Published online before print February 25, 2008
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* Centro Ricerche Biotecnologie Innovative and
Istituto Veneto Medicina Molecolare, Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Padova, Italy;
Rzeszów University of Technology, Faculty of Chemistry, Department of Biochemistry and Biotechnology, Rzeszów, Poland; and
Research Centre, Novartis Vaccines and Diagnostics S.r.l., Siena, Italy
1Correspondence: C.R.I.B.I.-University of Padova, Via G. Colombo 3, 35121 Padova, Italy. E-mail: emanuele.papini{at}unipd.it
ABSTRACT
Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-
and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA
351–405, a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1 and low levels of the main vasoactive mediators TNF- and IL-1. NadA351–405 also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.
Key Words: adhesion molecule • bacterial infection • inflammation • cytokines • chemokines
INTRODUCTION
The gram-negative bacterium Neisseria meningitidis is a naso-pharyngeal commensal widely diffused in the healthy human population that can give rise to septicaemia and meningitis [1
]. The mortality of these diseases is especially high in infants [2
] and is mostly a result in developed countries of the serotype B [1
]. When in the blood, N. meningitidis cells release outer-membrane vesicles, which contain a high amount of lipooligosaccharide (LOS), a shorter variant of gram-negative LPS. LOS is related to shock, as it stimulates monocytes to secrete powerful vasoactive cytokines such as IL-1 and TNF- [3
, 4
]. However, it has been proposed that some superficial virulence factors target the immune cells during tissue and blood invasion by meningococcus [5
, 6
]. Complete information about these factors is particularly important for serotype B, as unlike other virulent serotypes (A, C, Y, and W135), it is not affected by presently available vaccines [7
, 8
].
Bioinformatic analysis of the genome of a virulent N. meningitidis B strain [9 ] allowed the identification of previously unknown surface proteins [10 ], among which is the 45-kDa N. meningitidis Adhesin A (NadA). Structure prediction and homology comparison suggests that NadA belongs to the group of oligomeric coiled-coil adhesins (OCA) such as YadA of Yersinia enterocolitica and UspA2 of Moraxella catarrhalis [11 ]. Within these homotrimeric outer-membrane proteins, three structural regions are present: a conserved –COOH terminal membrane anchor, having a β structure; an intermediate coiled-coil stalk comprising a leucine zipper; and a –NH2 terminal region, forming the binding site(s) for target cell receptors [12 ].
NadA is a risk factor for the development of meningococcal disease, as it was found in 
50% of N. meningitidis strains isolated from patients and in only 5% of strains from healthy individuals [13
]. NadA has been implicated in the mucosal colonization by N. meningitidis B, as its expression enhances bacterial adhesion to and invasion of mucosal cells. Consistently, the soluble recombinant mutant (sr)NadA351–405, lacking the membrane anchor, binds to human conjunctival cells [14
]. NadA is an important candidate as an antimeningococcal vaccine as well. Therefore, we screened the specific association of NadA to human blood leukocytes encouraged by our recent finding that NadA induces the maturation of resting, monocyte-derived dendritic cells (DC) [15
]. Results demonstrated that NadA indeed binds to human monocytes and macrophages in a way similar to that observed in DC and Chang epithelial cells. Quantification of the pattern of cytokine secretion and of CD marker expression after acute and prolonged stimulation proved that LPS-free, clinical trial-grade NadA preparations induce an immune-modulatory activity on monocytes and determine their differentiation into a specific macrophage phenotype. NadA effects were clearly different from those induced by gram-negative endotoxin, as there is a tendency toward more chemokines secretion. Our data suggest that NadA interacts with and stimulates monocyte/macrophage/DC during N. meningitidis infection.
MATERIALS AND METHODS
NadA and anti-NadA antibodies
srNadA was designed and purified as described previously [11
]. The sequence of nada allele 3 from the hypervirulent N. meningitidis B strain 2996, encoding for the deletion mutant NadA351–405, devoid of the membrane anchor, was cloned into a pET21b vector (Novagen, Madison, WI, USA). The protein secreted in the extracellular medium of the transformed Escherichia coli BL21(DE3)-NadA351–405 strain was purified by Q Sepharose XL and Phenyl Sepharose 6 Fast Flow (Pharmacia, Uppsala, Sweden) chromatography. LPS contamination (tested by Limulus test kit from Sigma Chemical Co., St. Louis, MO, USA) was ablated to less than 0.005 EU/µg protein by a further passage on a hydroxyl apatite ceramic column (HA Macro Prep). Reverse-phase separation of NadA351–405 preparation in denaturating conditions shows a single peak homogeneously corresponding to the 1–315 sequence of the mature NadA protein, as determined by MALDI-TOF-mass spectrometry analysis. No E. coli antigens were detected by Western immunoblot analysis with a rabbit polyclonal antibody raised against whole E. coli cells (Dako, Denmark), and bacterial DNA contamination was 0.6–0.7 pg/µg protein [15
]. Purified NadA351–405 shows a single 35-kDa band after SDS-PAGE and silver-staining, consistent with the predicted molecular weight, and is a homotrimer assessed by light-scattering analysis, a structural condition typical of OCA, which suggests a proper folding of the protein [14
]. Aliquots of protein solution (2 mg/ml in PBS, pH 7.4) were frozen in liquid nitrogen and stored at –80°C. Mouse anti-NadA antiserum was obtained as described already [11
].
