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The Journal of Immunology, 2007, 179, 2477 -2484
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Novel Role for the Bactericidal/Permeability Increasing Protein in Interactions of Gram-Negative Bacterial Outer Membrane Blebs with Dendritic Cells1

Hendrik Schultz*,{dagger}, Janet Hume*,{ddagger},§, De Sheng Zhang{dagger}, Theresa L. Gioannini*,{dagger} and Jerrold P. Weiss2,*,{dagger},{ddagger}

* Inflammation Program, and {dagger} Department of Internal Medicine, University of Iowa and Iowa City Veterans Affairs Medical Center, and {ddagger} Department of Microbiology, University of Iowa, Iowa City, IA 52242; and § Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72202-3591


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The bactericidal/permeability-increasing protein (BPI) is thought to play an important role in killing and clearance of Gram-negative bacteria and the neutralization of endotoxin. A possible role for BPI in clearance of cell-free endotoxin has also been suggested based on studies with purified endotoxin aggregates and blood monocytes. Because the interaction of BPI with cell-free endotoxin, during infection, occurs mainly in tissue and most likely in the form of shed bacterial outer membrane vesicles ("blebs"), we examined the effect of BPI on interactions of metabolically labeled ([14C]-acetate) blebs purified from Neisseria meningitidis serogroup B with either human monocyte-derived macrophages or monocyte-derived dendritic cells (MDDC). BPI produced a dose-dependent increase (up to 3-fold) in delivery of 14C-labeled blebs to MDDC, but not to monocyte-derived macrophages in the presence or absence of serum. Both, fluorescently labeled blebs and BPI were internalized by MDDC under these conditions. The closely related LPS-binding protein, in contrast to BPI, did not increase association of the blebs with MDDC. BPI-enhanced delivery of the blebs to MDDC did not increase cell activation but permitted CD14-dependent signaling by the blebs as measured by changes in MDDC morphology, surface expression of CD80, CD83, CD86, and MHC class II and secretion of IL-8, RANTES, and IP-10. These findings suggest a novel role of BPI in the interaction of bacterial outer membrane vesicles with dendritic cells that may help link innate immune recognition of endotoxin to Ag delivery and presentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The bactericidal/permeability-increasing protein (BPI)3 and the LPS-binding protein (LBP) are essential components of the innate immune system repertoire against infections with Gram-negative bacteria (1). Although both proteins belong to a conserved family of lipid transfer proteins (2) and share a high degree of homology (3), they have different effects on interactions of the host with Gram-negative bacteria.

LBP is produced and secreted from hepatocytes and, consequently, is present in circulation and at lower levels in tissue. At these low doses, LBP can play a major role in sensing endotoxin, especially early after Gram-negative bacteria invasion when bacteria and endotoxin are present in minute amounts (4). Endotoxin, LPS, or lipo-oligosaccharides (LOS) comprise a group of unique and abundant glycolipids that are present on the surface of Gram-negative bacteria (5). LBP binds to endotoxin-rich lipid-water interfaces and catalyzes CD14-dependent disaggregation and transfer of single endotoxin molecules to soluble or membrane-bound CD14 (6). Endotoxin can be efficiently transferred from monomeric endotoxin-CD14 complexes to MD-2/TLR4 and thereby potently initiates an inflammatory response (6, 7, 8).

BPI is found most abundantly in azurophilic granules of neutrophil granulocytes and to a lesser extent in eosinophils, fibroblasts, and certain mucosal epithelial cells (9, 10, 11, 12). Binding of BPI to endotoxin in the Gram-negative bacteria outer membrane (OM) triggers sublethal and lethal effects on Gram-negative bacteria and neutralization of the proinflammatory activity of endotoxin (13, 14, 15, 16). In contrast to the effects of low concentrations of LBP, binding of BPI inhibits transfer of endotoxin to CD14 and, thus, TLR4-mediated signaling (17). However, BPI can promote opsonization of whole Gram-negative bacteria and their ingestion by neutrophil granulocytes as well as CD14-independent clearance of purified endotoxin by CD14+ blood monocytes, the latter apparently without causing proinflammatory reactions (17, 18).

Experimental animal models and preclinical and clinical studies in humans have demonstrated that exogenously administered (i.v.) BPI can exert protective effects in the bloodstream (19, 20, 21, 22). However, as yet, little is known about the interactions and fate of complexes including endotoxin and BPI in tissue settings where most of the initial encounters between Gram-negative bacteria and the host occur (4). Presentation of endotoxin in these settings most likely occurs as an integral component of the Gram-negative bacterial OM. This arrangement includes OM vesicles ("blebs") that are shed constitutively, but more prolifically, under adverse conditions including sublethal bacterial envelope injury as occurs early in infection, e.g., during interactions with BPI (23, 24, 25). The presence of innate immune-stimulating glycolipids (e.g., LPS/LOS, porins, lipoproteins) and other envelope antigenic components (e.g., OM proteins) may make these important vehicles for delivery of antigenic material to APCs as well as targets for clearance and resolution of endotoxin-driven inflammation (4).

