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Departments of
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Internal Medicine and
Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235;
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121;
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Medical Intensive Care Unit, University of Geneva, Geneva, Switzerland;
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Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and
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Department of Molecular Medicine, North Shore University Hospital/Cornell University Medical College, Manhasset, NY 11030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Investigation of the immunomodulatory properties of spirochetal lipoproteins is complicated by the difficulty in obtaining adequate quantities of these molecules free of endotoxin contamination. As an extension of findings by Bessler and co-workers (6, 8), we have shown that synthetic lipohexapeptides corresponding to the N termini of spirochetal lipoproteins have in vitro proinflammatory properties that mimic those of their full-length, acylated counterparts (11, 14, 15, 16, 18). Moreover, following intradermal injection, synthetic lipopeptides elicited histopathologic changes in mice and rabbits that closely resembled those observed during natural or experimental syphilis and Lyme disease, further substantiating their utility as lipoprotein surrogates (20).
Cells of the innate immune system exhibit an intrinsic ability to recognize the cell wall constituents of bacterial and fungal pathogens (21, 22, 23, 24, 25, 26, 27). In this regard, the biologic effects of LPS have been intensively investigated because of the central role played by this highly potent glycolipid in the pathophysiology of sepsis and septic shock by Gram-negative bacteria (28). According to the current paradigm, activation of monocytes/macrophages is initiated when LPS binds to membrane CD14 (mCD14)4 (29, 30), a 55-kDa glycosylphosphatidylinositol-anchored protein that lacks both transmembrane and cytoplasmic domains (31). The serum component known as LPS-binding protein (LBP) acts in a catalytic fashion to facilitate the binding of LPS to CD14 (29, 30, 32, 33). A signaling cascade ensues when the CD14-bound LPS presumably interacts with an as yet uncharacterized signal transducing element (27, 34). In addition to its membrane-bound form, which is found only on cells of myeloid lineage, CD14 exists as a soluble serum protein (sCD14) that mediates LPS signaling in cells lacking CD14 (35, 36, 37).
While CD14 has been shown to mediate the activation of monocytic cells by bacterial cell wall constituents other than LPS (21, 22, 23, 24, 25, 26, 27), its contribution to lipoprotein/lipopeptide signaling is unclear. Supporting its involvement is the observation that maturation of human myelomonocytic THP-1 cells (38) with 1,25-dihydroxyvitamin D3 (vitamin D3) markedly enhanced their responsiveness to lipoproteins and lipopeptides just as it does for LPS (16). It had previously been shown that vitamin D3 maturation enhanced the responsiveness of THP-1 cells to LPS at least in part by inducing the surface expression of CD14 (39, 40). Arguing against a role for CD14 is the observation that transfection with CD14 rendered murine pre-B 70Z/3 cells exquisitely sensitive to LPS (41) but failed to confer responsiveness to spirochetal lipoproteins and lipopeptides (16).
The objective of the present study was to clarify the role of CD14 in the activation of monocytic cells by spirochetal lipoproteins and lipopeptides. Herein, we report that cellular activation by these spirochetal constituents proceeds predominantly via CD14, although a CD14-independent pathway also was discernible. Of particular interest was our finding that fundamental differences exist in the CD14-dependent signaling pathways induced by LPS and spirochetal lipoproteins/lipopeptides. In addition to providing new insights into the pathogenesis of syphilis and Lyme disease, these findings are potentially relevant to mechanisms of immune effector cell activation by other non-LPS bacterial proinflammatory agonists.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Salmonella minnesota R5 LPS (Sigma, St. Louis, MO), suspended in PBS containing 0.03% BSA (low endotoxin; catalogue no. A4919; Sigma) or S. minnesota wild-type LPS (List Biologics, Campbell, CA) were used as positive controls in cell stimulation assays. E. coli LCD25 LPS was enzymatically deacylated (dLPS) as previously described (42). 1,25-Dihydroxyvitamin D3 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). G-418 sulfate was purchased from Mediatech (Herndon, VA). Great care was taken throughout to minimize contamination by environmental LPS during the preparation of all buffers and reagents by using baked glassware, disposable plasticware, and pyrogen-free H2O.