Cell preparation and culture conditions
Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, from human conjunctiva) and HeLa cells were maintained in DMEM (Gibco-BRL, Grand Island, NY, USA) supplemented with gentamycine (50 µg/ml) and 10% (v/v) heat-inactivated FCS. Raw 246.7 cells were maintained in RPMI (Gibco-BRL) supplemented with gentamycine (50 µg/ml) and 10% (v/v) heat-inactivated FCS. PBMC were isolated from buffy coats of healthy donors by centrifugation over a Ficoll-Hypaque (Amersham Corp., Arlington Heights, IL, USA) step gradient and suspended in RPMI 1640 (Gibco-BRL) supplemented with antibiotic and 10% FCS. Residual T and B cells were removed from the monocyte fraction by plastic adherence. The purity of preparations (percentage of CD14-positive cells) and cell viability (using the Trypan blue exclusion test) were higher than 98%. All cells were kept at 37°C in a humidified atmosphere containing 5% (v/v) CO2 unless otherwise specified. Macrophages were differentiated from monocytes by treating plastic adherent cells with RPMI-1640 medium without serum for 1 h and by a further incubation for 6 days in the same medium as above [16
].
Bacterial strains and growth conditions
The E. coli BL21(DE3) strain transformed with a pET21b plasmid bearing allele 3 of the nada gene and expressing full-length NadA associated to the outer membrane (E. coli-NadA) and its control carrying the pET21b plasmid with no insert (E. coli-pET) were obtained as described [14
]. They were cultured at 37°C in Luria-Bertani medium supplemented with 75 µg/ml ampicillin for 5 h until they reached the late logarithmic growth phase (OD 0.8) to warrant optimal NadA expression [14
], harvested, washed in PBS, and then used in experiments. The actual bacterial titers of recovered suspensions were subsequently checked after serial agar plating and quantified as cfu/ml.
Spectrofluorimetric measurement
NadA-Alexa (4.7 µg protein) was diluted in 1 ml PBS solution at pH 5.5 and pH 7.0. It was then incubated for 24 h at room temperature, with or without 21.8 µg/ml proteinase K. Alexa Fluor 488 dye-labeled proteins have absorption and fluorescence emission maxima of 494 nm and 519 nm, respectively. In addition, the fluorescence of Alexa Fluor 488 dye is insensitive to pH between pH 4 and 10. Emission fluorescence was collected over a range from 600 to 800 nm. The emission and excitation slit widths were 3 nm, and the scan rate was 120 nm min–1. All measurements were carried out at room temperature on a Shimadzu (Tokyo, Japan) RF-540 fluorescence spectrophotometer. Three runs were averaged for each sample. The same samples were resolved by a SDS-PAGE (12%) and stained with Coomassie blue.
Confocal immunofluorescence and epifluorescence microscopy
Semiconfluent Chang, HeLa, and Raw 246.7 cells were suspended by trypsin-EDTA treatment, seeded onto 13 mm-diameter coverslips, placed inside 24-well culture plates (Falcon) at the density of 6 x 104 per well, and incubated for 24 h. Lympho-monocytes (3x106 per well) or macrophages (106 per well) were plated onto 13 mm-diameter coverslips, placed inside 24-well culture plates (Falcon) in FCS-free RPMI, and incubated for 1 h at 37°C, 5% CO2. After three washings, cells were incubated with purified NadA351–405 for 3 h at 37°C or at 0°C. After three washings with PBS, cells were fixed in 4% paraformaldehyde for 20 min, washed three times with 0.38% glycine, 0.27% NH4Cl in PBS, and permeabilized with PBS containing 0.2% saponin and 0.5% BSA for 30 min at room temperature. Before addition of primary antibodies, Ig binding ability of monocytes was saturated by treatment with pooled human IgG (Sigma Chemical Co., 10 mg/ml in the permeabilization buffer). Monocytes, macrophages, and Chang cells were incubated for 1 h with mouse polyclonal anti-NadA antibody, washed five times, and incubated further for 30 min with secondary Texas red-labeled anti-mouse IgG antibody (Chang) or Texas red-labeled anti-mouse IgG F(ab)'2 (monocytes) diluted in permeabilization buffer. After five washings with PBS plus 0.5% BSA, three washings in PBS, and one wash in distilled water, coverslips were mounted in PBS containing 90% glycerol and 3% N-propyl-gallate and were analyzed with a confocal microscope (Bio-Rad, Hercules, CA, USA, MRC1024ES). Alternatively, Chang cells, macrophages, and monocytes attached to coverslips were incubated with Alexa-NadA351–405, diluted in RPMI plus 10% FCS and 50 µg/ml gentamycin or with no ligand for controls for different time spans (3–24 h), and washed rapidly four times with PBS. Fluorescence associated to cells was directly analyzed in PBS after mounting the coverslips on the chamber of a Leica DM IRB fluorescence microscope equipped with a CD camera. Representative pictures from confocal and epifluorescence microscopy were collected as Tiff files and processed with standard imaging programs.