We now show that, in addition to its potent antibiotic and endotoxin-neutralizing functions, BPI delivers OM vesicles to peripheral blood monocyte-derived dendritic cells (MDDC) where they are internalized but not to macrophages. Although BPI-dependent delivery of OM blebs to MDDC does not increase activation of these cells, it still permits LBP/CD14-dependent signaling. These findings raise the possibility that BPI and LBP have complementary rather than antagonistic roles in dendritic cell (DC) interactions with OM vesicles: BPI promotes delivery of OM Ags to MDDC, whereas LBP promotes MDDC activation needed for subsequent Ag processing, presentation, and T cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Recombinant human BPI and LBP were provided by XOMA. 4-Bromo-calcium ionophore (A23187) obtained from Calbiochem was used as a positive control in MDDC stimulation experiments. Recombinant human GM-CSF and IL-4 were obtained from R&D Systems. FBS for cell cultures was purchased from HyClone Laboratories. Fluorescently labeled mAbs against human CD1a, CD14, CD80, CD83, CD86, or HLA-DR and corresponding isotype controls were all from BD Biosciences and used according to the manufacturer’s instruction. The mouse anti-human lysosome-associated membrane protein-1 (LAMP-1) mAb (H4A3) was from the Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA). Tetramethylrhodamine isothiocyanate (TRITC)-conjugated IgG F(ab')2 secondary Ab was from Jackson ImmunoResearch Laboratories. ELISA kits (BD OptEIA; BD Biosciences) were used according to the manufacturer’s instructions to quantitate levels of human IL-8 and IP-10. For detection of human RANTES, a capture ELISA was established using the monoclonal mouse anti-human RANTES Ab (MAB678) as capture Ab and biotinylated anti-human RANTES polyclonal goat Ab for detection. Recombinant human RANTES was used as a standard (BD Biosciences). The RANTES ELISA was developed with the same reagents as the BD OptEIA ELISA kit.

Metabolic labeling and isolation of meningococcal LOS aggregates and blebs

An acetate auxotroph of Neisseria meningitidis serogroup B strain (26) was metabolically labeled using [14C]-acetic acid during growth as recently described (27). Bacteria were harvested at late log phase/early stationary phase and [14C]-LOS was purified from the bacterial pellet by hot phenol-water extraction, ethanol precipitation, followed by ultracentrifugation and resuspension of pelleted [14C]-LOS in water. 14C-labeled blebs were isolated from the conditioned bacterial culture medium by sterile filtration (0.22 µm), followed by ultrafiltration using a 100-kDa membrane cut-off and gel sieving chromatography of the retentate using a Sephacryl S-500 column (1.5 cm x 17 cm) (27). Purified [14C]-LOS was stored at 4°C; purified blebs were stored at –80°C and used only after one freeze-thaw cycle.

Fluorescent labeling of OM blebs

Freshly thawed intact 14C-labeled blebs were repurified by Sephacryl S-500 chromatography in PBS and concentrated by ultracentrifugation to a concentration of ~450 µg LOS/ml. The concentrated blebs were diluted in 0.1 M Na2CO3 buffer (pH 9.2), mixed with Alexa Fluor 488 tetrafluorophenyl (40 mg/ml in DMSO; Invitrogen Life Technologies) to a final concentration of 2 mg/ml Alexa Fluor 488 tetrafluorophenyl and then incubated for 1 h at 25°C with continuous stirring. Fluorescently labeled blebs were separated from free fluorescent dye by using a nucleic acid purification NAP-25 desalting column (Amersham Biosciences) equilibrated in PBS without calcium and magnesium. Fractions (0.5 ml) were collected and assessed for [14C]-LOS radioactivity by liquid scintillation spectroscopy and for Alexa Fluor 488 by absorbance at 488 nM to determine the concentration of [14C]-LOS and Alexa Fluor 488 in these fractions. The Alexa Fluor-labeled blebs contained ~1 µM Alexa Fluor 488 per micromole LOS. Labeled blebs were stored at 4°C in the dark until use.