Purification of native OspA (nOspA) from B. burgdorferi and recombinant, nonacylated OspA
B. burgdorferi strain TI1-EV, generously provided by
Jorge L. Benach (State University of New York, Stony Brook, NY), was
used as the source for nOspA. Lipoprotein was affinity purified as
previously described (16) and was stored in 33 mM Tris (pH 7.4), 1.6 mM
NaCl, and 20 mM n-octyl-ß-glucoside at -70°C.
Preparations of nOspA contained 
12 pg LPS/µg of protein as measured
by the QCL-1000 quantitative chromogenic Limulus amebocyte
lysate assay (BioWhittaker, Walkersville, MD). Nonacylated recombinant
OspA was generated by PCR amplification of the ospA gene
from B. burgdorferi TI1-EV and cloned into the expression
vector pGEX-2T as previously described (43). The recombinant OspA was
recovered following cleavage of the glutathione
S-transferase fusion protein with thrombin.
Synthetic hexapeptides and lipohexapeptides corresponding to the N termini of the spirochetal lipoproteins
Hexapeptides corresponding to the N termini of the B.
burgdorferi strain B31 OspA lipoprotein (Cys-Lys-Gln-Asn-Val-Ser)
(44) and the T. pallidum subspecies pallidum
47-kDa lipoprotein (Cys-Gly-Ser-Ser-His-His) (45) were synthesized on
an Applied Biosystems (Foster City, CA) 430A peptide synthesizer using
standard 9-fluorenylmethoxycarbonyl chemistry as recommended by the
manufacturer. For use in cell stimulation assays, quantities of
lyophilized hexapeptides were solubilized by vortexing in sterile,
pyrogen-free H2O. Lipohexapeptides corresponding to the
acylated N termini of the lipoproteins were synthesized as
tripalmitoyl-S-glycerylcysteine derivatives using a solid
phase synthesis procedure (11). Hexapeptides and lipopeptides contained
undetectable levels of endotoxin (1 pg LPS/µg protein) as measured
by the QCL-1000 quantitative chromogenic Limulus amebocyte
lysate assay.
Cell lines
The human promyelomonocytic cell line THP-1 (38) was maintained in RPMI 1640 medium (Mediatech) containing 2 mM L-glutamine and supplemented with 10% heat-inactivated FBS (HIFBS; heated for 30 min at 56°C; Mediatech), 100 U of penicillin/ml, and 100 µg of streptomycin/ml. In some experiments, THP-1 cells were preincubated with 50 nM vitamin D3 for 72 to 96 h before stimulation by LPS, nOspA, OspA-L, and 47-L. In others, THP-1 cells stably transfected with either the cloning vector pRc/RSV or pRc/RSV containing a cDNA encoding human CD14 were used.
Chinese hamster ovary (CHO) cells transfected with either the cloning vector pKoNeo or pKoNeo containing a cDNA encoding human CD14 (46) were provided by Douglas T. Golenbock (Boston University School of Medicine, Boston, MA). These cells were maintained in Ham’s F-12 medium (Mediatech) containing 2 mM L-glutamine and supplemented with 10% HIFBS, 100 U of penicillin/ml, and 100 µg of streptomycin/ml.
For experiments, cells were seeded in 6- or 24-well flat-bottom tissue culture plates (Becton Dickinson Labware, Lincoln Park, NJ) at a density of 1 x 106 or 5 x 105 cells/ml/well, respectively, and were grown to confluence at 37°C in a humidified atmosphere of 5% CO2 and air. All transfected cells were cultured in the continuous presence of 0.5 mg/ml (active drug) of the aminoglycoside G-418 sulfate to ensure the maintenance of stably transfected DNA conferring neomycin resistance. G-418 sulfate was removed 24 h before experimentation, cells were washed twice with appropriate medium, and LPS or spirochetal lipoproteins/lipopeptides were added in 10-µl volumes.