Labeling of NadA351–405
Radioiodination of NadA351–405 with 125I was carried out by using the Bolton and Hunter reagent (Amersham Corp.). Before use, NadA351–405 (50 µg) in 100 µl 0.1 M borate buffer at pH 8.5 was added to the N2-dried Bolton and Hunter reagent (500 µCi). The reaction was allowed to proceed for 45 min at room temperature and stopped by the addition of 100 mM glycine at pH 7.4 (final concentration). NadA was conjugated to the fluorochrome Alexa 488 using an N-hydroxysuccinimidyl derivative according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA). Alexa- and 125I-labeled NadA351–405 were separated from remaining reagents by size exclusion chromatography using Sephadex G25 (Sigma Chemical Co.) columns pre-equilibrated and eluted with PBS at room temperature.
Cell-binding experiments
In some cases, prior to experiments, cells were incubated at 37°C for 1 h with FCS/DMEM FCS/RPMI containing 200 nM Bafilomycin A1 (BafA1). After three washings with RPMI supplemented with 10 mM Hepes, 10% FCS, and 200 nM BafA1, cells were incubated at 37°C or 0°C for the indicated times with different concentrations of 100 nM Alexa-NadA351–405 or 30 nM 125I-NadA351–405 in the absence or in the presence of nonlabeled NadA351–405 (up to 5 µM). Cells were subsequently washed six times with PBS plus 0.5% BSA. Washed monocyte pellets were suspended in FACS buffer for FACS analysis or lysed with 0.5% SDS, and Chang cells were detached by treatment with 5 mM EDTA solution for FACS analysis or lysed with 0.5% SDS. Cell autofluorescence was subtracted from mean fluorescence intensity (MFI) values obtained from FACS analysis. Cell-associated radioactivity was measured by 
-counting.
Screening of NadA-specific binding to human leukocytes
Citrated whole blood from healthy human donors served as a source of human leukocytes. After erythrocyte lysis by hypotonic shock in 155 mM NH4Cl, 10 mM KHCO3, and 100 mM Na2EDTA at pH 7.4 for 3 min at room temperature, remaining leukocytes were washed two times with lysis buffer and incubated further with 600 nM Alexa-NadA351–405 in RPMI medium supplemented with 10% FCS and 10 mM HEPES on a rocking platform for 3 h at 37°C before analysis. Cells incubated with no ligand were used as negative control. Cells were then washed twice with PBS, pH 7.2 (Gibco-BRL), containing 1% FCS and 0.1% NaN3 (FACS buffer) at 4°C and then incubated further with the proper dilution of anti-CD mAb conjugated to PE for 30 min on ice to identify the main leukocyte populations. mAb against CD3, CD16, CD19, and isotype control IgG1 were obtained from BD PharMingen (San Diego, CA, USA); mAb against CD14 and isotype control IgG2 were obtained from Caltag (S. San Francisco, CA, USA). After a wash with FACS buffer, propidium iodide was added to exclude dead cells, and cell fluorescence intensities of the gated populations were measured with a FACSCalibur flow cytometer and analyzed with CellQuest software (Becton Dickinson, San Jose, CA, USA).
Measurement of TNF- and IL-8
Purified, adherent monocytes (3x106) were infected for 1 h at 37°C with E. coli-NadA or E. coli-pET (5–40 cfu/monocyte) in DMEM, without carbonate, supplemented with 20 mM Na-Hepes, pH 7.4, with no FCS or with 10% (v/v) heat-decomplemented FCS. Monocytes were washed thoroughly and incubated further for 3 h in DMEM with or without FCS. The concentrations of TNF- and IL-8 in the cell-free supernatants were determined with ELISA kits according to the manufacturer’s instructions; TNF- and IL-8, Biosource, San Diego, CA, USA).
Binding of E. coli-NadA and E. coli-pET to monocytes
Monocytes infected for 15–60 min with E. coli-NadA or E. coli-pET as described above were washed with PBS and dissolved by treatment for 2 min with 0.06% Niaproof 4/Tergitol (Sigma Chemical Co.) in water at 4°C. Released bacterial cells were counted after serial agar-plating. Alternatively, monocytes were infected for 30 min, washed as above, further incubated in the same medium for different time spans (from 0 to 60 min), and dissolved with Niaproof 4/Tergitol. Surviving cfu/monocytic cells were counted by serial agar plating as above.
Bio-Plex multiplex cytokine assay
Human adherent monocytes/macrophages were incubated for 24 h in RPMI plus 10% FCS, with or without NadA (1.5 or 3 µM) or E. coli LPS (0.2 µg/ml), and then culture supernatants were collected. In some experiments, solutions of purified NadA351–405 were heated at 95°C for 30 min or incubated with 20 µg/ml of an affinity-purified mouse polyclonal antibody to NadA, plus Protein A-Sepharose (50 µl wet matrix per 200 µl protein solution), centrifuged for 5 min at 13,000 rpm. The supernatant was used to stimulate cells. Antibody pairs directed against different, noncompeting epitopes of a given cytokine were purchased from Bio-Rad. Calibration curves from recombinant cytokine standards were prepared with fourfold dilution steps in RPMI-1640 medium containing 10% FBS. All assays were carried out in 96-well sterile, prewetted filter plates at room temperature and protected from light. A mixture containing 5000 microspheres per cytokine was incubated together with standards or samples in a final volume of 50 µl for 30 min under continuous shaking (300 rpm). After three washes by vacuum filtration with Bio-Plex washing buffer, a cocktail of biotinylated antibodies diluted in Bio-Plex detection antibody diluent was added (25 µl to each well). After 30 min incubation and washing, Streptavidin-PE diluted in Bio-Plex assay buffer was added (50 µl per well). At the end of 10 min incubation, under continuous shaking and after washing, the fluorescence intensity of the beads was measured in a final volume of 125 µl Bio-Plex assay buffer. Data analysis was done with Bio-Plex Manager software using a five-parametric curve-fitting. The detection limit of the assay for all antigens was 1 pg/ml.