Generation of monocyte-derived macrophages (MDM) and MDDC

Human MDM and MDDC were isolated and cultured following established methods (28, 29). Briefly, venous blood was drawn from healthy, adult volunteers in accordance with the University of Iowa Institutional Review Board guidelines. PBMC were isolated by density gradient centrifugation on Ficoll-Hypaque. For generation of MDM, PBMC were cultured in RPMI 1640 and 20% autologous serum in Teflon wells for 5 days at 37°C in 5% CO2. MDM were purified by adherence to tissue culture flasks for 2 h or more at 37°C in RPMI 1640, 20 mM HEPES (pH 7.4), and 10% autologous serum. MDM monolayers were washed twice with RPMI 1640 and then trypsinized, recovered in RPMI 1640, and transferred to Teflon wells (4 x 106 cells/ml) or 12-well tissue culture plates (5 x 105 cells/ml) for additional experiments. To obtain immature MDDC, purified PBMC were incubated in a 75-cm3 flask in RPMI 1640 for 1.5 h in a humidified incubator at 37°C. Nonadherent cells were removed by washing with warm RPMI 1640 three times, and adherent cells were cultured in DC medium (RPMI 1640 with L-glutamine (pH 7.35), essential and nonessential amino acids, 20% FBS), GM-CSF (100 ng/ml), and IL-4 (20 ng/ml). On day 5, immature DC were harvested using trypsin/EDTA (Cambrex). To confirm the purity and phenotype of the MDDC preparations used in our experiments, MDDC were incubated with mAb to CD1a, CD14, or CD83 or with isotype-matched control Abs. Cells were analyzed by flow cytometry on a FACSCalibur and examined by microscopy (Axioplan 2; Zeiss) (Fig. 1). Consistent with previous studies, CD1a was detected on all immature DC, whereas CD14 and CD83 surface expression was low. All results shown using MDM and MDDC represent data from cells derived from at least three different donors.


Figure 1
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  		FIGURE 1. Characterization of MDM and MDDC. Cells were generated as described in Materials and Methods. Results shown are representative of at least three experiments with three different donors. A, Light microscopy (x40 original magnification, Axioplan 2; Zeiss) of unstimulated MDM, MDDC, and MDDC incubated with 10 ng of LOS/ml for 24 h. B, Flow cytometry analysis was performed as described in Materials and Methods. Isotype control (gray line histogram) are shown in each panel. Unstimulated cells are shown for CD14 and CD1a (black line histogram) and for CD83 (dotted line histogram), and stimulated cells (10 ng of LOS/ml) are shown for CD83 (black line histogram).

 
Cell association of [14C]-LOS or 14C-labeled blebs

Immature MDDC and MDM were obtained as described, trypsinized, washed in RPMI 1640, and resuspended in small Teflon wells or 12-well tissue culture plates at a concentration of 4 x 106 cells/ml in 1 ml of RPMI 1640 supplemented with 20 mM HEPES, 1% human serum albumin (HSA) that was sterile-filtered through a 0.22-µm syringe filter before use. After recovery for 30 min, the cells were incubated with [14C]-LOS or blebs (with or without BPI or LBP in various concentrations in the presence or absence of 20% FBS, as indicated) in a total volume of 1.0 ml in small Teflon wells or 12-well tissue culture plates. After incubation for various times at 37°C on an orbital shaker in a 5% CO2 incubator, the cell suspensions were placed on ice for 30 min to facilitate detachment of MDDC and MDM from the surface of the wells. The cell suspension was removed from the wells, and the wells were washed with PBS (pH 7.4) supplemented with 20 mM HEPES and 1% HSA. The cell suspension and wash were combined in a polypropylene tube and centrifuged at 500 x g for 10 min at 4°C. The supernatant was immediately frozen at –80°C for subsequent detection of chemokines by ELISA. The cell pellet was washed once with 1 ml of PBS supplemented 20 mM HEPES and 1% HSA, resuspended in 200 µl of 5% SDS and 10 mM EDTA, and boiled for 10 min. Radioactivity in the reaction medium, cell washes, and cell lysate was determined by liquid scintillation spectroscopy (LS 6500 scintillation counter; Beckman Coulter), and the percentage of cell-associated [14C]-LOS or 14C-labeled blebs was calculated. Recovery of radioactivity was typically >90%.

Fatty acid analysis

To determine whether interaction of 14C-labeled blebs with MDDC resulted in delivery of [14C]-phospholipids and/or [14C]-LOS to MDDC, fatty acid analysis of associated radiolabeled material was performed as previously described (27). Briefly, 14C-labeled blebs recovered in washed MDDC cell pellets were treated sequentially with 4 N HCl and 4 N NaOH at 90°C to release ester- and amide-linked 14C-labeled fatty acids from the parent lipids. The treated samples were then extracted by the Bligh-Dyer procedure and the 14C-labeled free fatty acids were recovered in the chloroform phase. Individual 14C-labeled fatty acids were resolved by reverse-phase thin layer chromatography (0.2-mm HPTLC, RP-18; Merck) using acetonitrile/acetic acid (1:1, v/v) as the solvent system. Fatty acids were identified by comigration with authentic fatty acid standards and quantified by image analysis using a Typhoon imager and ImageQuant software (GE Healthcare). The fatty acid compositions of [14C]-LOS and [14C]-phospholipids from meningococcal blebs are almost completely different: [14C]-LOS contain almost exclusively 14C 3-OH-12:0, 12:0, and 3-OH-14:0 fatty acids, whereas the [14C]-phospholipids contain almost exclusively 14C 16:0, 16:1, 18:0, and 18:1 fatty acids (30). Therefore, analysis of the 14C-labeled fatty acids derived from the [14C]-lipids of blebs associated with MDDC provides a sensitive and quantitative analysis of the amount of [14C]-LOS and [14C]-phospholipids associated with MDDC.