FACS analysis
Cell surface expression of CD14 was determined by staining 5 x 105 cells suspended in PBS containing 3% normal mouse serum (NMS) with a saturating concentration of FITC-conjugated mouse anti-human CD14 mAb (UCHM1, IgG2a; Sigma) or FITC-conjugated isotype-matched control mAb (UPC10; Sigma) for 30 min on ice. Cells were washed twice with PBS containing 3% NMS, and bound mAb was detected with a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
IL-8 ELISA
Levels of IL-8 in culture supernatants were measured in Immulon II 96-well U-bottom plates (Dynatech, Chantilly, VA) using the Duoset ELISA Development System for human IL-8 (Genzyme Diagnostics, Cambridge, MA). The lower limit of detection of IL-8 was 7.8 pg/ml.
Inhibition of macrophage activation by anti-CD14 polyclonal serum
Vitamin D3-matured THP-1 cells (1 x 106 cells in 0.1 ml) were chilled for 5 min on ice and then incubated for an additional 20 min with 1/5, 1/25, or 1/50 dilutions of mouse polyclonal Abs directed against a human CD14-IgG1 fusion protein (47). Cells were then stimulated for 3 h with LPS (10 ng/ml), nOspA (75 ng/ml), OspA-L (1 µg/ml), and 47-L (1 µg/ml), and the culture supernatants were assayed for IL-8 as described above. Cells incubated with 1/5 dilutions of NMS or mouse anti-human IgG1 ascites (Zymed Laboratories, South San Francisco, CA) were used as negative controls.
Electrophoretic mobility shift assay (EMSA)
Following stimulation of 1 x 106 THP-1 or
CHO cells for 1 h, nuclear extracts from cell lysates were
prepared as previously described (39). The active form of NF-
B,
translocated into the nuclei of stimulated cells, was detected by
incubating 5 µg of nuclear extract protein with a radiolabeled,
double-stranded NF-B oligonucleotide prepared using the sequences
5'-GTTCGACAGAGGGGACTTTCCGAGAGG-3' and
3'-TGTCTCCCCTGAAAGGCTCTCCGTTG-5' (bolded
text indicates the consensus NF-KB binding sequence).
Protein-DNA complexes were resolved in 4% native polyacrylamide gels that were dried onto paper and visualized by exposure to a phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 6 h. The intensity of protein-DNA complexes was quantified with a PhosphorImager SF (Molecular Dynamics) using the ImageQuant version 3.3 software package. Results are presented in arbitrary phosphorimage units.
Stimulation and analysis of responses by peritoneal macrophages from CD14-deficient and control mice
Female CD14-deficient mice (from the fifth backcross with
BALB/c) (48) and control mice (BALB/c; Harlan Sprague-Dawley) (8 wk
old) were injected i.p. with 3 ml of 3% (w/v) Brewer thioglycolate
broth (Difco, Detroit, MI). Four days later, cells were harvested by
peritoneal lavage with 5 ml of RPMI 1640 (Life Technologies,
Gaithersburg, MD) containing 2 mM L-glutamine and
supplemented with 100 U of penicillin/ml and 100 µg of
streptomycin/ml. The cells were washed twice, resuspended in the above
medium supplemented with 1% autologous serum, and then added to the
wells (5 x 105 cells/well) of a 24-well tissue
culture plate (Nunc, Naperville, IL). The cells were incubated for
3 h and then were washed twice with 1 ml of medium before
treatment with the various stimuli. S. minnesota wild-type
LPS, nOspA, OspA-L, and 47-L were diluted in medium to the indicated
concentrations and added to the adherent macrophages (0.5 ml/well).