Cell survival assay
Monocytes were treated with NadA351–405, E. coli LPS, or medium alone and cultured for different time spans. Cell survival was performed with Caspase-3 and MTT assay according to the manufacturer’s instructions (Sigma Chemical Co.).
Surface marker analysis by flow cytometry
Monocytes treated with NadA351–405, E. coli LPS, or medium alone (control) were cultured for 7 days. After 1, 2, 3, and 7 days, cells were stained with PE-conjugated mAb to human CD14, CD16, CD1a, CD80 (B7.1), CD86 (B7.2), and MHC II (HLA-DR) or with isotype-matched control mAb (BD PharMingen and Caltag). Cells were immunostained with the proper dilution of PE-conjugated anti-human mAb at 4°C for 30 min in 100 µl PBS at pH 7.2 (Gibco-BRL) containing 1% FBS and 0.1% NaN3 (FACS buffer). After washing, propidium iodide was added to exclude dead cells, and cell fluorescence intensities of the gated populations were measured with a FACSCalibur flow cytometer and analyzed with CellQuest software (Becton Dickinson). Data were collected on 10,000–20,000 events.
Phenotypical analysis
Monocytes were treated with NadA351–405, E. coli LPS, or medium alone (control) and cultured for 7 days in 24-well plates (Falcon). After 1, 2, 3, and 7 days, cell morphology was analyzed by light microscopy (Leica DM IRE2).
RESULTS
NadA351–405 preferentially associates to human circulating monocytes
Alexa-NadA351–405 cell labeling of a whole leukocyte population from human blood was performed in the presence of BafA1 to block endolysosomal degradation. Double-labeling with specific anti-CD antibodies and flow cytometry were subsequently used to search for specific NadA leukocyte targets. Results showed that a single subpopulation corresponding to 2–4% of leukocytes efficiently binds the adhesin. These cells largely corresponded to monocytes (CD14-positive), as negligible fractions of T lymphocytes (CD3-positive), B lymphocytes (CD19-positive), NK cells (strongly CD16-positive), and polymorphonuclear neutrophils (PMN; moderately CD16-positive) were Alexa-NadA-positive (Fig. 1A
). The association of NadA to prevalent, classical CD14++ CD16– monocytes was also confirmed by quantitative MFI analysis (Fig. 1B)
.
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Figure 1. Specific binding of NadA351–405 to human blood monocytes. Whole leukocyte fraction was isolated from fresh human blood, incubated for 3 h at 37°C, with or without 600 nM Alexa-NadA351–405. Cells were treated with anti-CD PE-labeled antibodies (CD3 for T lymphocytes, CD14 for monocytes, CD16 for NK cells, CD19 for B lymphocytes) and analyzed by flow cytometry. The top two panels show a typical dot-dispersion graph [side-scatter (SSC) vs. forward-scatter (FSC)] of isolated leukocytes (left panel) and the extent of viable cells [side-scatter vs. fluorescence 3 (FL3); right panel]. Numbers represent the percentage of cells counted in a certain quadrant (A). MFI as a result of Alexa-NadA351–405 labeling of the selected leukocyte types indicated was also quantified. Data are from an experiment representative of four, run in triplicate, and bars represent ranges (B).
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351–405 binding to monocytes and intracellular degradation in endolysosomes351–405 significantly decreased (50% inhibition) the binding of 125 nM Alexa-NadA. The intensity of this competition was similar to that observed in Chang cells, a line characterized by high NadA binding [13
]. The adhesin transport inside endolysosomes was probed, exploiting the observation that fluorescence of Alexa-NadA is increased by proteolysis, probably because of a gain in quantic yield. This feature of the Alexa-NadA conjugate was proved by in vitro experiments (Fig. 2B)
showing that the intrinsic fluorescence of the Alexa moiety was increased by full degradation of the linked protein by proteinase K, and acidity had no effect. The absence of BafA1 in the experiment, a condition allowing physiological endolysosomal acidification and degradation, determines a 2.5-fold increase of the fluorescence of monocytes incubated with labeled NadA only and a fourfold increase of the fluorescence of cells incubated with labeled NadA plus nonlabeled ligand, so that the difference between the two conditions was nonsignificant. On the contrary, MFI associated to Chang cells was slightly increased (+20%) by BafA1 omission after incubation with labeled NadA only. These data suggested that monocyte-bound NadA is subjected to an intracellular degradation in acidic endolysosomal compartments, apparently more efficient for nonspecifically bound NadA with a subsequent increase of the fluorescence of produced Alexa-labeled peptides. At variance, the same data suggest that NadA binding to Chang cells is not followed by significant ligand endosomal degradation.
