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The Journal of Immunology, 1998, 160: 5485-5492.
Copyright © 1998 by The American Association of Immunologists

The Role of CD14 in Signaling Mediated by Outer Membrane Lipoproteins of Borrelia burgdorferi 1

R. Mark Wooten*, Tom B. Morrison*, John H. Weis*, Samuel D. Wright{dagger}, Rolf Thieringer{dagger} and Janis J. Weis2,*

* Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132; and {dagger} Merck Research Laboratories, Rahway, NJ 07065


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Borrelia burgdorferi possesses membrane lipoproteins that exhibit stimulatory properties and, consequently, have been implicated in the pathology related to Lyme disease. As CD14 has been shown to mediate signaling by a number of lipid-modified bacterial products, the involvement of CD14 in signaling mediated by two B. burgdorferi lipoproteins, outer surface protein A (OspA) and OspC, was determined. Lipoprotein-mediated induction of nuclear factor-{kappa}B nuclear translocation and production of IL-8 and IL-6 in HUVEC was enhanced in the presence of serum or soluble rCD14. CD14-specific Abs that block LPS-mediated signaling also inhibited lipoprotein-dependent signaling in HUVEC and neutrophils. The formation of stable complexes between OspA and CD14 was demonstrated by native gel electrophoresis. LPS was found to compete with OspA for binding with CD14, suggesting that LPS and OspA bind similar regions on CD14. The similarity in binding was further supported by the finding that a mutant soluble CD14, lacking the LPS binding site, did not facilitate lipoprotein signaling, nor did it form a complex with OspA. Binding of OspA to CD14 was dependent on the lipid modification, as unlipidated OspA did not form a complex with CD14 or stimulate cells. In contrast, the lipopeptide remaining after proteinase K digestion both formed a complex with CD14 and retained stimulatory properties. These findings indicate that CD14 facilitates bacterial lipoprotein signaling in mammalian cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lyme disease is a multisystem disease caused by transmission of Borrelia burgdorferi spirochetes to a susceptible host via the bite of an infected tick (1). The primary disease is usually treatable with antibiotics; however, if misdiagnosed or left untreated, the localized infection may spread to a number of tissues, with joint, heart, and brain tissues showing the most frequent complications (2). In human and murine disease, pathology is usually related to the presence of bacteria in tissues (3, 4), suggesting that some bacterial product(s) might be responsible for eliciting the localized inflammation characteristic for the disease. B. burgdorferi have been shown to produce a number of membrane-associated lipoproteins that possess a tripalmitoyl-S-glycerylcysteine (Pam3Cys)3-modified amino terminus (5, 6, 7), a modification previously associated with stimulatory activities in a number of different bacterial lipoproteins (8, 9). While the stimulatory properties of one lipoprotein, OspA, have been studied extensively by several laboratories (10, 11, 12), it is presumed that other B. burgdorferi lipoproteins, many of which are preferentially expressed during infection of the mammal, possess similar activities (13, 14, 15). Several of these lipoproteins have been shown to directly stimulate a number of cell types responsible for mediating innate immune responses: these include induction of inflammatory mediators by macrophages (9, 10, 12, 16, 17), alterations in expression of adhesion molecules and priming for the production of superoxide by neutrophils (18, 19), induction of NF-{kappa}B nuclear translocation and up-regulation of downstream inflammatory events in endothelial cells (20, 21, 22, 23), activation of mast cells (24), and mitogenesis and Ig production in B cells (10, 17, 25). The inflammatory events induced by these lipoproteins suggest that the persistent localized presence of B. burgdorferi in tissues could lead to the characteristic pathology of Lyme disease.

Little is known regarding specific receptors that transduce the activation signals ascribed for bacterial lipoproteins, yet similarities between the cell types and inflammatory events, stimulated in response to bacterial LPS and B. burgdorferi lipoproteins, make it attractive to speculate whether common signaling mechanisms may exist for the two. B. burgdorferi do not contain LPS (26) and lipoprotein activity is not inhibited by polymyxin B (10, 20), indicating that the active moiety for B. burgdorferi lipoproteins differs from that of LPS. Additionally, cells from LPS-insensitive C3H/HeJ mice respond normally to OspA in vitro (10), and mice infected with B. burgdorferi develop arthritis, showing identical pathology and kinetics as that of C3H/HeN mice (4, 27). These findings suggest that OspA and LPS have distinct receptors; however, it is possible that they may utilize common factor(s) to facilitate receptor binding and cell activation.