Following a 4-h incubation, cell-free supernatants were collected and
assayed for TNF-
by ELISA according to the manufacturer’s
instructions (Genzyme Diagnostics). The lower limit of detection of
TNF- was 10 pg/ml.
Serum and LBP dependence experiments
To assess whether the stimulatory activity of spirochetal
lipoproteins/lipopeptides was dependent upon serum, THP-1 cells were
washed four times with PBS and cultured in Cellgro Complete serum-free
medium (Mediatech) for 24 h before experimentation. Following
incubation of 1 x 106 cells for 1 h with
various concentrations of LPS, nOspA, OspA-L, and 47-L, the
translocation of NF-B was assessed by EMSA as described above. To
determine the effects of exogenous LBP, cells were adapted to growth
under serum-free culture conditions for 6 wk in Cellgro Complete
serum-free medium. One-tenth milliliter of culture supernatant
(CHO-S-SFM II, Life Technologies) from CHO cells transfected with
either pRc/RSV or pRc/RSV containing a cDNA encoding human LBP
(provided by Peter Tobias, Scripps Research Institute, La Jolla, CA)
was added to wells containing 1 x 106 cells. The
cells were incubated for 1 h with LPS (10 ng/ml), nOspA (75
ng/ml), OspA-L (1 µg/ml), or 47-L (1 µg/ml) and then harvested for
assessment of NF-B translocation by EMSA as described above.
Deacylated LPS antagonism experiments
Vitamin D3-matured THP-1 cells were treated
with 10 nM dLPS (36 ng/ml) for 15 min before and during stimulation by
10 nM LPS (40 ng/ml) or various concentrations of nOspA, OspA-L, and
47-L. Following stimulation of 1 x 106 cells for
1 h, the translocation of NF-B was assessed by EMSA.
Experiments were conducted in the presence of 10% HIFBS to provide a
source of LBP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We reported previously that spirochetal lipoproteins and synthetic
lipopeptides induced murine and human monocytes/macrophages to produce
IL-1ß, IL-6, IL-12, and TNF- (9, 14, 16). One proinflammatory
cytokine not examined in these prior studies was IL-8, a potent
leukocyte chemoattractant (49, 50). Examination of IL-8 secretion by
monocytic cells was warranted for two reasons. First, recent evidence
supports the contention that local production of IL-8 at sites of
spirochetal infection promotes an infiltration of leukocytes that
produces the characteristic histopathologic changes of syphilis and
Lyme disease (51, 52). Second, because IL-8 is secreted relatively
rapidly (within 1–2 h) by activated monocytes/macrophages, its
presence in culture supernatants following short incubation periods
reflects the direct actions of the agonists under study as opposed to
the autocrine effects of subsequently released cytokines. To ensure
that the biologic activities under investigation were not limited to a
particular lipoprotein or lipopeptide, parallel studies were conducted
throughout using lipopeptides representing both T. pallidum
and B. burgdorferi lipoproteins (47-L and OspA-L,
respectively) as well as a purified, native lipoprotein, B.
burgdorferi OspA (nOspA).
As shown in Figure 1
, LPS, nOspA, OspA-L,
and 47-L induced the secretion of IL-8 by vitamin
D3-matured THP-1 cells in a dose-dependent manner. The
finding that vitamin D3 maturation markedly enhanced the
responsiveness to both LPS and spirochetal components (Fig. 1) was
consistent with earlier observations (16, 39, 40). However, interesting
differences in the dose-response curves for these compounds also were
observed (Fig. 1). First, the concentration ranges that induced IL-8
were considerably narrower for nOspA and lipopeptides than for LPS.
