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*
Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132; and
Merck Research Laboratories, Rahway, NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
(
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. 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-
was purchased from Genzyme (Cambridge, MA).
125I-labeled protein A was purchased from NEN Life
Sciences (Wilmington, DE). LPS from Salmonella typhi strain
14901 (
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. 9
). 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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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-B
Electrophoretic mobility shift assays (EMSA) for nuclear levels
of NF-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-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nuclear translocation of the transcription factor NF-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-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. 1). 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- 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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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-B nuclear
translocation. Incubation with My-4 or a matched isotype control Ab did
not cause NF-B translocation (Fig. 2A). LPS at 20 ng/ml
strongly induced NF-B translocation, and this level was reduced by
My-4, but not by the isotype control. In other experiments, NF-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-B translocation induced by OspA was
completely inhibited by My-4 (Fig. 2A); however,
partial inhibition was seen at concentrations greater than 100 ng/ml
(data not shown). TNF--mediated signaling was not affected by My-4,
indicating that TNF- 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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B. A panel of Abs
was tested for their effect on IL-8 and IL-6 production by HUVEC (Fig. 2
B). 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. 2B), 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- (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. 3). 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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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. 4). 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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57–64CD14) had no effect on
lipoprotein sensitivity compared with the full-length CD14 (Fig. 4).
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. 5). 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. 5, 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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In the previous experiment (Fig. 5), 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. 6). 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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To further determine whether LPS and OspA bind similar portions of
CD14, OspA was preincubated with 57–64CD14, which
is deficient in LPS binding, and the resulting complexes were assessed
by Western blot (Fig. 7). As shown
previously, both LPS and OspA form complexes with CD14; however,
neither formed complexes with 57–64CD14. This is in
agreement with the findings in Figure 4, in which
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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Earlier experiments (Figs. 4 and 5) 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. 8A). pk-OspA was also
able to interfere with the formation of LPS-CD14 complexes (Fig. 8B). 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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),
while neither uOspA (Fig. 9) 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-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
(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.
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Acknowledgments |
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Footnotes |