One candidate molecule for facilitating lipoprotein-mediated signaling is CD14, a receptor involved in LPS-mediated signaling (28, 29, 30). This receptor exists as a 55-kDa protein attached to the membrane of neutrophils and monocytes/macrophages (mCD14) via a glycosylphosphatidylinositol linkage, or as a soluble protein in serum (sCD14). LPS-sensitive cells that lack mCD14, such as endothelial cells, become activated in response to preformed complexes of LPS-sCD14 (31, 32, 33, 34). While activation of LPS-sensitive cells can occur at high LPS concentrations in the absence of CD14, studies indicate that blockage or removal of CD14 decreases the sensitivity of cells to LPS by 100- to 10,000-fold (35, 36, 37). Therefore, CD14 does not appear to represent the specific receptor responsible for LPS signaling, but more likely acts as a coreceptor to facilitate LPS-mediated cell activation. CD14 has recently been determined to mediate cell activation by a number of bacterial products other than LPS (38, 39, 40, 41, 42, 43, 44, 45, 46). Several of these agonists possess lipid moieties that are responsible for their activities, suggesting that CD14 may be a candidate for mediating stimulatory activities of B. burgdorferi lipoproteins. In this study, the presence of sCD14 increased HUVEC sensitivity to B. burgdorferi lipoproteins by 20-fold, while blocking CD14 activity resulted in a 20-fold suppression in sensitivity. Further studies indicated OspA can form complexes with CD14, and that OspA interacts, via its lipid moiety, with a similar portion of CD14 as that responsible for CD14-LPS complex formation. These studies suggest that CD14 plays a role in facilitating lipoprotein-mediated signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant proteins

Lipidated rOspA from the B31 strain of B. burgdorferi (47) was provided by Robert Huebner (Connaught Laboratories, Swiftwater, PA). Purified OspA is a 31-kDa molecule possessing stimulatory properties similar to that of native OspA purified from B. burgdorferi (11), and contains a lipid moiety analogous with the Pam3Cys-modified structure (48, 49). A truncated OspA (uOspA) that lacks the signal peptidase II cleavage site (50) was provided by John Dunn (Brookhaven National Laboratories, Brookhaven, NY). The uOspA lacks the stimulatory properties associated with the lipidated OspA (11). Lipidated rOspC, a 23-kDa molecule, and truncated nonlipidated OspC (uOspC) from the Pko strain of B. burgdorferi (51, 52) were also provided by Robert Huebner. rOspA contained less than 0.3 endotoxin U/500 ng of protein by Limulus amebocyte lysate assay (1 endotoxin U equals approximately 200 pg of a reference Escherichia coli LPS; Associates of Cape Cod, Woods Hole, MA), while OspC contained 3 endotoxin U/500 ng protein.

Recombinant human sCD14 was expressed and purified as described elsewhere (P. A. Detmers, D. Zhou, E. Polizzi, R. Thieringer, S. Vaidya, and V. S. Bansal, manuscript in preparation). Briefly, Schneider-2 insect cells were transfected with the entire open reading frame encoding human CD14. Purified CD14 was at least 95% pure, as judged by SDS-PAGE, and appeared as a mixture of differently glycosylated forms between 45 and 48 kDa possessing the same biologic properties as sCD14 (unpublished observations). Endotoxin was less than 0.3 ng of LPS/mg of CD14 using LPS from Salmonella minnesota R595 (List Biologics, Campbell, CA) as the standard. A deletion mutant of CD14 that lacks amino acids 57–64 of the mature protein ({Delta}57–64CD14) was constructed as described by Juan et al. (53), and was expressed and purified using the same conditions as for the unmodified soluble protein. {Delta}57–64CD14 lacks a site shown to be essential for LPS-CD14 complex formation and for mediating LPS signaling ((53) and unpublished observations). Only the above described OspA, OspC, and CD14 recombinant proteins are used in these studies.

Reagents

CD14-specific Abs 60bca (54) and 26ic (54), and the isotype (IgG2b) control Ab 3G9F3 were purified by ammonium sulfate precipitation from the supernatant of cell lines obtained from American Type Culture Collection (Rockville, MD). My-4 (CD14-specific) and its isotype control B3 (IgG2b) were purchased from Coulter (Hialeah, FL). MEM-18 (CD14-specific) Ab was purchased from Sanbio (Uden, The Netherlands). Paired mAbs and recombinant standards for assaying IL-6 and IL-8 were purchased from Endogen (Woburn, MA). Avidin-horseradish peroxidase was purchased from Vector Laboratories (Burlingame, CA). Phycoerythrin-labeled anti-CD11b (M1/70HL) Ab and isotype control (IgG2b) were purchased from Becton Dickinson (La Jolla, CA). Human rTNF-{alpha} was purchased from Genzyme (Cambridge, MA). 125I-labeled protein A was purchased from NEN Life Sciences (Wilmington, DE). LPS from Salmonella typhi strain 14901 ({approx}10 kDa), FMLP, polyclonal rabbit anti-mouse IgG, and purified mouse IgG1 were obtained from Sigma (St. Louis, MO).

