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B Activation and TNF Production Induced by Lipopolysaccharide and Group B Streptococcal Cell Walls1
*
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway;
The Maxwell Finland Laboratory for Infectious Diseases, Division of Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, MA 02118;
§
Institute of Microbiology, Faculty of Medicine and Surgery, University of Messina, Messina, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation induced by LPS and cell wall preparations from
group B streptococci type III (GBS). LPS and GBS caused TNF secretion
from human monocytes in a CD14-dependent manner, and soluble CD14, LPS
binding protein, or their combination potentiated both LPS- and
GBS-induced activities. Blocking of either CD14 or CD18, the common
ß-subunit of CR3 and CR4, decreased GBS-induced TNF release, while
LPS-mediated TNF production was inhibited by anti-CD14 mAb only.
Chinese hamster ovary cell transfectants (CHO) that express human CD14
(CHO/CD14) responded to both LPS and GBS with NF-B translocation,
which was inhibited by anti-CD14 mAb and enhanced by LPS binding
protein. While LPS showed fast kinetics of NF-B activation in
CHO/CD14 cells, a slower NF-B response was induced by GBS. LPS also
activated NF-B in CHO cells transfected with either human CR3 or CR4
cDNA, although responses were delayed and weaker than those of CHO/CD14
cells. In contrast to LPS, GBS failed to induce NF-B in CHO/CR3 or
CHO/CR4 cells. Both C3H/OuJ (Lpsn) and
C3H/HeJ (Lpsd) mouse peritoneal macrophages
responded to GBS with TNF production and NF-B translocation, whereas
LPS was active only in C3H/OuJ macrophages. Thus, LPS and GBS
differentially involve CD14 and CR3 or CR4 for signaling NF-B
activation in CHO cells and TNF release in human monocytes, and engage
a different set of receptors and/or intracellular signaling pathways in
mouse macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Complement receptors CR3 (also called Mac-1 and CD11b/CD18) and CR4
(also referred to as CD11c/CD18 and p150,95) are transmembrane
glycoproteins that belong to the ß2 integrin family. They
are expressed on the surface of neutrophils, monocytes, macrophages,
and NK cells and are involved in numerous cell-cell and cell-substrate
interactions (27). Patients with a deficiency in the expression of
ß-integrins are predisposed to life-threatening infections due to
impaired transendothelial emigration, intravascular adhesion,
phagocytosis, and target cell killing (28). CR3-deficient mice show a
significant delay in apoptosis of extravasated neutrophils (29),
demonstrating a role for CR3 in apoptotic cell death. In addition to
CD14, CR3 and CR4 have been reported to mediate cell activation
following LPS binding. Indeed, transfection of CR3 or CR4 cDNA into CHO
cells confers upon them the ability to respond to LPS, as evidenced by
NF-B translocation (30, 31). Furthermore, stimulation of neutrophils
with LPS in the presence of serum or LBP leads to association between
CR3 and CD14, with subsequent dissociation of CD14-CR3 complexes as
cells attach to substrates (32). This implies the possibility of
cross-talk between those two receptors in mediating LPS signaling.
However, PBMC obtained from CD18-deficient patients bind and respond to
LPS normally (33), suggesting that CD14, which is expressed on
CD18-deficient cells, can mediate LPS effects even when the CD18
expression is profoundly decreased. Yet, it remains unknown whether CR3
and CR4 are involved in mediating the effects of Gram-positive
bacteria.
In the present work we compared the capacities of LPS and group B
streptococci type III (GBS) cell wall preparations to induce NF-B
and to stimulate TNF release in human monocytes and mouse macrophages.
To dissect further the roles of CD14, CR3, and CR4, CHO transfectants
expressing those molecules were used to assess the NF-B-inducing
activity of LPS and GBS. In addition, blocking anti-CD14 and
anti-CD18 mAbs were used to evaluate the involvement of these
molecules in LPS- and GBS-induced TNF production from human monocytes.