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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:
3 Abbreviations used in this paper: Pam3Cys, tripalmitoyl-S-glycerylcysteine; EMSA, electrophoretic mobility shift assay; LBP, lipopolysaccharide-binding protein; mCD14, membrane CD14; NF-B, nuclear factor-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.
Received for publication November 14, 1997. Accepted for publication January 30, 1998.
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A. R. Cruz, M. W. Moore, C. J. La Vake, C. H. Eggers, J. C. Salazar, and J. D. Radolf Phagocytosis of Borrelia burgdorferi, the Lyme Disease Spirochete, Potentiates Innate Immune Activation and Induces Apoptosis in Human Monocytes Infect. Immun., January 1, 2008; 76(1): 56 - 70. [Abstract] [Full Text] [PDF]
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Z. Zhao, R. Fleming, B. McCloud, and M. S. Klempner CD14 Mediates Cross Talk between Mononuclear Cells and Fibroblasts for Upregulation of Matrix Metalloproteinase 9 by Borrelia burgdorferi Infect. Immun., June 1, 2007; 75(6): 3062 - 3069. [Abstract] [Full Text] [PDF]
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M. W. Moore, A. R. Cruz, C. J. LaVake, A. L. Marzo, C. H. Eggers, J. C. Salazar, and J. D. Radolf Phagocytosis of Borrelia burgdorferi and Treponema pallidum Potentiates Innate Immune Activation and Induces Gamma Interferon Production Infect. Immun., April 1, 2007; 75(4): 2046 - 2062. [Abstract] [Full Text] [PDF]
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J. J. Lazarus, M. J. Meadows, R. E. Lintner, and R. M. Wooten IL-10 Deficiency Promotes Increased Borrelia burgdorferi Clearance Predominantly through Enhanced Innate Immune Responses J. Immunol., November 15, 2006; 177(10): 7076 - 7085. [Abstract] [Full Text] [PDF]
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 J. M. Buckley, J. H. Wang, and H. P. Redmond Cellular reprogramming by gram-positive bacterial components: a review J. Leukoc. Biol., October 1, 2006; 80(4): 731 - 741. [Abstract] [Full Text] [PDF]
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 K. O. Omueti, J. M. Beyer, C. M. Johnson, E. A. Lyle, and R. I. Tapping Domain Exchange between Human Toll-like Receptors 1 and 6 Reveals a Region Required for Lipopeptide Discrimination J. Biol. Chem., November 4, 2005; 280(44): 36616 - 36625. [Abstract] [Full Text] [PDF]
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Q. Xu, S. V. Seemanapalli, L. Lomax, K. McShan, X. Li, E. Fikrig, and F. T. Liang Association of Linear Plasmid 28-1 with an Arthritic Phenotype of Borrelia burgdorferi Infect. Immun., November 1, 2005; 73(11): 7208 - 7215. [Abstract] [Full Text] [PDF]
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R. C. Garcia, R. Murgia, and M. Cinco Complement Receptor 3 Binds the Borrelia burgdorferi Outer Surface Proteins OspA and OspB in an iC3b-Independent Manner Infect. Immun., September 1, 2005; 73(9): 6138 - 6142. [Abstract] [Full Text] [PDF]
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 J. C. Salazar, C. D. Pope, M. W. Moore, J. Pope, T. G. Kiely, and J. D. Radolf Lipoprotein-Dependent and -Independent Immune Responses to Spirochetal Infection Clin. Vaccine Immunol., August 1, 2005; 12(8): 949 - 958. [Abstract] [Full Text] [PDF]
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S. Ghosh, A. C. Steere, B. D. Stollar, and B. T. Huber In Situ Diversification of the Antibody Repertoire in Chronic Lyme Arthritis Synovium J. Immunol., March 1, 2005; 174(5): 2860 - 2869. [Abstract] [Full Text] [PDF]
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M. R.-E.-I. Benhnia, D. Wroblewski, M. N. Akhtar, R. A. Patel, W. Lavezzi, S. C. Gangloff, S. M. Goyert, M. J. Caimano, J. D. Radolf, and T. J. Sellati Signaling through CD14 Attenuates the Inflammatory Response to Borrelia burgdorferi, the Agent of Lyme Disease J. Immunol., February 1, 2005; 174(3): 1539 - 1548. [Abstract] [Full Text] [PDF]
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T. Vasselon, P. A. Detmers, D. Charron, and A. Haziot TLR2 Recognizes a Bacterial Lipopeptide through Direct Binding J. Immunol., December 15, 2004; 173(12): 7401 - 7405. [Abstract] [Full Text] [PDF]