Proteinase K digestion

Proteinase K digestion was performed on OspA (pk-OspA) and uOspA (pk-uOspA), as previously described (10). Briefly, OspA or uOspA was suspended in 50 mM Tris-HCl, pH 8, containing 1 mM CaCl2, and proteinase K was added at 1 µg/2.5 µg of protein. After overnight incubation at 37°C, the reaction was stopped with 5 mM PMSF. Lipopeptide was recovered by chloroform-methanol extraction (5:10:4; chloroform:methanol:water), and the final product was resuspended in water. Recovery of lipopeptide was assumed to be 100%, which is supported by data showing that, on a molar basis, lipopeptide had the same activity as the initial intact lipoprotein (see Fig. 9Go). No intact lipoprotein was detected by silver staining of SDS-PAGE, nor was there a detectable lipopeptide (data not shown), suggesting that the remaining product was <=10 kDa.



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  		FIGURE 9. Effect of proteinase K-digested OspA on IL-8 production by HUVEC. The indicated modifications of OspA were added to HUVEC in medium containing 10% human serum. Supernatants were harvested after 24 h, and IL-8 levels were assessed by ELISA. Data points represent duplicate samples, and error bars are included unless obscured by symbols.

 
Cell culture

HUVEC were cultured as previously described (20). Briefly, HUVEC were obtained by digestion of umbilical veins with 0.1% collagenase (Worthington Biochemical, Freehold, NJ) and cultured in gelatin-coated 16-mm or 35-mm multiwell plates containing endothelial cell medium (medium 199 supplemented with 2 mM L-glutamine and 20% pooled human serum). Only primary cultures of monolayers that were tightly confluent were utilized in these studies.

Neutrophils were isolated from human blood as previously described (55). Briefly, erythrocytes were removed from heparinized blood by dextran sedimentation and hypotonic lysis. Polymorphonuclear leukocytes (PMN) were purified from the remaining cells by Ficoll-Paque density sedimentation and resuspended in HBSS containing 0.5% human serum albumin. Experiments involving neutrophils were performed within 4 h of PMN isolation.

All experiments involving cell culture were conducted in the presence of 10 µg/ml polymyxin B, except for samples stimulated with LPS.

Detection of nuclear NF-{kappa}B

Electrophoretic mobility shift assays (EMSA) for nuclear levels of NF-{kappa}B were performed as previously described (20). Briefly, HUVEC were treated with agonists for the indicated times before collecting the cells. Nuclei were subsequently isolated, and equal amounts of nuclear protein from each sample were incubated with a 32P-labeled NF-{kappa}B-specific probe (Promega, Madison, WI). The resulting complexes were resolved by electrophoresis on a 4% native polyacrylamide gel. Radiolabeled products were analyzed by autoradiography and quantified using a GS-250 molecular imager (Bio-Rad, Hercules, CA).

Detection of cytokines

HUVEC were treated with agonists for the indicated times before collecting supernatants. Supernatants were assayed for IL-8 and IL-6 content by sandwich ELISA, using paired mAbs. Cytokine levels were determined using a biotinylated detection Ab and avidin-horseradish peroxidase, and values were obtained using rIL-8 or rIL-6 standards.

Detection of surface CD11b

Agonists were added to 1 x 106 purified PMN for 1 h before stopping the reaction by adding ice-cold PBS containing 0.1% azide and 20% goat serum. Phycoerythrin-labeled anti-CD11b and isotype control were diluted in the same PBS solution and used to stain cells per the manufacturer’s recommendation. Cells were then fixed in 0.5% paraformaldehyde, and fluorescence was measured using a FACScan flow cytometer (Becton Dickinson). PMN were identified by forward and side scatter. Isotype controls showed no staining above background autofluorescence. Previous work has shown that neutrophils stimulated with these concentrations of LPS do not up-regulate CD11b in the absence of serum (19), presumably due to the absence of LBP. Therefore, 2% serum was added to LPS-stimulated cells, but not to those cells stimulated with OspA or FMLP.