Assays of stimulation of MDDC with [14C]-LOS or 14C-labeled blebs

MDDC (5 x 105/ml) in 12-well tissue culture plates were incubated for 24 h at 37°C, 5% CO2, and 95% humidity in RPMI 1640 with L-glutamine (pH 7.4), essential and nonessential amino acids, 20% FBS with various concentrations of [14C]-LOS or 14C-labeled blebs with or without BPI as indicated in the experiments. Cells were removed from the tissue culture plate using 1 mM EDTA and the recovered extracellular medium was stored at –80°C for later analysis of extracellular IL-8, RANTES, and IP-10 by ELISA. Live MDDC were stained with fluorescent Abs and isotype controls and kept at 4°C in the dark for assay of surface expression of MHC class II, CD83, CD80, and CD86 using flow cytometry (FACSCalibur; BD Biosciences) and FlowJo software (Tree Star). To block CD14 in serum, a mAb against CD14 (MY4; Beckman Coulter) was used at the indicated concentrations in DC medium containing 10% FBS. Serum was preincubated at 37°C for 30 min with MY4 or the corresponding murine isotype (IgG2b; eBioscience) before being added to the cell incubation medium.

Immunofluorescence microscopy and image processing

To evaluate internalization of blebs into DC, Alexa Fluor 488 14C-labeled blebs (100 ng of LOS/ml) were incubated with MDDC (4 x 106 cells/ml) in the presence or absence of BPI (100 nM) under conditions identical with the cell association assay described (2 h, 37°C, Teflon wells). Thereafter, the cells were washed twice in PBS to remove any unbound material, and MDDC were allowed to adhere to an eight-chamber microscopy slide for 20 min. Adherent MDDCs were washed in PBS, fixed in 10% formalin for 15 min at 25°C, and then permeabilized in 50% methanol/50% acetone for 5 min at 4°C as previously described (31). Fixed and permeabilized cells were rinsed in PBS supplemented with 0.5 g/L sodium azide and 5 g/L BSA, and then blocked in PBS containing 10% heat-inactivated normal horse serum (blocking buffer) for 1 h at 25°C (31). Following incubation with primary Abs (mouse anti-human LAMP-1 Ab (H4A3), rabbit polyclonal anti-BPI IgG (a gift from XOMA) or the corresponding murine or rabbit isotype diluted in blocking buffer) for 1 h at 25°C, cells were washed five times in PBS supplemented with 0.5 g/L sodium azide and 5 g/L BSA, and incubated with appropriate secondary Abs (TRITC-labeled anti-mouse or anti-rabbit IgG diluted in blocking buffer) for an additional hour. After five washes in PBS supplemented with sodium azide and BSA, the slide was detached from the chamber and mounted using the Molecular Probes SlowFade Light Antifade kit. Cells were viewed using a Zeiss LSM 510 confocal microscope.

Statistical analysis

Data from each experimental group were subjected to an analysis of normality and variance. Differences between experimental groups were analyzed for statistical significance using a two-tailed Student’s t test for paired samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BPI increases the delivery of N. meningitidis serogroup B LOS and OM blebs to MDDC, but not to MDM

We have previously demonstrated BPI-dependent association of purified endotoxin, LPS and LOS (17, 32), to CD14+ blood monocytes. Because interactions of endotoxin and BPI during Gram-negative bacteria infection occur mainly in tissue, we sought to extend the analyses to MDM and MDDC that should better resemble monocyte-derived cells in tissue. We compared cell association of purified aggregates of endotoxin, 14C-labeled meningococcal LOS, and 14C-labeled OM blebs shed from metabolically 14C-labeled meningococci during bacterial growth.

Fig. 2, A and B, shows that interactions with host cells of purified LOS and OM blebs with or without BPI were remarkably different in MDM and MDDC. In serum-free medium, both LOS aggregates and, to a lesser extent, OM blebs showed a level of cell association that was greater with MDM than with MDDC. Addition of BPI produced a dose-dependent inhibition of cell association of LOS aggregates with MDM but had little or no effect on interactions of OM blebs with MDM (Fig. 2A). In contrast, BPI increased association up to 3- to 5-fold of both LOS aggregates and OM blebs with MDDC (Fig. 2B). However, whereas the effect of BPI on interactions of LOS aggregates with MDDC was only seen at low BPI concentrations (i.e., low BPI/LOS molar ratio (32)), association of OM blebs with MDDC increased with increasing BPI concentrations. In contrast, to BPI, LBP did not increase cell association of OM blebs with MDDC and only modestly increased cell association of LOS aggregates (Fig. 2C). Similar results were seen following 1 or 2 h of incubation at 37°C. These findings demonstrate a selective effect of BPI in delivery of endotoxin-rich particles to host cells where MDDC are targeted more than MDM.


