the protein may have in infection. The method we used was intended to bind an equal number of organisms to the cells to observe the effect of MBL on the uptake and killing of the same inoculum size as in the absence of MBL with minimal addition of organisms from the supernatant. However, it was not possible to wash away all the nonadherent organisms after cold incubation, and the small numbers of remaining extracellular bacteria multiplied in the cell supernatants over 2 h to ultimately high concentrations. The available evidence suggests that in disease, meningococci propagate from relatively low numbers of initial organisms. The presence of MBL more than halved the number of organisms in the supernatant of live cells, which is likely the result of increased capture of organisms by macrophages as a result of the collectin.

A number of cell-surface molecules have been suggested as receptors or binding proteins for MBL [10 , 11 , 33 , 34 ]. The two main suggestions are calreticulin [33 ], operating through CD93 [10 ] and complement receptor 1 (CR1) [11 ]. In immobilized ligand studies, the interaction of MBL with CR1 or calreticulin appears to be a material-binding interaction [11 , 33 ], which can lead to the binding of MBL-opsonized targets to an immobilized receptor [11 ]. The evidence for whether MBL and C1q can compete with one another for binding to these proteins is mixed; total inhibition does not appear possible with calreticulin, but more complete inhibition appears possible with CR1. However, it has also been shown that C1q and MBL do not compete with each other for binding to macrophages [35 ]. Indeed, it has been shown that MBL binds very poorly to MDMs at 4°C [35 ]. We have not attempted to identify a MBL receptor in this study, but our results may suggest a reason for the difficulties in identifying such a receptor, as the lectin may not be responsible for the initial binding of microorganisms during phagocytosis, although it appears integral to the whole process.

We have begun to characterize the interaction of MBL with another of the innate receptors for meningococci, the SR-A [27 , 28 ]. Our data suggest that this receptor is important as an internalization receptor [28 ]. In the absence of specific blocking antibodies for human SR-A, we used poly(I) to inhibit the internalization of organisms by macrophages via this receptor. This treatment will nonspecifically block other SRs, but SR-A appears to be the major nonopsonic receptor for meningococcal phagocytosis by macrophages [27 ]. We observed no effect of poly(I) on internalization in the presence of MBL. This suggests that the lectin is able to direct meningococci from one potential receptor pathway to another and that the SR-A pathway may not be active when organisms are optimally coated with MBL. Although MBL is present at extravascular sites [36 ] and is present in the airways of mice infected by influenza virus [37 ], it is unlikely to be present at concentrations sufficient to saturate the meningococcal surface to the exclusion of SR-A in the environment of the tissue macrophage. In vivo, several mechanisms are likely to interact and influence phagocytosis, and this suggests that the uptake of meningococci in MBL-deficient and -sufficient individuals is likely to differ in character.

To conclude, MBL is an "opsonin", as it stimulates uptake of organisms by phagocytes. However, it differs from "classical" (or direct) opsonins such as Ig or complement in that it does not itself enhance binding of microorganisms to phagocytes. This is entirely consistent with reports that describe MBL alone enhancing opsonization, as many other microorganisms are able to bind directly to human cells, and phagocytosis would then be enhanced by the ability of MBL to promote uptake. The effect we describe is also consistent with the concept of "indirect" opsonization, in which other pathways are enhanced, such as Ig- and complement-mediated opsonization, as both will improve the binding of organisms to phagocytes. We suggest that dependent opsonin, that is, an opsonin dependent on other interactions to have an effect, is a more accurate description of the role of MBL in the phagocytosis of N. meningitidis than direct or indirect opsonin. The importance of this function of MBL in meningococcal disease is unknown but may be particularly pertinent in protection during the early stages of contact with the organism, when SRs also appear to be important.


   ACKNOWLEDGEMENTS
 
Grant support was from The Wellcome Trust, Project Grant No. 059247, Meningitis Research Foundation Grant No. 7/02, Action Research Project Grant SP3580, and the European Union (Contract No. QLRT-CT-2001-01039). Research at the Institute of Child Health and Great Ormond Street Hospital for Children National Health Service (NHS) Trust benefits from research and development funding received from the UK NHS executive. The excellent technical assistance of Anne Cook and Linda Goodwin is gratefully acknowledged.

Received June 16, 2004; revised October 29, 2004; accepted November 1, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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