Detection of CD14-containing complexes

CD14 was incubated with various compounds in PBS containing 5 mM EDTA overnight at 37°C. Complexes were detected after electrophoresis on 4 to 15% polyacrylamide (Bio-Rad) native gels. Proteins were transferred to polyvinylidene fluoride membranes, and Western blotting was performed using either 60bca (CD14) or H5332 (OspA) (56). Proteins were detected using polyclonal rabbit anti-mouse IgG and 125I-labeled protein A before visualizing by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum enhances the sensitivity of HUVEC to OspA

Nuclear translocation of the transcription factor NF-{kappa}B is a rapid event in human endothelial cell activation. While the B. burgdorferi lipoprotein OspA has been shown to potently and directly stimulate NF-{kappa}B translocation, we observed that removal of serum from HUVEC cultures followed by extensive washing decreased their ability to respond to OspA. Using EMSA analysis, a lipoprotein dose response indicated an increased sensitivity to OspA in the presence of 20% serum (Fig. 1Go). Phosphor image analyses of these gels indicated that HUVEC produce equivalent responses to 15- to 20-fold less OspA in serum-supplemented media compared with serum-free conditions. This was not due to a generalized decrease in responsiveness of these cells, as responses to TNF-{alpha} were not affected by the presence of serum. These results suggest that a component of serum increases the sensitivity of HUVEC to OspA.



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  		FIGURE 1. Effect of serum on OspA stimulation of HUVEC. HUVEC were washed five times with serum-free medium before adding the indicated amount of OspA for 70 min in the presence or absence of 20% human serum. Nuclear extracts were assayed for NF-{kappa}B content by EMSA, and results were analyzed by autoradiography. TNF-{alpha} was added at 500 U/ml.

 
CD14 Abs inhibit cell activation by B. burgdorferi lipoproteins

One serum component that has been shown to be important in endothelial cell signaling mediated by LPS and other bacterial products is sCD14; endothelial cells lack the membrane form of this molecule. The role of sCD14 was initially tested by incubating serum with a CD14-specific Ab, My-4, and determining its effect on NF-{kappa}B nuclear translocation. Incubation with My-4 or a matched isotype control Ab did not cause NF-{kappa}B translocation (Fig. 2GoA). LPS at 20 ng/ml strongly induced NF-{kappa}B translocation, and this level was reduced by My-4, but not by the isotype control. In other experiments, NF-{kappa}B translocation induced by 1 ng/ml LPS could be completely inhibited by My-4 (data not shown), consistent with reports indicating that responses to lower concentrations of LPS are more sensitive to the availability of CD14 (31). NF-{kappa}B translocation induced by OspA was completely inhibited by My-4 (Fig. 2GoA); however, partial inhibition was seen at concentrations greater than 100 ng/ml (data not shown). TNF-{alpha}-mediated signaling was not affected by My-4, indicating that TNF-{alpha} signaling is not mediated by CD14 and that the suppression seen in LPS- and OspA-mediated signaling was not a generalized effect. These results suggest that CD14 is the serum factor that increases sensitivity to OspA by HUVEC.



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  		FIGURE 2. Effects of CD14-specific Abs on endothelial cell activation. In A, the effect of CD14-specific blocking Ab on NF-{kappa}B nuclear translocation was determined. Medium containing 20% serum that had been preincubated without addition of Ab (-), with 5 µg/ml of an isotype control Ab (anti-CD22; c), or with 5 µg/ml of the CD14-specific Ab (My-4; {alpha}CD14) was added to washed HUVEC. Cells were then incubated with either medium alone, LPS (20 ng/ml), OspA (100 ng/ml), or TNF-{alpha} (500 U/ml) for 70 min. Nuclear extracts were prepared and assayed for NF-{kappa}B content by EMSA, and visualized by autoradiography. In B, the effect of CD14-specific Abs on IL-8 and IL-6 production by HUVEC was assessed. HUVEC were washed before adding medium containing 10% serum that had been preincubated with 10 µg/ml of either an isotype control (IgG1), a CD14-specific Ab that does not block LPS activation (26ic), or one of the CD14-specific Abs that block LPS activation (60bca and MEM-18). The indicated concentration of either OspA (upper) or OspC (lower) was added to these cells for 24 h before harvesting supernatants and assaying for IL-8 (left) or IL-6 (right) content by ELISA. These panels represent separate experiments performed on HUVEC from different donors and are representative of six experiments. Data points represent the average of duplicate wells, and error bars are indicated unless obscured by symbols.