Second, as found in prior studies (14, 16), the lipopeptides were
considerably less potent on a molar basis than nOspA. LPS was
approximately 1 log more potent than nOspA and 4 logs more potent than
the lipopeptides with respect to activation of the vitamin
D3-matured cells. These results compare quite favorably
with previously reported potencies derived using a murine macrophage
cell line (RAW 264.7 cells) (14) and with studies using
lipopentapeptide analogues of E. coli lipoprotein and
peritoneal macrophages from C3H/He mice (53). Lastly, although the
thresholds for responsiveness to nOspA and the lipopeptides were
considerably higher than that to LPS, the peak cellular responses were
comparable (Fig. 1). As previously observed (9, 13, 14, 15, 17, 20), the
stimulatory activities of both nOspA and the lipopeptides were
dependent upon lipid modification and were not due to LPS
contamination, as determined by the Limulus amebocyte lysate
assay and by insensitivity to polymyxin B (10 µg/ml; data not
shown).
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To determine whether CD14 contributes to the activation of THP-1
cells by spirochetal lipoproteins and lipopeptides, mouse antiserum
directed against recombinant human sCD14 was used in functional
blocking studies. Polyclonal Abs were used in lieu of mAbs because of
the possibility that LPS and lipoproteins/lipopeptides interact with
different CD14 epitopes. Incubation of vitamin
D3-matured cells with various dilutions of the
antiserum before incubation with LPS, nOspA, OspA-L, and 47-L inhibited
secretion of IL-8 in a dose-dependent manner (Fig. 2). A 1/5 dilution of the antiserum
reduced the cellular response to each compound by an average of 91
± 3, 85 ± 2, 96 ± 2, and 91 ± 3%, respectively.
Substantial inhibition also was observed with 1/50 dilutions of the
antiserum. In contrast, NMS (Fig. 2) and anti-human IgG1 ascites (a
control for the IgG1 portion of the CD14 fusion protein used to
generate the antiserum; data not shown) were incapable of blocking the
responses to these immunomodulators.
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Ulevitch and co-workers recently noted that undifferentiated THP-1
cells become highly responsive to LPS when stably transfected with CD14
(J. Pugin and R. J. Ulevitch, unpublished observations). The use
of this transfected cell line (designated THP-1-CD14) seemed
advantageous, as it would enable us to examine CD14 interactions with
LPS and spirochetal lipoproteins/lipopeptides in a myeloid background
without the pleiotropic effects of vitamin D3
maturation (54). At the outset, flow cytometry was used to compare
surface expression of CD14 by transfected cells and that of cells that
had undergone vitamin D3 maturation. These studies
confirmed that vitamin D3 maturation induces a marked
up-regulation of surface CD14 (39, 40, 55). Compared with their vitamin
D3-matured counterparts, THP-1-CD14 cells expressed
slightly higher levels of CD14 and also expressed the Ag more uniformly
(Fig. 3). In contrast, nontransfected
immature cells expressed extremely low levels of CD14, which were
indistinguishable from those on the cells transfected with the cloning
vector alone (designated THP-1-RSV; data not shown).
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A,
THP-1-CD14 cells responded to much lower doses of LPS and produced
considerably more IL-8 at each LPS concentration tested than did the
THP-1-RSV cells. THP-1-CD14 cells also were more responsive to nOspA
and the lipopeptides, showing a shift in sensitivity as well as an
increase in the overall amounts of IL-8 secreted (Fig. 4, B–D). Interestingly, concentrations of nOspA >150
ng/ml and concentrations of OspA-L and 47-L >0.6 µg/ml induced
secretion of IL-8 by the THP-1-RSV cells (Fig. 4, B–D). This potential CD14-independent pathway was
most prominent with 47-L (Fig. 4D). Finally, in
comparing the responsiveness of THP-1 cells to LPS and spirochetal
lipoprotein/lipopeptides, it was noted that the CD14-transfected cells
consistently showed a higher threshold of sensitivity to both LPS and
lipoprotein/lipopeptides than did their vitamin D3-matured
counterparts and, for each concentration of immunomodulator tested,
produced considerably less IL-8 (compare Figs. 1
and 4, which show
experiments conducted in parallel). Given the comparable levels of CD14
expressed on both cell populations (Fig. 3), the difference in
responsiveness may relate to the differentiation state of the
cells.