The data suggest that while LPS and GBS share CD14, they differentially
use CR3 and CR4 for triggering NF-B activation in CHO cells and TNF
production in human monocytes. Furthermore, macrophages derived from
LPS-hyporesponsive C3H/HeJ mice that also express a defect in the
sphingomyelin pathway responded to GBS, but not to LPS, by TNF
production and NF-B translocation. This suggests that, in contrast
to LPS, GBS does not use the sphingomyelin pathway for signaling TNF
release and NF-B activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GBS cell wall preparations were obtained by mixing whole lyophilized bacteria (strain H738, 3 mg/ml in distilled water) with equal volumes of 8% SDS and boiling for 30 min. After overnight incubation at room temperature with agitation, the suspension was centrifuged (30,000 x g for 15 min), and the pellet was extracted twice by boiling with 4% SDS and washed by centrifugation at 20°C four times with water, twice with 2 N NaCl, and again with water. The resulting GBS cell wall preparation had an LPS content of 14 ng/mg, as measured by Limulus amebocyte lysate assay. Recombinant sCD14 and LBP were provided by Dr. H. Lichenstein (Amgen, Thousand Oaks, CA). Recombinant bactericidal permeability-increasing protein (BPI) was supplied by Dr. M. Marra (Incyte, Palo Alto, CA). Anti-human CD14 mAb 3C10 and anti-human CD18 mAb HB203 (mouse IgG2) were purified on Sepharose goat anti-mouse IgG, as described by the manufacturer, from supernatants of the respective hybridoma cell lines purchased from American Type Culture Collection (Rockville, MD). The mAb 6H8, which recognizes a widely distributed 180-kDa glycoprotein (T. Espevik and B. Naume, unpublished observation), was used as a control. Rough LPS from Salmonella minnesota R595 (ReLPS) and LPS from Pseudomonas aeruginosa were purchased from Sigma (St. Louis, MO). Phenol/water-extracted Escherichia coli K235 LPS was prepared as previously described (34) and was protein free. Human rTNF (sp. act. of 7.6 x 107 U/mg of protein) was supplied by Genentech (South San Francisco, CA).
Cell lines and culture conditions
The CHO/NEO and CHO/CD14 cell lines were obtained by a stable transfection as described by Golenbock et al. (10). The CHO/CR3 and CHO/CR4 cell lines were engineered by cotransfection of a CHO-K1 cell line with human CR3 or CR4 cDNA and CD18 cDNA, as described previously (30, 31). Transfectants were maintained in Ham’s F-12 medium supplemented with 10% FCS (HyClone, Logan, UT) and 1 mg/ml G418 (Sigma) in a 5% CO2 humidified atmosphere at 37°C.
Isolation of human monocytes and mouse macrophages
Monocytes were isolated from human A+ buffy coats (The BloodBank, Norwegian University of Science and Technology, Trondheim, Norway) as previously described (35). Adherent cell monolayers were cultured in 24-well plates (Costar, Cambridge, MA) in either AIM serum-free medium (Life Technologies, Grand Island, NY) supplemented with 1% L-glutamine and 40 µg/ml gentamicin or in AIM medium containing 25% human A+ serum. Monocytes were stimulated for 8 h at 37°C with the indicated preparations. Thereafter, supernatants were collected and stored at -80°C until use. C3H/OuJ and C3H/HeJ mice (female, 5 wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME). Peritoneal exudate macrophages were isolated by peritoneal lavage 3 days after i.p. injection of 3 ml of sterile 3% thioglycolate broth. After washing, cells were resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 0.3% sodium bicarbonate, and 2% FBS. For preparation of nuclear extracts, cells were plated in six-well plates (4 x 106 cells/well), incubated overnight, washed three times with prewarmed RPMI 1640, and treated with the indicated stimuli in a total volume of 2 ml. To produce TNF, macrophages (0.5 x 106 cells/well in 24-well plates) were incubated for 4 h at 37°C in a 5% CO2 atmosphere, washed, and stimulated with LPS or GBS in 1 ml of culture medium. Supernatants were collected and stored at -80°C until use.