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Figure 2. Binding of NadA351–405 to monocyte and Chang human conjunctival cells. Chang cells or freshly isolated monocytes were treated with 100 nM Alexa-NadA351–405 in the absence (solid columns) or in the presence of 5 µM nonlabeled NadA351–405 (open columns) and 200 nM BafA1 for 3 h in complete medium. Adherent cells were detached by treatment with an EDTA-containing solution. Alexa MFI associated to monocyte and Chang cells was determined by flow cytometry. Data represent the mean (±SE) of three experiments run in triplicate (*, P 0.05; A). Emission fluorescence spectra of a 70-nM Alexa-NadA351–405 solution in PBS, pH 7.2 or 5.5, were measured ( ex=494 nm) after incubation for 24 h at room temperature, with or without proteinase K (21.8 µg/ml). Protein samples from a representative experiment (corresponding to an initial 4 µg) were also resolved by 12% SDS-PAGE and stained with Coomassie blue (B).
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351–405 was clearly found on the plasma membrane of nonfixed Chang cells after a 3-h incubation at 37°C (Fig. 3A)
. Consistently, confocal microscopic analysis under the same conditions using a nonlabeled adhesin showed that the NadA351–405 signal was found uniformly on the plasma membrane of Chang cells (Fig. 3C)
. In living, adherent monocytes (Fig. 3E)
, the Alexa-NadA351–405 signal was more clustered compared with that seen in Chang cells and could be seen on the periphery of the cell and in intracellular vesicles. Confocal microscopic analysis confirmed the presence of NadA clusters and an intense perinuclear staining (Fig. 3G)
. In macrophages, Alexa-NadA351–405 was found mostly intracellularly and in particular, associated with perinuclear compartments, in agreement with confocal microscopic analysis (Fig. 3I
and 3K)
. NadA did not associate significantly to HeLa cells (Fig. 3M)
, in agreement with previous observations [14
], but was found in large, intracellular structures in the stable macrophage murine cell line Raw 246.7 (Fig. 3O)
. Both approaches confirmed that the meningococcal adhesin binds in a cell-specific way to monocytes/macrophages and is then internalized more intensely compared with in Chang epithelial cells.
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Figure 3. Direct epifluorescence and indirect immunofluorescence of Chang human conjunctival cells, monocytes, and macrophages treated with Alexa 488-labeled NadA351–405. Adherent Chang cells, monocytes, and macrophages were incubated for 3 h with culture medium containing 600 nM Alexa 488-labeled NadA351–405 at 37°C. Cells were then analyzed by direct epifluorescence with a Leica DM IRB microscope (A, E, and I). Alternatively, cells grown on glass coverslips were incubated with 600 nM NadA351–405 for 3 h, washed extensively, fixed with paraformaldehyde, and permeabilized with saponin. Cells were then subjected to indirect immunostaining with specific anti-NadA mouse antibodies and with Texad red-labeled antimouse IgG antibodies and analyzed by confocal microscopy (Bio-Rad MRC1024ES; C, G, and K). (B, F, and J and D, H, and L) Alive, control cells treated with no Alexa 488-labeled NadA351–405 or fixed cells treated with no NadA351–405. As controls, HeLa and Raw cells were treated with 600 nM NadA351–405, as described previously (M and O). (N and P) Fixed cells treated with no NadA351–405.
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351–405 revealed the presence of specific binding sites on monocytes and macrophages detectable at 37°C but not at 0°C. Adhesin binds to these sites with an apparent dissociation constant (Kd) of –2 to 3 µM, based on the monomer molecular weight, or of –1 µM, based on the trimer molecular weight (Fig. 4A
and 4B
). It has to be emphasized that binding of Alexa-NadA351–405 to other leukocyte types, PMN, and T lymphocytes was not inhibited by nonlabeled NadA351–405 at 37°C and 0°C and therefore, had to be regarded as nonspecific (Fig. 4C)
. To measure the affinity of NadA-specific interaction with Chang cells in an accurate way, we were forced to use 125I-NadA351–405 and adherent Chang cells to avoid the critical step of cell detachment necessary for FACS analysis, which affects MFI (see Fig. 2
). In both cases, the signal variations reflected the binding of the same nonlabeled ligand to specific sites. Data showed that the binding competition curve as a result of increasing doses of nonlabeled NadA351–405 was similar to the one observed in monocytes and compatible with binding sites with an affinity of 2–3 µM for adhesion (monomer molecular weight; Fig. 4D
). In monocytes/macrophages and Chang cells, specific binding was clearly detected at 37°C but not at 0°C.
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Figure 4. Binding of Alexa-NadA351–405 to monocytes, macrophages, PMN, lymphocytes, and Chang human conjunctival cells. Monocytes, PMN, and lymphocytes in suspension were incubated for 3 h with 100 nM Alexa-NadA351–405 in the presence of increasing concentrations of nonlabeled NadA351–405 and 200 nM BafA1 at 0°C (open symbols) or 37°C (solid symbols). Adherent macrophages were treated as above and scraped before analysis. MFI of selected cell populations were quantified by flow cytometry. Data are from an experiment representative of four, and bars represent ± SE (A–C). Chang cells were treated at 37°C (solid symbols) or at 0°C (open symbols) with 30 nM 125I-NadA351–405 and increasing concentrations of nonlabeled NadA for 3 h in the presence of BafA1. Cells were then washed, dissolved in 0.5% SDS, and radioactivity was measured by -counting. Values represent mean (±SE) of data from two experiments run in triplicate (D).