 
We also determined the effect of CD14-specific Abs on the production of proteins whose transcription is dependent on NF-{kappa}B. A panel of Abs was tested for their effect on IL-8 and IL-6 production by HUVEC (Fig. 2GoB). Isotype controls and 26ic (an Ab that binds CD14, but does not block LPS activation of cells) (28, 31) had no effect on IL-8 production. Two CD14-specific Abs known to block LPS-mediated activation of cells, 60bca (28, 31) and MEM-18 (53), showed approximately 10-fold inhibition of OspA activation of HUVEC. A second B. burgdorferi lipoprotein, OspC, was also able to induce IL-8 and IL-6 production by endothelial cells (Fig. 2GoB), and was inhibited by the CD14-blocking Abs. Interestingly, 26ic partially inhibited the production of both IL-8 and IL-6 in response to OspC, although not to the extent of 60bca or MEM-18. This may reflect the fact that much of OspC is found as a disulfide-linked dimer (R. Huebner, personal communication), and is, therefore, bulkier and more susceptible to steric interference than the monomeric OspA. None of the CD14-specific Abs had any effect on induction of IL-8 or IL-6 by TNF-{alpha} (data not shown). These results indicate that blocking CD14 function decreases the sensitivity of HUVEC to Borrelia lipoproteins.

We have found that B. burgdorferi lipoproteins also potently stimulate murine macrophages and human neutrophils, both cell types that express mCD14 and are not dependent on serum for activation. The role of mCD14 in activation of neutrophils by B. burgdorferi lipoprotein was determined by assessing the effect of CD14 Abs on the up-regulation of surface-expressed CD11b (Fig. 3Go). LPS (<=100 ng/ml) activation of neutrophils was blocked completely by 60bca, consistent with its dependence on CD14 in this dose range. OspA activation was suppressed by approximately 10-fold using the blocking Ab 60bca, while the nonblocking Ab 26ic did not block neutrophil activation. Neutrophil activation by FMLP, another microbial agonist, was not affected by the addition of either Ab (data not shown). These findings suggest that lipoprotein-sensitive cells that possess mCD14 demonstrate a CD14-mediated sensitivity to OspA that is similar to that seen in endothelial cells. Also, LPS activation appears to be more dependent on CD14 than OspA activation, as LPS activation was blocked completely by 60bca, while OspA activation was only inhibited. Additionally, our previous observation that OspA activates neutrophils in the absence of serum suggests that LBP has no role in OspA-mediated signaling (19).



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  		FIGURE 3. Effects of CD14-specific Abs on CD11b expression by neutrophils. Purified neutrophils were resuspended in serum-free medium for OspA activation or medium containing 2% human serum for LPS activation. Cells were incubated with 5 µg/ml of the indicated CD14-specific Abs and then stimulated with the indicated concentrations of LPS or OspA for 1 h. Induced expression of CD11b was determined by flow cytometry after staining with phycoerythrin-conjugated Abs and compared with unstimulated controls. Symbols represent single data points from one of three separate experiments.

 
CD14 facilitates cell activation by B. burgdorferi lipoproteins

To determine whether CD14 is responsible for the serum-mediated enhancement of HUVEC responses to lipoproteins, rCD14 was added to HUVEC stimulated with either OspA or OspC in serum-free medium. CD14 alone increased sensitivity to OspA or OspC by 10- to 20-fold, as assessed by IL-8 ELISA (Fig. 4Go). LBP at concentrations up to 500 ng/ml had no effect on lipoprotein responses (data not shown), indicating that LBP does not provide a similar role for lipoproteins as that seen with LPS. Truncated recombinant OspA (uOspA) and OspC (uOspC), lacking only the lipid modification, did not stimulate IL-8 production by HUVEC (data not shown), irrespective of the presence of CD14. These findings confirm that CD14 is responsible for increasing the relative sensitivity of HUVEC to stimulation by lipoproteins; however, CD14 is not essential for signaling.



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  		FIGURE 4. Effects of rCD14 on lipoprotein-induced production of IL-8 by HUVEC. HUVEC were washed five times before adding the indicated concentrations of OspA or OspC in either serum-free medium, medium containing 1 µg/ml CD14, or 1 µg/ml {Delta}57–64CD14 ({Delta}CD14). After 24 h, supernatants were harvested and assayed for IL-8 content by ELISA. Data points represent duplicate samples, and error bars are included unless obscured by symbols. These panels represent separate experiments performed using HUVEC from different donors, and are representative of results from six experiments.

 
Addition of a deletion mutant of CD14 that lacks a sequence crucial to LPS binding ({Delta}57–64CD14) had no effect on lipoprotein sensitivity compared with the full-length CD14 (Fig. 4Go). This finding suggests that CD14 interacts with these lipoproteins in a similar fashion as with LPS.