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secretion by thioglycolate-elicited peritoneal
macrophages from control (CD14+) and CD14-deficient BALB/c
mice. In these experiments, the macrophages were incubated in medium
supplemented with 1% autologous serum to eliminate potential
activation of the CD14-deficient macrophages by sCD14-LPS complexes
(48, 56). As found previously (48, 56, 57), cells from the
CD14-deficient animals produced considerably less TNF- in response
to LPS, although cytokine secretion was noted at higher concentrations
(Fig. 5A). Macrophages
from CD14-deficient animals also were less responsive to nOspA and the
two lipopeptides than were cells from CD14+ mice (Fig. 5, B–D). Only a minimal response by the CD14-deficient
macrophages was detected at the highest concentration of nOspA tested
(Fig. 5B), whereas a CD14-independent component was
more evident with the two lipopeptides and with 47-L in particular
(Fig. 5, C and D). It is worth noting
that, despite the different cytokines studied, the responses of the
CD14+ and the CD14-deficient macrophages roughly paralleled
those of the THP-1-CD14 and THP-1-RSV cells (compare Figs. 4 and 5).
Furthermore, the use of autologous serum in these experiments suggests
that sCD14 from FBS played a nominal role in lipopeptide-mediated
activation of undifferentiated and RSV-transfected THP-1 cells
(Figs. 1 and 4, respectively).
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The mCD14-mediated recognition of low concentrations of LPS
(typically <40 ng/ml) is enhanced by the serum component LBP (29, 30, 32, 33). Having shown that mCD14 can potentiate the response of
monocytes/macrophages to spirochetal lipoproteins and lipopeptides,
experiments next were conducted to examine the potential involvement of
LBP or other serum components in this process. Here we assessed cell
activation by nuclear translocation of NF-B, a transcriptional
activator implicated in cytokine induction in LPS- and
lipoprotein/lipopeptide-stimulated immune cells (16, 17, 58, 59). The
rapid kinetics of NF-B translocation (within 15 min of stimulation)
should preclude autocrine effects due to subsequently secreted
cytokines (39).
In one series of experiments, vitamin D3-matured THP-1
cells were washed extensively and maintained in serum-free medium
for 24 h. They then were incubated in the absence or the
presence of 10% HIFBS with various concentrations of LPS, nOspA, or
lipopeptides. As predicted (29, 30, 32, 33), the cellular response to
LPS differed markedly depending upon the absence or the presence of
serum; the serum dependence of the LPS response was particularly
striking at LPS concentrations 10 ng/ml (Fig. 6A). In contrast,
nearly identical dose-response curves for nOspA and the two
lipopeptides were obtained in the absence or the presence of serum
(Fig. 6, B–D). Similar results were obtained when
measuring secretion of IL-8 or when parallel experiments were conducted
with THP-1-CD14 cells (data not shown).
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while LBP dramatically enhanced responsiveness to LPS, it had no
effect on the responses of the cells to nOspA or lipopeptides. It
should be noted that immunomodulator concentrations were employed that
generate submaximal responses to ensure that the ability of LBP to
augment cellular responses would not be masked.
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Previously, we reported that mouse pre-B 70Z/3 cells transfected
with CD14 responded to low concentrations of LPS, but not to nOspA or
spirochetal lipopeptides even at concentrations as high as 1 and 15
µg/ml, respectively (16). One potential explanation for this
dichotomy between LPS and lipoprotein/lipopeptide responsiveness is
that these nonmyeloid cells possess the putative LPS signal transducer
but lack a comparable element required for CD14-dependent
lipoprotein/lipopeptide signaling. To test this hypothesis, we
investigated whether responses to LPS and lipoproteins/lipopeptides
could be dissociated in other nonmyeloid cells. Golenbock and
co-workers showed that CHO cells, which are normally LPS nonresponsive,
become highly responsive to LPS following transfection with human CD14
(46). It was of interest, therefore, to evaluate the responses of these
cells to spirochetal lipoproteins and lipopeptides. As previously
observed, CHO cells transfected with CD14 were exquisitely sensitive to
LPS (Fig. 8A). In
contrast, the responses of these same cells to nOspA, OspA-L, and 47-L
were not significantly different (P < 0.05, by
ANOVA) from those of cells transfected with the cloning vector alone
(Fig. 8, B–D, respectively).