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S. Noubir, Z. Hmama, and N. E. Reiner Dual Receptors and Distinct Pathways Mediate Interleukin-1 Receptor-associated Kinase Degradation in Response to Lipopolysaccharide: INVOLVEMENT OF CD14/TLR4, CR3, AND PHOSPHATIDYLINOSITOL 3-KINASE J. Biol. Chem., June 11, 2004; 279(24): 25189 - 25195. [Abstract] [Full Text] [PDF]
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X. Zhou, D. Q. Gao, J. Michalski, J. A. Benitez, and J. B. Kaper Induction of Interleukin-8 in T84 Cells by Vibrio cholerae Infect. Immun., January 1, 2004; 72(1): 389 - 397. [Abstract] [Full Text] [PDF]
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 D. Heumann, R. Lauener, and B. Ryffel The dual role of LBP and CD14 in response to Gram-negative bacteria or Gram-negative compounds Innate Immunity, December 1, 2003; 9(6): 381 - 384. [Abstract] [PDF]
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J. L. Coleman and J. L. Benach The Urokinase Receptor Can Be Induced by Borrelia burgdorferi through Receptors of the Innate Immune System Infect. Immun., October 1, 2003; 71(10): 5556 - 5564. [Abstract] [Full Text] [PDF]
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J. C. Salazar, C. D. Pope, T. J. Sellati, H. M. Feder Jr, T. G. Kiely, K. R. Dardick, R. L. Buckman, M. W. Moore, M. J. Caimano, J. G. Pope, et al. Coevolution of Markers of Innate and Adaptive Immunity in Skin and Peripheral Blood of Patients with Erythema Migrans J. Immunol., September 1, 2003; 171(5): 2660 - 2670. [Abstract] [Full Text] [PDF]
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 S. Cepok, D. Zhou, F. Vogel, B. Rosche, V. Grummel, N. Sommer, and B. Hemmer The Immune Response at Onset and During Recovery From Borrelia burgdorferi Meningoradiculitis Arch Neurol, June 1, 2003; 60(6): 849 - 855. [Abstract] [Full Text] [PDF]
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L. A. Augusto, M. Synguelakis, J. Johansson, T. Pedron, R. Girard, and R. Chaby Interaction of Pulmonary Surfactant Protein C with CD14 and Lipopolysaccharide Infect. Immun., January 1, 2003; 71(1): 61 - 67. [Abstract] [Full Text] [PDF]
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J. H. Wang, M. Doyle, B. J. Manning, Q. Di Wu, S. Blankson, and H. P. Redmond Induction of Bacterial Lipoprotein Tolerance Is Associated with Suppression of Toll-like Receptor 2 Expression J. Biol. Chem., September 20, 2002; 277(39): 36068 - 36075. [Abstract] [Full Text] [PDF]
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J. Anguita, S. W. Barthold, R. Persinski, M. N. Hedrick, C. A. Huy, R. J. Davis, R. A. Flavell, and E. Fikrig Murine Lyme Arthritis Development Mediated by p38 Mitogen-Activated Protein Kinase Activity J. Immunol., June 15, 2002; 168(12): 6352 - 6357. [Abstract] [Full Text] [PDF]
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D. Diament, M. K. C. Brunialti, E. C. Romero, E. G. Kallas, and R. Salomao Peripheral Blood Mononuclear Cell Activation Induced by Leptospira interrogans Glycolipoprotein Infect. Immun., April 1, 2002; 70(4): 1677 - 1683. [Abstract] [Full Text] [PDF]
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G. H. Giambartolomei, V. A. Dennis, B. L. Lasater, P. K. Murthy, and M. T. Philipp Autocrine and Exocrine Regulation of Interleukin-10 Production in THP-1 Cells Stimulated with Borrelia burgdorferi Lipoproteins Infect. Immun., April 1, 2002; 70(4): 1881 - 1888. [Abstract] [Full Text] [PDF]
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T. Vasselon, W. A Hanlon, S. D Wright, and P. A. Detmers Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38 J. Leukoc. Biol., March 1, 2002; 71(3): 503 - 510. [Abstract] [Full Text] [PDF]
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R. N. Fichorova, A. O. Cronin, E. Lien, D. J. Anderson, and R. R. Ingalls Response to Neisseria gonorrhoeae by Cervicovaginal Epithelial Cells Occurs in the Absence of Toll-Like Receptor 4-Mediated Signaling J. Immunol., March 1, 2002; 168(5): 2424 - 2432. [Abstract] [Full Text] [PDF]