OspA forms a stable complex with CD14

To determine whether CD14 and OspA could form stable complexes, equal concentrations of proteins (by mass) were incubated overnight and complexes were detected by electrophoresis on native gels and Western blotting using CD14-specific Ab. LPS or OspA alone was not observed by CD14-specific Western blot, and CD14 alone was seen as a diffuse band on native gels (Fig. 5Go). In the presence of LPS, CD14 formed a complex that migrated further into the gel, presumably due to the greater negative charge provided by LPS (34). OspA also formed a complex with CD14 that ran slower in the gel than LPS-CD14. Parallel blots developed using an OspA-specific Ab confirmed that OspA was present in the slower migrating complex and that the complex migrated distinct from uncomplexed OspA (data not shown). Neither uOspA nor human serum albumin formed complexes with CD14, suggesting that OspA-CD14 binding was specific and required the lipid moiety of OspA. The addition of an equal concentration of uOspA (Fig. 5Go, last lane) had no effect on the formation of the OspA-CD14 complex. In contrast, LPS at an equal concentration appeared to totally block CD14-OspA complex formation, resulting in presence of an LPS-CD14 complex only. These results indicate that OspA does form a complex with CD14 and that the lipid moiety is required for OspA-CD14 interaction. These results also suggest that LPS and OspA bind in a mutually exclusive fashion, perhaps to the same site.



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  		FIGURE 5. Complex formation between OspA and CD14. Compounds indicated above the gel (LPS, OspA, CD14, uOspA, or human serum albumin (HSA)) were prepared as 100 µg/ml stock solutions. Fifteen microliters of each were added to a reaction tube, as indicated (+). The total volume for each mixture was brought to 45 µl with buffer and incubated overnight. Twenty-five microliters of each mixture were electrophoresed on a native gel, and CD14-containing complexes were visualized by Western blotting using 60bca.

 
LPS and OspA compete for binding to CD14

In the previous experiment (Fig. 5Go), when LPS and OspA were added at equal concentrations based on mass, all of CD14 migrated as an LPS-CD14 complex. To address these relative binding affinities, equimolar amounts of OspA and CD14 were mixed in the presence of increasing amounts of LPS (Fig. 6Go). When an eightfold molar excess of LPS was added to an equimolar amount of OspA and CD14, all CD14 appeared to migrate at a rate consistent with an LPS-CD14 complex. As less LPS was added, the quantity of CD14 complexed with LPS lessened and an OspA-CD14 complex appeared. Phosphor image analysis of these complexes indicates that, when OspA, LPS, and CD14 were mixed at a 1:1:1 ratio, CD14 was roughly split between LPS-CD14 and OspA-CD14. These findings imply that LPS and OspA bind to a similar portion of CD14, and their binding affinities may be in the same range.



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  		FIGURE 6. Inhibition of OspA-CD14 complex formation by LPS. Complexes of OspA and CD14 were generated by mixing 10 µl each of a 2 µM solution, where indicated. LPS was also added as 10 µl per reaction mixture, but at a molar concentration ranging from 8-fold to 0.25-fold of that of OspA and CD14, as indicated. Reaction mixtures were incubated overnight before electrophoresing 25 µl of each sample on a native gel. CD14-containing complexes were visualized by Western blot using 60bca, and data are represented by autoradiography.

 
LPS and OspA bind a related site on CD14

To further determine whether LPS and OspA bind similar portions of CD14, OspA was preincubated with {Delta}57–64CD14, which is deficient in LPS binding, and the resulting complexes were assessed by Western blot (Fig. 7Go). As shown previously, both LPS and OspA form complexes with CD14; however, neither formed complexes with {Delta}57–64CD14. This is in agreement with the findings in Figure 4Go, in which {Delta}57–64CD14 was unable to facilitate lipoprotein signaling. These findings are consistent with OspA and LPS binding to the same or closely related sites on CD14.



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  		FIGURE 7. Importance of the LPS binding site on CD14-OspA complex formation. Ten microliters of 2 µM LPS or OspA were added as indicated to 10 µl of either 2 µM CD14 (+) or 2 µM {Delta}57–64CD14 ({Delta}) and incubated overnight (final volume = 20 µl). Mixtures were electrophoresed on a native gel, and CD14-containing complexes were visualized by Western blot using 60bca.

 
Lipid modification is essential for OspA binding to CD14

Earlier experiments (Figs. 4Go and 5Go) indicated that lipoproteins lacking only their Pam3Cys-modified amino terminus were unable to activate cells or form complexes with CD14, suggesting that the lipid modification was responsible for activity. To determine whether the lipid-modified peptide is sufficient for CD14 interaction, OspA and uOspA were digested with proteinase K (pk-OspA and pk-uOspA, respectively) and incubated with CD14 or LPS-CD14; the resulting complexes were assessed by Western blot. The addition of a fivefold molar excess of pk-OspA to CD14 resulted in a complex that migrated further into the gel than CD14 alone (Fig. 8GoA). pk-OspA was also able to interfere with the formation of LPS-CD14 complexes (Fig. 8GoB). pk-uOspA formed no detectable complex with CD14, nor was it able to inhibit LPS-CD14 complex formation. These results suggest that the lipid portion of OspA is responsible for OspA-CD14 complex formation.