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, 10 nM dLPS (36 ng/ml), a concentration
well below the Kd for binding of dLPS to CD14
(100 ng/ml) (60), completely blocked cell activation by an equimolar
concentration of LPS. In contrast, the same concentration of dLPS had
no inhibitory effect on nOspA-mediated activation of macrophages, even
when used in a fourfold molar excess (10 nM dLPS to 2.5 nM nOspA).
Similarly, dLPS was unable to block cell activation by either of the
two lipopeptides (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that bacterial lipoproteins and lipopeptides with diverse N-terminal sequences can activate monocytes/macrophages (6, 14, 53, 63) has been difficult to reconcile with the presumption that these immunomodulators exert their effects via specific receptor-ligand interactions. Also difficult to explain has been the finding that the biologic activities of these immunomodulators are dependent upon lipid modification (13, 14, 15, 17, 64). We now postulate that there must be a region in CD14 that recognizes an amphipathic structural motif at the N terminus of bacterial lipoproteins. Additional support for this conjecture derives from recent native gel electrophoresis experiments in which it was found that acylation was necessary for the binding of recombinant OspA to sCD14 (65). Using both natural and synthetic bacterial lipopeptides, Jung and co-workers have shown that the presence of ester-bound fatty acids is a prerequisite for biologic activity, whereas the amide-bound fatty acid is dispensable (63, 66); thus, it can be inferred that the amide-linked fatty acid is of limited importance for CD14 binding.
The idea that proinflammatory activity can be regarded as a generic property of spirochetal lipoproteins and lipopeptides has important implications for syphilis and Lyme disease pathogenesis. A large proportion of the membrane immunogens of both T. pallidum and B. burgdorferi are lipid modified (67, 68, 69), and it is reasonable to propose that these molecules act in concert to promote the inflammatory processes that culminate in clinical manifestations. Moreover, there is now a substantial body of evidence that B. burgdorferi does not express OspA and OspB following tick transmission (70, 71, 72, 73), while other antigenically unrelated lipoproteins are selectively expressed in the mammalian host (71, 72, 73, 74). Promiscuous binding by CD14 would permit differentially expressed lipoproteins to assume the immunomodulatory roles that have been proposed for OspA and OspB based upon in vitro studies (9, 10, 12, 13, 14, 15, 16, 17, 19, 75). Consistent with this idea is our finding that synthetic lipopeptides derived from the N termini of two lipoproteins expressed during infection, OspC and the OspF homologue BbK2.10 (74), have in vitro proinflammatory activities comparable to those of OspA and OspB lipopeptides (T. J. Sellati and J. D. Radolf, unpublished observations).
One of the most important findings reported here is that the CD14-dependent signaling pathways used by LPS and spirochetal lipoproteins/lipopeptides differ in at least two fundamental respects. In contrast to LPS, activation by spirochetal lipoproteins was not facilitated by LBP or other serum components. This finding is not without precedent; Wright and co-workers have shown recently that Staphylococcus aureus cell wall extract stimulates human PBMC in a CD14-dependent, LBP-independent fashion (24). LBP is thought to enhance LPS responsiveness by transferring LPS monomers out of LPS aggregates to a binding site(s) on CD14 (32). The lack of involvement of LBP in cell activation by lipoproteins and lipopeptides suggests that these amphiphilic compounds either interact with CD14 as aggregates or that monomers bind to CD14 unassisted by a serum intermediary. The latter scenario, rather than lower binding affinities, could explain the ostensibly lower potencies of lipoproteins and lipopeptides, compared with LPS, inasmuch as the amphiphilic spirochetal constituents (especially the lipopeptides) are extremely insoluble and undoubtedly exist in a highly aggregated state in an aqueous environment.