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and IL-8 secretion by human monocytes and IL-8 release in the extracellular medium. Data showed that the secretion of TNF- was increased significantly upon NadA-positive E. coli infection (twofold increase with respect to the parental strain), only at a multiplicity of infection (MOI) higher than 25 cfu/cell (Fig. 5C)
. IL-8 release induced by NadA-expressing bacteria was more intense (fivefold increase with respect to the parental strain), even at a low MOI (12.5 cfu/cell) compared with the control strain (Fig. 5D)
. In conclusion, increased cytokine/chemokine secretion was not the result of stronger bacterial-monocyte association or persistence mediated by NadA but appeared to be a NadA-dependent activation.
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Figure 5. Monocyte adhesion, survival, and stimulation by a NadA-expressing E. coli strain. Isolated, adherent human monocytes were incubated with NadA+ (solid symbols) and NadA– (open symbols) E. coli with an infection ratio of 20 cfu/monocytes in DMEM with no antibiotics. After 1 h, monocytes were lysed with Tergitol (A). Alternatively, after 1 h incubation with both strains (20 cfu/monocytes), cells were washed and incubated further for different time spans, as indicated (B). Live bacteria associated to cells were determined after serial dilution, agar-plating, and cfu counting. Values represent mean (±SE) of five experiments run in triplicate. Isolated adherent human monocytes were incubated with NadA+ (solid columns) and NadA– (open columns) E. coli at the indicated infection ratio in DMEM with no antibiotics, supplemented with 10% (v/v) FCS. After 1 h incubation, monocytes were washed and incubated further for 3 h with culture medium. The amount of TNF- (C) and of IL-8 (D) present in the extracellular medium was measured by ELISA. Data represent mean (±SE) of four experiments run in triplicate (*, P0.05).
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351–405 induces cytokine and chemokine secretion by human monocytes351–405 and a maximally stimulating E. coli LPS dose (0.2 µg/ml) as a positive control (Fig. 6
). IL-1, IL-1β, and TNF- were slightly induced by meningococcal adhesin, and IL-6 and the chemokines IL-8, MCP-1, MIP-1, and RANTES were secreted efficiently (60–100% of maximal LPS effect). Vasoactive cytokines IL-1 and TNF- were not induced by 1.5 µM NadA and were moderately secreted after stimulation with a higher dose (3 µM).The secretion of IL-8, already present in nonstimulated monocytes, was increased further by NadA. No IL-12p70 or IL-23 (the latter measured in a separate ELISA assay; not shown) or few IL-10 were induced. A significant release of GM-CSF, effectively secreted upon LPS stimulation, was not induced by NadA. IFN-inducible protein 10 (IP-10), eotaxin, IFN-, IL-2, IL-4, IL-5, IL-7, IL-13, IL-15, and IL-17 were not detected, even after LPS stimulation (not shown). NadA351–405 preparations pretreated at 95°C for 30 min or immunodepleted of NadA with affinity-purified, anti-NadA mouse antibodies linked to protein A-Sepharose beads lost more than 90% of their activity (not shown), suggesting that indeed, this is a result of NadA and not LPS or other active contaminants. In addition, NadA effects were not synergized by suboptimal doses of purified E. coli LPS (0.01–10 ng/ml; not shown).
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Figure 6. Secretion of cytokines and chemokines by monocytes treated with NadA351–405 and E. coli LPS. Human adherent monocytes were incubated for 24 h in RPMI plus 10% FCS, with or without NadA or E. coli LPS as indicated, and then culture supernatants were collected. The amounts of the indicated, secreted cytokines and chemokines were determined with a Bio-Plex multiplex cytokine assay. Values are the mean from three experiments run in duplicate ± SE.
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Figure 7. Survival of NadA-treated monocytes. Freshly isolated monocytes were cultured for up to 7 days in RPMI with 10% FCS alone in the presence of 0.5 µM NadA or 200 ng/ml E. coli LPS. The activation of Caspase 3 was measured after 24 h at the indicated concentrations of NadA or LPS. Data represent the mean (±SE) of four experiments and compared with controls (*, P0.05; A and B). Cell survival was determined by MTT assay at the indicated time. Mean values (±SE) of data from two experiments run in triplicate are presented as percentage of control on the first day (C).
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Figure 8. Surface marker expression of monocytes subjected to further differentiation by NadA. Adherent monocytes were cultured for 7 days in the presence of 0.5 µM NadA, 200 ng/ml E. coli LPS, or medium alone as negative control. After 1, 2, 3, and 7 days, cells were collected, and the indicated markers were quantified with PE-labeled, anti-CD-specific antibodies and quantified by FACS. Data are expressed as MFI and are the mean of four experiments run in triplicate. Bars are ± SE.