Figure 2
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  		FIGURE 2. Dose-dependent effects of BPI on cell association of Neisseria meningitidis serogroup B (NMB) LOS and OM blebs with MDM and MDDC. MDM (A) and immature MDDC (B and D) were incubated at a concentration of 4 x 106 cells/ml in the standard incubation medium with [14C]-LOS ({circ}) or 14C-labeled OM blebs (•) containing 10 ng of LOS/ml at 37°C for 2 h with or without the indicated concentrations of BPI or LBP (C). D, Incubations contained 20% serum. Cell association of [14C]-LOS or 14C-labeled OM blebs was measured as described in Materials and Methods. Results shown represent the mean ± SEM of data from at least three independent experiments, each in duplicate. E, Measurement of [14C]-LOS and [14C]-phospholipids (PL) from 14C-labeled OM blebs (100 ng of LOS/ml) associated with MDDC following incubation at 37°C for 2 h in the standard incubation medium ±20% serum. Cell associated [14C]-LOS and [14C]-phospholipids were assayed by fatty acid analysis as described in Materials and Methods. Incubation conditions were identical with those in A–D. Data shown represent the results from one experiment, representative of three similar experiments. Data are expressed as arbitrary OD units. Total cell-associated [14C]-lipids in the absence of BPI was set at 1. Experimental conditions in which the cell association of LOS aggregates or OM blebs in the presence of BPI were significantly (*, p < 0.05) different from those observed in the absence of BPI.

 
These initial experiments were conducted in serum-free medium containing 1% HSA to facilitate detection of BPI-dependent interactions. However, when BPI is mobilized following Gram-negative bacteria invasion of tissue, other endotoxin-interactive proteins, including LBP and soluble CD14, also accumulate and so potentially compete with BPI for interaction with the cell-free endotoxin-rich particles. We tested the effects of 20% serum to roughly approximate levels of plasma proteins transudated to tissue during local inflammatory responses (33). Under these experimental conditions, the stimulatory effects of BPI on association of LOS aggregates and OM blebs to MDDC were blunted at low (≤1 nM) BPI concentrations. However, at higher (≥10 nM) concentrations, BPI produced a significant dose-dependent increase in the association of OM blebs, but not the association of purified LOS, with MDDC that was comparable to the effects of similar concentrations of BPI in the absence of serum (Fig. 2, compare D to B). Serum alone had no effect on association of LOS aggregates or OM blebs with MDDC. Both [14C]-phospholipids and [14C]-LOS derived from the blebs were recovered in MDDC pellets (Fig. 2E), suggesting delivery of the full membrane content of the blebs to MDDC. The ratio of 14C-hydroxylated fatty acid (3-OH-C12:0 and 3-OH-C14:0) to 14C-nonhydroxylated fatty acid (C12:0) residues of LOS recovered in the MDDC pellet at 1 or 2 h was the same as the ratio present in the added blebs (data not shown), indicating little or no deacylation of LOS by acyloxyacyl hydrolase (34) during this time.

Internalization of OM blebs by MDDC in the presence of BPI

To determine whether blebs that become associated with MDDC in the presence of BPI were internalized, we used fluorescently labeled (Alexa Fluor 488) OM blebs and examined the localization of the cell-associated blebs after 2-h incubation with MDDC in the presence of BPI. As shown in Fig. 3, virtually all MDDC bound and internalized Alexa Fluor 488-labeled OM blebs. Analysis of serial (i.e., Z-series) sections of individual cells containing OM blebs confirmed colocalization of Alexa Fluor blebs with LAMP-1 (Fig. 3D). BPI was also internalized by MDDC under these conditions (Fig. 3C), apparently co-internalized with the OM blebs.


Figure 3
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  		FIGURE 3. Internalization of OM blebs by MDDC in the presence of BPI. MDDC were incubated in the standard incubation medium at a concentration of 4 x 106 cells/ml with Alexa Fluor 488-labeled OM blebs (green) (100 ng of LOS/ml) in the presence or absence of BPI (100 nM) as described in Materials and Methods. After spinning and washing, cells were allowed to attach to an eight-chamber glass slides at a final concentration of 5 x 105 cells/ml. Fixed and permeabilized MDDC were stained for LAMP-1 (A, B, and D) or BPI (C) using a TRITC-coupled anti-mouse secondary Ab (red). Cells were examined by confocal immunofluorescence microscopy using a Zeiss LSM 510 confocal microscope (original magnification, x60). Localization of OM blebs in MDDC by confocal microscopy shows detection of Alexa Fluor 488-labeled OM blebs (green) in the top left quadrant (A–C), localization of LAMP-1 (A and B) or BPI (C) (red) in the bottom left quadrant, and a merged image (A–C) in the bottom right quadrant. MDDC are shown using Normarski optics (top right quadrant). D, Merged images of Alexa Fluor 488-labeled blebs and LAMP-1 in a Z-series of a single cell are shown.