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  		FIGURE 8. Interaction of proteinase K-digested OspA with CD14. A, Ten microliters of a 2 µM stock solution of CD14 were added to an equal or fivefold excess molar amount of either pk-OspA or pk-uOspA. After incubating overnight, mixtures were electrophoresed on a native gel and visualized by Western blot using 60bca. B, Same as in A, except that 10 µl of 2 µM LPS was added to each mixture.

 
Finally, since the lipid-modified amino terminus of OspA is sufficient for CD14 binding, we wanted to ascertain whether pk-OspA could activate HUVEC. At each concentration tested, OspA and pk-OspA appeared to elicit similar levels of IL-8 (Fig. 9Go), while neither uOspA (Fig. 9Go) nor pk-uOspA (data not shown) was able to induce these cytokines. These data suggest that the lipopeptide is sufficient both for interaction with CD14 and activation of lipoprotein-sensitive cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Lyme disease is characterized by inflammatory pathology that can affect multiple sites in the host (2). B. burgdorferi possess inherent stimulatory properties, believed to be mediated by spirochetal lipoproteins, which may be responsible for eliciting much of the pathology of Lyme disease (57, 58). As these lipoproteins stimulate similar cell types and induce similar inflammatory events to those mediated by LPS, we surmised that these bacterial products might share common mechanisms for signaling in sensitive cells. In this study, we determined whether the LPS coreceptor, CD14, might be involved in mediating the signaling events elicited by B. burgdorferi lipoproteins. This study is the first to report that bacterial lipoproteins can activate sensitive cells via pathways mediated by CD14. Addition of CD14-specific Abs that block LPS-mediated signaling decreased the sensitivity of HUVEC to stimulation by B. burgdorferi lipoproteins OspA and OspC 20-fold. This suppression appears to affect both early signaling events, such as NF-{kappa}B nuclear translocation, as well as late events, such as IL-8 and IL-6 production. CD14 appears to be solely responsible for this increased sensitivity, as addition of rCD14 under serum-free conditions increased the sensitivity of HUVEC to OspA and OspC 20-fold, similar to the increase seen for NF-{kappa}B nuclear translocation in the presence of serum. Cells that possess mCD14 also seem to mediate lipoprotein signaling via CD14, as neutrophils showed a reduced sensitivity to OspA after addition of CD14-blocking Ab. Therefore, both HUVEC (sCD14) and neutrophils (mCD14) appear to attain a greater sensitivity to B. burgdorferi lipoproteins by utilizing CD14 for signaling. However, both of these cell types were activated by lipoproteins when CD14 was either absent or blocked, albeit at increased lipoprotein concentrations. This indicates that CD14 does facilitate lipoprotein signaling, but it is not the ligand-specific receptor responsible for activation of lipoprotein-sensitive cells.

These studies also indicate that OspA directly interacts with CD14 in vitro, forming a unique complex that migrates independently of free OspA and CD14 on native gels. Complex formation with CD14 exhibited some degree of specificity and appeared to be lipid mediated, as uOspA was unable to bind CD14. The protein structure necessary for OspA to interact with CD14 appeared to be minimal, as proteinase K-digested OspA was able to efficiently complex with OspA, as well as inhibit LPS-CD14 complex formation; proteinase K-digested uOspA had no effect. This lipopeptide structure also was sufficient to stimulate HUVEC at similar concentrations as intact OspA.

Several aspects of OspA binding to CD14 appeared to be similar to LPS-CD14 interaction. OspA competed with LPS binding to CD14, and titration studies suggested that the relative binding affinities were similar. A CD14 deletion mutant that is deficient in LPS binding ({Delta}57–64CD14) (53) was also unable to bind OspA. In addition, a number of CD14-specific Abs known to inhibit cell activation in response to LPS also decreased the sensitivity of cells to Borrelia lipoproteins, while a CD14 Ab that does not inhibit LPS signaling had little effect on lipoprotein sensitivity. These findings indicate that LPS and OspA bind to similar portions of CD14 via their lipid moieties, resulting in an increased sensitivity to stimulation by sensitive cells. Recently, a number of other bacterial products have been shown to mediate their stimulatory activities through CD14 (39, 40, 41, 42, 43, 44, 45, 46). While the precise structural features responsible for recognition by CD14 are not known, nearly all of these microbial components contain lipid modifications. This observation is consistent with the finding that CD14 binds a wide spectrum of lipid species, including membrane phospholipids (59), and that CD14 can act as a phospholipid transfer protein (59) by shuttling mammalian phospholipids, as well as bacterial LPS, between various lipoproteins (60) and plasma membranes (61). In this capacity, it would be feasible for CD14 to bind spirochetal lipoproteins and transfer these molecules to the cell membranes of sensitive cells, where they are recognized by specific receptors capable of directly mediating lipoprotein signaling.