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and IL-1β and higher levels of chemokines such as IL-8, MIP-1, and MCP-1 (Fig. 6)
. This feature was also observed when monocyte-derived macrophages were treated with NadA (Fig. 9A
). On the contrary, monocyte stimulation by LPS determined a higher release of vasoactive cytokines (TNF- and IL-1β), and macrophage stimulation with the same agonist was characterized by a higher secretion of chemokines (Fig. 9B)
. Interestingly, cells that survived after NadA activation secreted a pattern of immune mediators resembling that released by macrophages (high chemokines/low inflammatory cytokines) when stimulated with LPS (Fig. 9D)
. This again suggests that NadA induces the differentiation of monocytes into a macrophage-like cell type. When compared with LPS-induced differentiation, NadA differentiation was clearly different. In fact, endotoxin-induced differentiation down-regulates the secretion of some cytokines and chemokines after a subsequent LPS challenge (Fig. 9E
and 9F)
. On the contrary, cells differentiated by NadA still remain reactive upon a second stimulation and secrete the same pattern of soluble mediators (Fig. 9D)
. NadA not only induces a relatively low level of proinflammatory cytokines but induces a cell phenotype that secretes a cytokine/chemokine pattern typical of macrophage cells when restimulated by LPS.
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Figure 9. Pattern of cytokines/chemokines secretion by cells differentiated from monocytes with NadA. Freshly isolated monocytes or monocyte-derived macrophages were stimulated with 0.5 µM NadA or 200 ng/ml E. coli LPS for 24 h, and then culture supernatants were collected (A and B). Alternatively, adherent monocytes were incubated with medium alone (C, negative control), 0.5 µM NadA (D), or 1–200 ng/ml LPS (E and F, positive control); after 3 or 7 days, monocytes were treated for 24 h with 200 ng/ml LPS, and then culture supernatants were collected. The amounts of secreted cytokines and chemokines were determined with a Bio-Plex multiplex cytokine assay (IL1-β, TNF-, IL-6, IL-10, IL-12p70, IL-8, MCP-1, MIP-1, RANTES, and IP-10). Data are representative of three experiments run in duplicate.
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In the first part of this work, we successfully used Alexa derivatization of soluble adhesin to screen for leukocyte targets of NadA. We found that NadA specifically binds, not only to epithelial cells such as conjunctival Chang cells but also to monocytes and monocyte-derived macrophages as well, and PMN, NK, and lymphocytes showed variable, nonspecific binding. We observed that NadA binds monocytes/macrophages and Chang cells more intensely and specifically at 37°C compared with 0°C. The affinity of NadA association to monocyte/macrophage and Chang cell receptors is estimated to correspond to Kd 3 µM, based on the molecular weight of the NadA monomer, but can be more appropriately calculated as 1 µM, given that recombinant NadA is a homotrimer [3
]. This is in keeping with our recent detection of specific low-affinity receptors for NadA on monocyte-derived DC, although in these cells, a smaller fraction of high-affinity titers could also be identified [15
]. Bacterially expressed NadA, which forms a superficial array like other OCA, is, however, expected to have an improved affinity for the plasma membrane of target cells.
Functional and morphological experiments confirmed the association of isolated NadA with monocytes/macrophages. However, compared with conjunctival cells, endocytosis and intracellular degradation was evident in these immune cells. Specific reception on monocytes/macrophages, using Alexa-labeled ligand, could only be detected at 37°C and in the presence of an agent that blocks V-ATPase-dependent endolysosomal degradation. This is a result of the high endocytic and degradation rate of the ligand by these cells compared with epithelial cells, which alters the quantic yield of the fluorescent probe.
The presence on Chang cells and monocytes/macrophages of specific NadA receptors suggests that this putative adhesin may be involved, not only in mucosal colonization and invasion but also in tissue and blood invasion. Although N. meningitidis LOS has been long proposed as a major stimuli for inflammatory cells, studies performed with LOS-defective N. meningitidis strains suggest that surface proteins can also induce monocytes to secrete cytokines [17 , 18 ], and NadA may be one of this superficial virulence factor with immune-modulatory activity.