 
Effects of BPI on activation of MDDC by purified LOS or OM blebs

To examine the effect of BPI-enhanced delivery of N. meningitidis serogroup B OM blebs on the MDDC phenotype, we measured both cell surface and secretory MDDC activation markers. We compared the effects of purified LOS aggregates and OM blebs in the presence or absence of BPI in 20% serum on MDDC activation. This method permitted analysis of the effects of BPI on cell signaling that was either associated (OM blebs) or not associated (LOS aggregates) with BPI-enhanced-dependent delivery of the particles to MDDC. Fig. 4A shows that, at the equivalent of 10 ng of LOS/ml added, both purified LOS aggregates and OM blebs increased surface expression of CD80, CD86, HLA-DR, and CD83 on MDDC. The increased cell association of OM blebs induced by added BPI did not result in increased MDDC activation, as measured by surface expression of CD80, CD86, HLA-DR and CD83. In fact, the increased surface expression of these proteins induced by either LOS aggregates or by OM blebs was partially inhibited by 100 nM BPI but not by a lower (10 nM) BPI concentration (data not shown) that increased MDDC-OM bleb interactions to a similar extent (Fig. 2D). Activation of MDDC was further increased by exposure to higher concentrations of LOS aggregates (100 ng of LOS/ml, data not shown) or OM blebs (Fig. 4B). Under these conditions, addition of 100 nM BPI had no inhibitory effect on activation of MDDC induced by LOS aggregates (data not shown) or by the OM blebs (Fig. 4B). Similarly, both LOS aggregates and OM blebs (10 ng of LOS/ml) induced synthesis and secretion of IP-10, IL-8, and RANTES that was also partially inhibited at higher BPI concentrations (Fig. 5). BPI alone in the absence of endotoxin did not have any effect on the expression of CD83, HLA-DR, CD80, and CD86 or on chemokine secretion (data not shown).


Figure 4
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  		FIGURE 4. Comparison of activation of MDDC by purified LOS aggregates or OM blebs in the presence or absence of BPI. A, Immature MDDC were incubated either alone or with LOS or OM blebs (10 ng of LOS/ml) in the presence or absence of 100 nM BPI at a concentration of 5 x 105 cells/ml for 24 h followed by assaying cell surface expression of CD80, CD83, CD86, and HLA-DR by flow cytometry using Abs as described in Materials and Methods. Isotype control (thin gray histogram), unstimulated MDDC (black line histogram), and stimulated MDDC treated with LOS or blebs in the presence or absence of BPI (dotted line histogram) are shown. B, MDDC were incubated with OM blebs (100 ng of LOS/ml) in the presence or absence of 100 nM BPI for 24 h followed by assaying cell surface expression of CD80, CD83, CD86, and HLA-DR. Data shown represent one of at least three similar experiments. Unstimulated cells (black line histogram), cells treated with OM blebs (100 ng LOS/ml) (dotted line histogram), and cells treated with OM blebs (100 ng LOS/ml) and 100 nM BPI (thick gray line histogram), each stained with fluorescent Ab to the indicated surface marker as described in Materials and Methods, are shown.

 

Figure 5
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  		FIGURE 5. Activation of MDDC by purified LOS aggregates or OM blebs by chemokine production. MDDC at a concentration of 5 x 105 cells/ml were incubated with LOS and OM blebs (10 ng of LOS/ml) at the indicated BPI concentration for 24 h. Cells were harvested, and supernatants were collected and analyzed by ELISA as described in Materials and Methods. The results shown represent the mean ± SEM of at least three independent experiments. When LOS and OM blebs were used at a higher dose (100 ng of LOS/ml; data not shown) the presence of 100 nM BPI did not reduce cell activation. Statistically significant (*, p < 0.05) differences in cell responses between treated cells and untreated controls are indicated.

 
Activation of MDDC by OM blebs is CD14-dependent in the absence and presence of BPI

The results shown in Figs. 4 and 5 strongly suggest that although BPI promotes uptake of bacterial OM blebs by MDDC, this BPI-dependent interaction does not promote MDDC activation. However, it is possible that there is BPI-induced MDDC activation that is not appreciated because of coincident BPI-dependent inhibition of LBP/CD14-dependent signaling. To test whether BPI promotes CD14-independent activation of MDDC by OM blebs, we compared activation of MDDC exposed to OM blebs in the presence and absence of BPI in serum after pretreatment of serum with control or neutralizing Ab to CD14. As shown in Fig. 6, the activation of MDDC (i.e., increased surface CD83/86 expression and secretion of IL-8) induced by OM blebs in the presence or absence of BPI was nearly completely blocked by blocking CD14 function, strongly suggesting that BPI does not promote MDDC activation.