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R. M. Wooten, Y. Ma, R. A. Yoder, J. P. Brown, J. H. Weis, J. F. Zachary, C. J. Kirschning, and J. J. Weis Toll-Like Receptor 2 Is Required for Innate, But Not Acquired, Host Defense to Borrelia burgdorferi J. Immunol., January 1, 2002; 168(1): 348 - 355. [Abstract] [Full Text] [PDF]
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P. O. Neilsen, G. A. Zimmerman, and T. M. McIntyre Escherichia coli Braun Lipoprotein Induces a Lipopolysaccharide-Like Endotoxic Response from Primary Human Endothelial Cells J. Immunol., November 1, 2001; 167(9): 5231 - 5239. [Abstract] [Full Text] [PDF]
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A. O. Aliprantis, D. S. Weiss, J. D. Radolf, and A. Zychlinsky Release of Toll-Like Receptor-2-Activating Bacterial Lipoproteins in Shigella flexneri Culture Supernatants Infect. Immun., October 1, 2001; 69(10): 6248 - 6255. [Abstract] [Full Text] [PDF]
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A. O. Aliprantis, D. S. Weiss, and A. Zychlinsky Toll-like receptor-2 transduces signals for NF-{kappa}B activation, apoptosis and reactive oxygen species production Innate Immunity, August 1, 2001; 7(4): 287 - 291. [Abstract] [PDF]
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R. R. Ingalls, E. Lien, and D. T. Golenbock Membrane-Associated Proteins of a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis Activate the Inflammatory Response through Toll-Like Receptor 2 Infect. Immun., April 1, 2001; 69(4): 2230 - 2236. [Abstract] [Full Text] [PDF]
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S. D. C. Pfannes, B. Müller, S. Körner, W. G. Bessler, and P. Hoffmann Induction of soluble antitumoral mediators by synthetic analogues of bacterial lipoprotein in bone marrow-derived macrophages from LPS-responder and -nonresponder mice J. Leukoc. Biol., April 1, 2001; 69(4): 590 - 597. [Abstract] [Full Text]
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T. Sugiyama and S. D. Wright Soluble CD14 Mediates Efflux of Phospholipids from Cells J. Immunol., January 15, 2001; 166(2): 826 - 831. [Abstract] [Full Text] [PDF]
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D. A. Bouis, T. G. Popova, A. Takashima, and M. V. Norgard Dendritic Cells Phagocytose and Are Activated by Treponema pallidum Infect. Immun., January 1, 2001; 69(1): 518 - 528. [Abstract] [Full Text] [PDF]
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P. K. Murthy, V. A. Dennis, B. L. Lasater, and M. T. Philipp Interleukin-10 Modulates Proinflammatory Cytokines in the Human Monocytic Cell Line THP-1 Stimulated with Borrelia burgdorferi Lipoproteins Infect. Immun., December 1, 2000; 68(12): 6663 - 6669. [Abstract] [Full Text] [PDF]
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R. R. Ingalls, E. Lien, and D. T. Golenbock Differential roles of TLR2 and TLR4 in the host response to Gram-negative bacteria: lessons from a lipopolysaccharide-deficient mutant of Neisseria meningitidis Innate Immunity, October 1, 2000; 6(5): 411 - 415. [Abstract] [PDF]
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S. F. Porcella, C. A. Fitzpatrick, and J. L. Bono Expression and Immunological Analysis of the Plasmid-Borne mlp Genes of Borrelia burgdorferi Strain B31 Infect. Immun., September 1, 2000; 68(9): 4992 - 5001. [Abstract] [Full Text] [PDF]
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N. W. J. Schroder, B. Opitz, N. Lamping, K. S. Michelsen, U. Zahringer, U. B. Gobel, and R. R. Schumann Involvement of Lipopolysaccharide Binding Protein, CD14, and Toll-Like Receptors in the Initiation of Innate Immune Responses by Treponema Glycolipids J. Immunol., September 1, 2000; 165(5): 2683 - 2693. [Abstract] [Full Text] [PDF]
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 D. A. Haake Spirochaetal lipoproteins and pathogenesis Microbiology, July 1, 2000; 146(7): 1491 - 1504. [Full Text]
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M. D. Cunningham, R. A. Shapiro, C. Seachord, K. Ratcliffe, L. Cassiano, and R. P. Darveau CD14 Employs Hydrophilic Regions to ""Capture"" Lipopolysaccharides J. Immunol., March 15, 2000; 164(6): 3255 - 3263. [Abstract] [Full Text] [PDF]