To investigate the functional effects of NadA on monocytes, we exploited an E. coli model expressing surface full-length NadA that previously allowed the discovery of the adhesion and invasion properties of NadA in epithelial cells [14
]. Different from the epithelial model, we found here that expression of NadA on the surface E. coli does not enhance its association to monocytes or its intracellular survival. This observation indicates that the presence of NadA is less relevant for bacterial binding to the monocyte, a cell already equipped with receptors for microbes. Interestingly, however, infection for 1 h with the NadA+ strain stimulated a higher secretion of TNF- and IL-8 in the following 3 h compared with the NadA-parental strain. Interestingly, at a low bacteria:monocyte ratio, TNF- secretion induced by NadA-positive bacteria was similar to the control strain, and IL-8 secretion was significantly more intense. This suggests that NadA, although not a determinant for binding to monocytes, triggers an intracellular signal leading to their activation. To better define the immunomodulatory activity of NadA, we stimulated adherent, isolated monocytes with purified NadA and analyzed the pattern of cytokine/chemokine secretion after 24 h with a 22-Plex array. Data were compared with the secretion induced by a strong dose of LPS, the main proinflammatory agonist of gram-negative bacteria. Results suggest that NadA can stimulate the monocyte to increase their synthesis and release of a wide range of cytokines and chemokines. However, different from LPS, the secretion of vasoactive shock causing cytokines IL-1, IL-1β, and TNF- was relatively low and could be detected only at relatively high concentrations (3 µM). On the other hand, IL-6, the systemic, acute-response cytokine endowed with anti-inflammatory activity and Th2-polarizing properties [19
], was induced by NadA more efficiently than IL-1β and TNF- (50–60% of LPS effect). As for chemokines, release of IL-8 and of monocyte chemoattractant MCP-1, MIP-1, and RANTES was comparable with that induced by LPS. It appears that the NadA stimulus, compared with LPS, generates a mixture of signals that is moderately inflammatory, especially in terms of endothelial activation. A similar tendency is evident when NadA+ E. coli cell effects are compared with NadA–. In fact, although TNF- production is clearly induced, at low bacteria concentration, only the chemokine IL-8 is induced to a significantly high level in a NadA-dependent way. It is important to emphasize that cytokines such as IL-1β and TNF-, less efficiently induced by NadA, directly increase endothelial-leukocyte adhesion, microcirculation blood-flow alterations, and permeabilization. On the other hand, IL-6, a cytokine induced to significant levels by NadA (60% of LPS effect), is now recognized to have a systemic and local, anti-inflammatory [20
] and antiseptic effect in animal models [21
]. Although induction of IL-8, MCP-1, RANTES, and MIP-1 by NadA is predicted to favor endothelial transmigration of monocyte, lymphocyte, and other immune cells, evidence obtained in vivo suggests that MCP-1 has an anti-inflammatory action. In fact, MCP-1 favors Th2 lymphocyte differentiation through IL-4 induction [22
], inhibits the production of IL-12 and TNF-, increases the production of IL-10 in sepsis induced in animal models, and prevents shock-related death [23
].
Monocyte treatment for days with NadA overactivates the antigen-presenting machinery: the expressions of HLA-DR and CD86, necessary for efficient T lymphocyte activation, were in fact increased. However, differently from LPS-differentiated monocytes, CD80 was not induced. The up-regulation of CD14, the coreceptor of LPS and lipoteichoic acid, suggests that NadA, like LPS, improves the innate binding capability of bacterial microbes and of their products. In addition, NadA, unlike LPS, as well, increases the expression of FcRIII-CD16 and seems therefore also to improve the binding capacity of microbes and microbial products that are mediated by antibodies. The NadA stimulus appears to determine a typical, matured macrophage cell endowed with an increased innate and immune-mediated microbe capture and antigen-presenting efficacy. Although CD14++ CD16– monocytes are the preponderant cell-type target of NadA, our data cannot exclude that NadA-differentiated cells are heterogeneous and also derive from different monocyte subpopulations, in particular, from the less-represented CD14+ and CD16+ monocyte subpopulation, and further investigations are required to address this interesting possibility.
NadA differentiation induced a cell phenotype, which upon LPS stimulation, secretes a pattern of cytokines/chemokines closer to that released by bona fide macrophages, which is tending toward chemokine production at the expenses of IL-1β/TNF-.
In conclusion, we discovered that NadA from N. meningitidis interacts in a similar way with epithelial cells and monocytes/macrophages. This suggests that NadA may be involved, not only in mucosal colonization, as already suggested, but also in eliciting tissue defences after the NadA+ N. meningitidis strains’ crossing of the epithelial barrier. NadA, a candidate for an antimenigococcus B vaccine, was also proven to be biologically active on monocytes and macrophages inducing a profile of extracellular signals that favors increased monocyte recruitment and a low, proinflammatory profile. In addition, NadA can support the differentiation of monocytes into a macrophage-like cell type characterized by increased expression of CD14 and the receptor for antigen-antibody complex CD16. This is expected to increase their innate and antibody-mediated antigen-binding and presentation. It has already been proven that NadA is a good immunogen for vaccine formulations with self-adjuvating properties [15 ]. In this study, we showed that this adhesin is also endowed with an intrinsic, proimmune effect useful to counteract infection without exacerbating, vasoactive, potentially dangerous effects.
ACKNOWLEDGEMENTS
This work was supported by PRIN 2002 and 2003 grants from Italian MIUR and is in partial fulfillment of the Ph.D. degree in Biotechnology of C. M. and S. F. M. S. is supported by a grant from the University of Padova. We are especially indebted to the personnel of the Centro Trasfusionale of the Hospital of Padova (ULSS 16) for providing buffy coats from human donors. We further thank Dr. Maurizio Morandi and Dr. Eugenia Ciccopiedi for NadA351–405 purification.
Received December 5, 2007; revised January 18, 2008; accepted January 29, 2008.
REFERENCES
and R-848 dependent activation of human monocyte-derived dendritic cells by Neisseria meningitidis adhesin A J. Immunol. 179,3904-3916This article has been cited by other articles:
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  R. Tavano, S. Franzoso, P. Cecchini, E. Cartocci, F. Oriente, B. Arico, and E. Papini The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without further stimulating their proinflammatory activity on circulating monocytes J. Leukoc. Biol., July 1, 2009; 86(1): 143 - 153. [Abstract] [Full Text] [PDF]
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