Preparation of nuclear extracts
Nuclear extracts were prepared according to the method of Dignam et al. (36). Briefly, adherent cell monolayers were washed with ice-cold PBS, harvested using a rubber policeman, transferred to Eppendorf tubes, and centrifuged (800 x g, 10 min, 4°C). Cells were resuspended in 0.5 ml of ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM benzamidine, and 0.5 mM PMSF), incubated on ice for 15 min, and lysed by adding Nonidet P-40 to a final concentration of 0.5%. Nuclei were pelleted (1,000 x g, 10 min, 4°C) and resuspended in 50 µl of ice-cold buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM DTT, 1 mM benzamidine, and 0.5 mM PMSF). After a 30-min incubation on ice, the tubes were centrifuged (10,000 x g, 10 min, 4°C), and supernatants were collected and stored at -80°C. The protein concentration was measured by the Bio-Rad protein assay with BSA as a standard (Bio-Rad Laboratories, Hercules, CA).
Electrophoretic mobility shift assay (EMSA)
The NF-B-specific oligonucleotide probe
5'-AGTTGAGGGGACTTTCCCAGGC-3' (from Promega (Madison, WI) or
synthesized by the BIC Synthesis and Sequencing Facility, Uniformed
Services University of the Health Sciences, Bethesda, MD) from the
murine Ig B light chain gene enhancer was 32P end
labeled with T4 polynucleotide kinase (Promega). Nuclear extracts (4
µg) were incubated with 0.2 ng of DNA probe in a binding buffer
containing 2 µg of poly(dI-dC) (Pharmacia Fine Chemicals, Uppsala,
Sweden), 20 mM HEPES (pH 7.9), 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.25
mg/ml BSA, and 4% glycerol for 30 min at room temperature. The
DNA-protein complex was separated from free oligonucleotide by
electrophoresis in a 6% polyacrylamide gel (0.25x Tris borate-EDTA,
150 V, 2 h). The gels were dried (80°C, 2 h) and exposed to
x-ray film (X-OMAT AR, Eastman Kodak, Rochester, NY).
TNF assay
TNF activity in supernatants from human monocytes and mouse macrophages was measured in the WEHI 164 clone 13 bioassay as described previously (37). The lower limit of detection in this assay was 0.35 pg/ml TNF.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first series of experiments, we compared the capacities of
LPS and GBS to induce TNF from human monocytes and evaluated the roles
of membrane and sCD14 as well as LBP in mediating this response. As
shown in Figure 1
, A and
B, LPS was 50- to 100-fold more potent than GBS in
stimulating TNF release in both the presence and the absence of normal
human A+ serum. In the presence of serum, addition of
anti-CD14 mAb, 3C10, resulted in a shift of the LPS dose-response
curve to an approximately 100-fold higher LPS concentration, whereas
under serum-free conditions, 3C10 completely inhibited LPS-induced TNF
release at LPS concentrations of 
100 ng/ml (Fig. 1A). 3C10 also decreased GBS-induced TNF production,
shifting the GBS dose-response curve about 2- to 5-fold to the right
(Fig. 1B). Neither an irrelevant mAb, 6H8 (Fig. 1),
nor a nonblocking anti-CD14 mAb, 26ic (data not shown), affected
either LPS- or GBS-induced TNF production. High concentrations of LPS
and GBS overrode the blocking ability of the anti-CD14 mAb 3C10
(Fig. 1, A and B), suggesting a
CD14-independent mechanism of cell stimulation. As shown in Figure 1, A and B, human serum significantly potentiated
not only LPS-stimulated but also GBS-stimulated TNF production. To
delineate the mechanism by which human serum enhances GBS-induced TNF
release, monocytes were stimulated with LPS and GBS under serum-free
conditions in the absence or the presence of human sCD14, rLBP, or
both. Figure 1C demonstrates that recombinant LBP and, to a
lesser extent, sCD14 alone increased both LPS- and GBS-induced TNF
production from human monocytes, and the combination of LBP and sCD14
further enhanced the responses. In the absence of LPS or GBS, neither
sCD14, LBP, nor their combination induced TNF to levels higher than
those detected in monocyte cultures incubated with medium alone (data
not shown). The LPS inhibitor, BPI (38), used at a concentration of 100
ng/ml, reduced LPS-induced TNF secretion in monocytes from 2932 ±
274 to 1258 ± 66 pg/ml. However, GBS-induced TNF secretion
(698 ± 62 pg/ml) was not inhibited by BPI (744 ± 32 pg/ml),
suggesting that GBS-mediated TNF release is not due to small levels of
contaminating LPS in GBS preparations. Taken together, these data
support the involvement of both membrane and soluble CD14 in LPS- and
GBS-mediated TNF production as well as enhancement of the response
by LBP.