Figure 6
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  		FIGURE 6. Activation of MDDC by OM blebs is CD14-dependent in the absence or presence of BPI. MDDC at a concentration of 5 x 105 cells/ml were incubated in the standard incubation medium containing serum with or without OM blebs (100 ng of LOS/ml) in the presence or absence of BPI (100 nM) for 24 h as indicated. Surface expression of CD86 (mean fluorescence in unstimulated control: 3.3) (A and B) and of CD83 (mean fluorescence in unstimulated control: 126) (B) or extracellular accumulation of IL-8 (C) was determined. Where indicated, the anti-CD14 Ab MY4 or the corresponding isotype control was used at a concentration of 30 µg/ml as described in Materials and Methods. Data shown represent the mean ± SEM (*, p < 0.05) of either three independent experiments (B and C) or one experiment that is representative of three similar experiments (A). The stimulation of CD83/CD86 surface expression and the extracellular accumulation of IL-8 by the added OM blebs were virtually the same in the presence or absence of BPI. Statistically significant (p < 0.05) differences between bracketed samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrate a novel function of BPI: in an apparently selective manner, BPI increases delivery of extruded Gram-negative bacteria OM vesicles (blebs) to and uptake by human MDDC (Figs. 2 and 3). This effect of BPI resembles previously demonstrated effects with CD14+ human blood monocytes (17, 32). Remarkably, cultured macrophages derived from the same CD14+ monocytes as those yielding MDDC were apparently not targets of BPI-coated endotoxin-rich particles, either purified endotoxin aggregates or OM blebs (Fig. 2A). "Substrate" selectivity was also apparent, with BPI-dependent interactions with MDDC observed with OM blebs but not purified LOS aggregates over a broad BPI dose range (32) (Fig. 2B) and in the presence of serum (Fig. 2D). Further studies will be needed to determine the molecular basis of this cellular and substrate selectivity and whether this selectivity is apparent with primary populations of macrophages (35, 36) and DC (37).

BPI-dependent delivery of purified endotoxin, OM blebs, and intact Gram-negative bacteria is CD14-independent (17, 18), compatible with the very low levels of membrane-bound CD14 in immature DC (38) (Fig. 1). These very low levels and the lower efficiency of LBP interactions with OM-embedded vs purified endotoxin (27) may help explain the absence of LBP-dependent delivery of the OM blebs to MDDC, when LBP was added alone (Fig. 2C) or as a component of serum (Fig. 2D). The ability of BPI to promote OM bleb-MDDC interaction in the presence of 20% serum suggests that a similar capacity for BPI may be possible at tissue sites of infection and inflammation where potentially competing endotoxin-binding proteins such as LBP will also be present (30, 33, 39, 40). This potential likely reflects the very high affinity binding properties of BPI toward the Gram-negative bacterial OM that distinguishes it from LBP and other known host endotoxin-binding proteins (41, 42, 43). Extracellular BPI concentrations sufficient for a significant increase in the uptake of OM blebs by MDDC (≥10 nM) (Fig. 2D) have been documented in an experimental model of peritoneal inflammation (44).

Previous studies with CD14+ monocytes showed that CD14-independent delivery by BPI of purified endotoxin and OM blebs did not stimulate cell activation and could significantly inhibit the activation induced in the absence of BPI (17, 32). In contrast, uptake of BPI-opsonized intact bacteria stimulated luminol-ECL by phagocytosing neutrophils, reflecting coincident induction of the cellular respiratory burst and degranulation (18). Our findings in this study are consistent with and extend the former studies. However, because assays of MDDC activation required coculture of the cells with serum, we also observed an unexpected persistence of serum and LOS-dependent cellular activation even at relatively high BPI concentrations (Figs. 4 and 5). This activation, with or without BPI present, was CD14-dependent (Fig. 6), strongly suggesting that even with sufficient BPI present to increase association of the blebs with MDDC, (LBP)/CD14-dependent signaling remained possible. Although the OM blebs could contain other CD14-dependent agonists (45), the close similarity in the dose dependence (i.e., LOS) and CD14 dependence of MDDC activation by purified LOS and blebs and (partial) inhibition at higher BPI/LOS (bleb) ratios (Fig. 4) is most consistent with a predominant role of OM LOS in MDDC activation by the blebs (43, 46). The similar potency of purified LOS and blebs in these experiments contrasts with earlier experiments with endothelial cells and purified LBP and soluble CD14 in which LOS aggregates were 3–10 times more potent than the OM blebs (normalized by LOS content) in inducing cell activation (27). This difference may reflect a greater responsiveness of DC to other bioactive constituents of the OM blebs (e.g., OM proteins (47)) and/or a role of other serum components in activation of host cells by Gram-negative bacterial OM vesicles (48, 49, 50, 51).