The role of CD14 in signaling by B. burgdorferi lipoproteins has previously been addressed by Norgard et al. (62), using a 70Z/3 pre-B cell line (CD14-) that had been transfected with human CD14 (CD14+). While 70Z/3 cells were shown to be unresponsive to LPS, OspA, or synthetic lipopeptides, the expression of CD14 allowed these cells to become responsive to LPS, but not OspA or synthetic lipopeptides. This indicates that CD14 alone was not sufficient to mediate lipoprotein signaling. Our findings confirm that CD14 is not the receptor directly responsible for transducing lipoprotein-mediated signaling, but rather suggests that CD14 works as it does for LPS by shuttling lipoproteins to cell membranes, where they can subsequently interact with the ligand-specific receptor(s). Therefore, any cell that lacks the lipoprotein-specific machinery responsible for signaling would remain unresponsive, even in the presence of CD14. Interestingly, this group has recently demonstrated that transfection of CD14 into a responsive cell does greatly increase the responsiveness to lipopeptides and lipoproteins (68).

Recently, a number of additional genes encoding the signal sequence for peptidase II cleavage and Pam3Cys-lipid modification have been identified for B. burgdorferi (14, 15), suggesting that these spirochetes can produce many distinct lipoproteins. Work by Schwan (13) and others suggests that these genes can be differentially expressed during various stages of infection (14, 15, 63, 64, 65, 66), subsequently changing the Ags present on the spirochete. Theoretically, even if antigenically distinct lipoprotein-encoding genes are selectively expressed during various stages of infection, they all should possess similar activities, as the lipid modification has been shown to be the moiety responsible for the stimulatory properties of OspA and other bacterial lipoproteins (10, 11, 47). This theory is strengthened by this study, the first to report that OspC, a lipoprotein that is up-regulated during the tick’s bloodmeal and expressed early in mammalian infection, possesses similar stimulatory properties as OspA for cells involved in inflammatory responses. The ability to change the antigenic makeup of lipoproteins on its surface while maintaining their stimulatory properties is one possible explanation for how B. burgdorferi could evade the host immune response while eliciting the inflammatory pathology characteristic of Lyme disease.

Interestingly, OspA is currently in trials as a vaccine candidate for B. burgdorferi infection (67). Lipid-modified OspA is highly immunogenic without requirement for further adjuvant, whereas the unlipidated recombinant is a poor immunogen (47). The findings presented in this work suggest a mechanism for the immunogenicity of OspA and other bacterial lipoproteins: interaction with mCD14 or sCD14 may facilitate Ag presentation by targeting to APCs or directly to B lymphocytes.


   Acknowledgments
 
We thank Robert Huebner for recombinant OspA, OspC, and uOspC; John Dunn for recombinant uOspA; Guy Zimmerman for providing human tissues; and Ying Ma for technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI-32223 (J.J.W.), AR-43521 (J.J.W.), and AI-24158 (J.H.W.), Grant 5P30-CA-42014 to the University of Utah, and an Arthritis Foundation Postdoctoral Fellowship (R.M.W.). The project described was also supported in part by an award from the American Lung Association (J.H.W.). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the American Lung Association. Back

2 Address correspondence and reprint requests to Dr. Janis J. Weis, University of Utah School of Medicine, Division of Cell Biology and Immunology, Department of Pathology, 50 North Medical Drive, Salt Lake City, UT 84132. E-mail address: Back

3 Abbreviations used in this paper: Pam3Cys, tripalmitoyl-S-glycerylcysteine; EMSA, electrophoretic mobility shift assay; LBP, lipopolysaccharide-binding protein; mCD14, membrane CD14; NF-{kappa}B, nuclear factor-{kappa}B; Osp, outer surface protein; pk-OspA, proteinase K-digested outer surface protein A; pk-uOspA, proteinase K-digested unlipidated outer surface protein A; PMN, polymorphonuclear leukocyte; sCD14, soluble CD14; uOsp, unlipidated outer surface protein. Back

Received for publication November 14, 1997. Accepted for publication January 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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