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A. M. Soler-Rodriguez, H. Zhang, H. S. Lichenstein, N. Qureshi, D. W. Niesel, S. E. Crowe, J. W. Peterson, and G. R. Klimpel Neutrophil Activation by Bacterial Lipoprotein Versus Lipopolysaccharide: Differential Requirements for Serum and CD14 J. Immunol., March 1, 2000; 164(5): 2674 - 2683. [Abstract] [Full Text] [PDF]
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I. G. Scragg, D. Kwiatkowski, V. Vidal, A. Reason, T. Paxton, M. Panico, A. Dell, and H. Morris Structural Characterization of the Inflammatory Moiety of a Variable Major Lipoprotein of Borrelia recurrentis J. Biol. Chem., January 14, 2000; 275(2): 937 - 941. [Abstract] [Full Text] [PDF]
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 Z. Hmama, D. Nandan, L. Sly, K. L. Knutson, P. Herrera-Velit, and N. E. Reiner 1{alpha},25-Dihydroxyvitamin D3-Induced Myeloid Cell Differentiation Is Regulated by a Vitamin D Receptor-Phosphatidylinositol 3-Kinase Signaling Complex J. Exp. Med., December 6, 1999; 190(11): 1583 - 1594. [Abstract] [Full Text] [PDF]
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E. Lien, T. J. Sellati, A. Yoshimura, T. H. Flo, G. Rawadi, R. W. Finberg, J. D. Carroll, T. Espevik, R. R. Ingalls, J. D. Radolf, et al. Toll-like Receptor 2 Functions as a Pattern Recognition Receptor for Diverse Bacterial Products J. Biol. Chem., November 19, 1999; 274(47): 33419 - 33425. [Abstract] [Full Text] [PDF]
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J. P. Brown, J. F. Zachary, C. Teuscher, J. J. Weis, and R. M. Wooten Dual Role of Interleukin-10 in Murine Lyme Disease: Regulation of Arthritis Severity and Host Defense Infect. Immun., October 1, 1999; 67(10): 5142 - 5150. [Abstract] [Full Text] [PDF]
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M. Hirschfeld, C. J. Kirschning, R. Schwandner, H. Wesche, J. H. Weis, R. M. Wooten, and J. J. Weis Cutting Edge: Inflammatory Signaling by Borrelia burgdorferi Lipoproteins Is Mediated by Toll-Like Receptor 2 J. Immunol., September 1, 1999; 163(5): 2382 - 2386. [Abstract] [Full Text] [PDF]
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T. J. Sellati, D. A. Bouis, M. J. Caimano, J. A. Feulner, C. Ayers, E. Lien, and J. D. Radolf Activation of Human Monocytic Cells by Borrelia burgdorferi and Treponema pallidum Is Facilitated by CD14 and Correlates with Surface Exposure of Spirochetal Lipoproteins J. Immunol., August 15, 1999; 163(4): 2049 - 2056. [Abstract] [Full Text] [PDF]
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R. I. Tapping, S. L. Orr, E. M. Lawson, K. Soldau, and P. S. Tobias Membrane-Anchored Forms of Lipopolysaccharide (LPS)-Binding Protein Do Not Mediate Cellular Responses to LPS Independently of CD14 J. Immunol., May 1, 1999; 162(9): 5483 - 5489. [Abstract] [Full Text] [PDF]
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S. Sugawara, R. Arakaki, H. Rikiishi, and H. Takada Lipoteichoic Acid Acts as an Antagonist and an Agonist of Lipopolysaccharide on Human Gingival Fibroblasts and Monocytes in a CD14-Dependent Manner Infect. Immun., April 1, 1999; 67(4): 1623 - 1632. [Abstract] [Full Text] [PDF]
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J. Talkington and S. P. Nickell Borrelia burgdorferi Spirochetes Induce Mast Cell Activation and Cytokine Release Infect. Immun., March 1, 1999; 67(3): 1107 - 1115. [Abstract] [Full Text] [PDF]
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G. H. Giambartolomei, V. A. Dennis, B. L. Lasater, and M. T. Philipp Induction of Pro- and Anti-Inflammatory Cytokines by Borrelia burgdorferi Lipoproteins in Monocytes Is Mediated by CD14 Infect. Immun., January 1, 1999; 67(1): 140 - 147. [Abstract] [Full Text] [PDF]
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J. Garcia, B. Lemercier, S. Roman-Roman, and G. Rawadi A Mycoplasma fermentans-derived Synthetic Lipopeptide Induces AP-1 and NF-kappa B Activity and Cytokine Secretion in Macrophages via the Activation of Mitogen-activated Protein Kinase Pathways J. Biol. Chem., December 18, 1998; 273(51): 34391 - 34398. [Abstract] [Full Text] [PDF]
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