Particularly intriguing was the finding that the LPS and lipoprotein/lipopeptide signals appear to be transduced via distinct transmembrane elements. Indirect evidence for this was the observation that CHO cells transfected with CD14 were exquisitely sensitive to LPS but were insensitive to lipoproteins and lipopeptides, a result that parallels earlier findings with 70Z/3 cells (16). Additional evidence was provided by the observation that substoichiometric concentrations of an LPS antagonist were unable to block lipoprotein and lipopeptide signaling in vitamin D3-matured THP-1 cells under the same conditions in which LPS signaling was completely ablated. Interestingly, transfection of THP-1 cells with CD14 did enhance responsiveness to lipoproteins and lipopeptides, suggesting that, unlike 70Z/3 and CHO cells, THP-1 cells constitutively express the putative lipoprotein/lipopeptide transducer. Previously, we showed that spirochetal lipoproteins and lipopeptides activate macrophages from LPS-nonresponsive C3H/HeJ mice (9, 14); the existence of distinct LPS and lipoprotein/lipopeptide signal transducers is one plausible explanation for this dichotomy. Nevertheless, because the responses of monocytic cells to lipoproteins/lipopeptides so closely resemble those elicited by LPS (14), we believe that these two CD14-dependent pathways subsequently must overlap and/or converge. Golenbock and co-workers also have proposed that LPS and lipoarabinomannan from Mycobacterium tuberculosis share CD14 as a binding receptor but differ with respect to downstream elements necessary for specific cellular activation (26, 76). However, in contrast to spirochetal lipoproteins and lipopeptides, signaling by lipoarabinomannan can be inhibited by LPS partial structures and is LBP dependent (26).
CD14-independent as well as CD14-dependent pathways have been described for bacterial products other than LPS; these pathways tend to be engaged at higher immunomodulator concentrations than those needed for CD14-dependent stimulation (24, 25, 77). Thus, it was not entirely surprising that CD14-independent responses became apparent at progressively higher concentrations of the spirochetal constituents. Two observations regarding this CD14-independent effect were noteworthy. First, it was observed only in the cellular backgrounds (i.e., THP-1 cells and murine peritoneal macrophages) in which CD14 expression enhanced lipoprotein/lipopeptide responsiveness, suggesting that CD14-dependent and -independent signaling are mechanistically interrelated and potentially nondissociable. Second, the CD14-independent response was more prominent with the synthetic lipopeptides, most notably 47-L. Thus, while lipoproteins and lipopeptides induce qualitatively similar responses in different effector cells of innate immunity and appear to exert these effects through highly similar mechanisms, data presented in this report raise the possibility that differences in biologic activity may exist between lipoproteins and their synthetic analogues. Further studies should clarify this issue while delineating the structural features of lipoproteins and lipopeptides that influence the engagement of CD14-dependent and -independent pathways.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Periodontics, University of Washington, Seattle, WA 98195-7444.
3 Address correspondence and reprint requests to Dr. Justin D. Radolf, Division of Infectious Diseases, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9113. E-mail address:
4 Abbreviations used in this paper: mCD14, membrane CD14; LBP, lipopolysaccharide-binding protein; sCD14, soluble CD14; dLPS, deacylated lipopolysaccharide; nOspA, native outer surface protein A; HIFBS, heat-inactivated fetal bovine serum; OspA-L, lipopeptide corresponding to the N terminus of the outer surface protein A lipoprotein of Borrelia burgdorferi; 47-L, lipopeptide corresponding to the N terminus of Treponema pallidum; RSV, Rous sarcoma virus; NMS, normal mouse serum; EMSA, electrophoretic mobility shift assay.
Received for publication November 7, 1997. Accepted for publication January 30, 1998.
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