Previous studies of BPI and endotoxin have focused on the cytotoxic and endotoxin-neutralizing functions of BPI and predicted an additional role of BPI in endotoxin clearance that would help resolve endotoxin-driven inflammation. However, the apparent absence of BPI-dependent delivery to macrophages and the limited (≤10% of total) BPI-dependent delivery of OM blebs to DC without accompanying endotoxin deacylation by acyloxyacyl hydrolase (34, 52) sufficient to antagonize cell signaling seems incompatible with a prominent role of BPI in bulk clearance and detoxification of remnants of Gram-negative bacteria. The selective delivery of OM blebs to DC suggests, instead, a different function for BPI, which is that of promotion of delivery of bacterial antigenic material to APCs. In this way, innate immune recognition of endotoxin by BPI may enhance the efficiency of induction of Ag-specific immune responses and thus promote the transition from innate to adaptive immune responses to Gram-negative bacterial infection. The demonstration, that the OM blebs are internalized into LAMP-1-positive compartments of MDDC in the presence of BPI (Fig. 3) is compatible with a route that leads ultimately to not only bacterial Ag uptake but also its processing and presentation. Recent studies have suggested the importance of delivery of antigenic material to the same compartment in which signaling is initiated for subsequent Ag presentation (53). Although BPI has much greater affinity than LBP for the Gram-negative bacterial OM (18, 39, 41, 54), we believe that the large number of endotoxin molecules/OM vesicles (each bleb contains hundreds of thousands of molecules of endotoxin; (27)) and the remarkable potency of endotoxin signaling (55) may permit interaction of both BPI and LBP (and soluble CD14) with the vesicle surface triggering both increased Ag uptake and CD14/MD-2/TLR4-dependent signaling from the same site (Fig. 7).


Figure 7
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  		FIGURE 7. Model of the role of BPI in the interaction of Gram-negative bacterial (GNB) OM blebs with MDDC. BPI promotes CD14-independent delivery of OM blebs to MDDC. In parallel, LBP and soluble CD14 (sCD14) promote activation of MDDC by extraction and delivery of endotoxin to MD-2/TLR4. The ability of BPI and LBP/sCD14 to interact with the same bleb could permit coincident enhancement of Ag delivery by BPI and LBP/sCD14-dependent cell activation and thereby promote subsequent Ag-presenting functions of the DC.

 
Autoantibodies to BPI have been documented in a select set of pathological circumstances, most prominently in cystic fibrosis, TAP deficiency, and inflammatory bowel diseases (56, 57, 58). Each of these conditions are associated with or preceded by highly acute or recurrent Gram-negative bacterial infection and neutrophilic inflammation. We speculate that the BPI-dependent interactions described in this study could result not only in increased bacterial Ag presentation when infection and host inflammatory responses are self-limited, but also presentation of BPI by activated DC when Gram-negative bacterial infection and neutrophilic inflammation are less well controlled. The extent to which extruded OM vesicles, as opposed to intact bacteria, are important vehicles for BPI-assisted Ag delivery and LBP/CD14-dependent DC activation awaits further study.


   Acknowledgments
 
We are indebted to Reitu Agrawal, PhD, Tara Hermann, PhD, Christopher Thompson, PhD, and Miranda Curtiss, BA, for help and support with macrophage and DC cultures and FACS analysis. We also gratefully acknowledge the help and support of Grant Schulert, BA, Yoko Nakano, PhD, and Lee-Ann Allen, PhD, with confocal microscopy and Tom Nelson with figures. We also thank Meta Kuehn, PhD, and Susanne Bauman, Department of Biochemistry, Duke University (Durham, NC) for help with OM bleb-labeling procedures.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants PO144642 and AI59372 from the U.S. Public Health Service (to J.P.W.). Back

2 Address correspondence and reprint requests to Dr. Jerrold P. Weiss, Roy J. and Lucille A. Carver College of Medicine, University of Iowa and the Veterans Affairs Medical Center, Inflammation Program, Oakdale Research Campus, 2501 Crosspark Road, Coralville, IA 52241. E-mail address: Jerrold-Weiss{at}uiowa.edu Back

3 Abbreviations used in this paper: BPI, bactericidal/permeability-increasing protein; LBP, LPS-binding protein; HSA, human serum albumin; LOS, lipo-oligosaccharide; MDM, monocyte-derived macrophage; DC, dendritic cell; MDDC, monocyte-derived DC; OM, outer membrane; LAMP-1, lysosome-associated membrane protein-1; TRITC, tetramethylrhodamine isothiocyanate. Back

Received for publication May 16, 2007. Accepted for publication May 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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