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B by both LPS and GBS
Monocytes and macrophages express several LPS binding and/or
receptor molecules, e.g., CD14, the leukocyte integrins CR3 and CR4,
and a 80-kDa LPS binding protein (11, 30, 39), whose potential
contributions to GBS signaling cannot be easily separated in this
system. To analyze the individual role of CD14, we used a CHO cell line
genetically engineered to express human CD14 (10). Figure 2A illustrates that
stimulation of CHO/CD14 cells with LPS or GBS in a serum-free medium,
AIM, strongly activated NF-B, whereas no NF-B translocation was
seen in control CHO/NEO transfectants. In CHO/CD14 cells, detectable
NF-B translocation was seen after 15 min of stimulation with LPS,
which reached a plateau following 30 to 60 min and declined by 180 min
(Fig. 2B). In contrast, GBS induced NF-B
activation only after 60 min, and it persisted at a similar level for
the 180-min period of stimulation (Fig. 2B). As shown
in Figure 3B, as little as 10
ng/ml of LPS caused a moderate NF-B stimulation, which was
prominently enhanced as the concentration of LPS was increased from 10
to 100 and 1000 ng/ml. Consistent with their TNF-inducing potencies
(Fig. 1
, A and B), 250- to 500-fold higher
concentrations of GBS were needed to observe NF-B induction
comparable to that detected in LPS-treated CHO/CD14 cells (Fig. 3, A and B). Importantly, combining
anti-CD14 mAb 3C10 with either LPS or GBS led to a drastic
inhibition of their capacity to induce NF-B (Fig. 3A). As was the case for human monocytes (Fig. 1C), recombinant LBP markedly potentiated NF-B
activation caused by both LPS and GBS (Fig. 3B),
resulting in a 100-fold shift of the dose-response curves toward lower
concentrations of the stimuli.
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B in both CHO/CR3 and CHO/CR4
transfectants
To examine a possible role for the leukocyte integrins CR3
and CR4 in mediating LPS and GBS activities, we analyzed the kinetics
and dose responses of NF-B translocation in CHO/CR3 and CHO/CR4
cells treated with these preparations under serum-free conditions.
Figure 4, A and B,
shows that LPS markedly induced NF-B in both CHO/CR3 and CHO/CR4
cell lines, but not until after 60 min, and the maximal response was
reached following 120 to 180 min of LPS stimulation. As depicted in
Figure 4, A and C, a high concentration of LPS
(10 µg/ml) was required to achieve strong NF-B binding activity in
both cell lines. One microgram per milliliter of LPS induced moderate
responses, and only a weak NF-B translocation was induced by 100
ng/ml LPS (Fig. 4, A and C). These
concentrations of LPS, however, did not induce NF-B in control
CHO/NEO cells (Fig. 2 and data not shown). In contrast, kinetic and
dose-response experiments revealed that GBS failed to activate NF-B
in either CHO/CR3 or CHO/CR4 cells, even when used at 200 µg/ml.
These results indicate that while LPS can use CR3 or CR4 for signaling
NF-B activation in CHO cells, the expression of these leukocyte
integrins does not render CHO cells responsive to GBS.
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In the next series of experiments, the involvement of
CD14 and CD18, the common ß-subunit of human leukocyte integrins
CD11a/CD18, CR3, and CR4 (27), in LPS- and GBS-mediated TNF production
from human monocytes was studied. To this end, anti-CD14 mAb, 3C10,
or anti-CD18 mAb, HB 203, were added to monocytes for 30 min to
block the respective receptor structures, followed by stimulation of
cells with either LPS or GBS under serum-free conditions. Figure 5 shows that anti-CD14 mAb markedly
decreased the capacities of both LPS and GBS to stimulate TNF release.
Interestingly, anti-CD18 mAb caused an even greater suppression of
GBS-mediated TNF production than did the anti-CD14 mAb (Fig. 5). In
contrast, LPS-induced TNF secretion was not inhibited by anti-CD18
mAb. A control mAb, 6H8, did not influence either LPS- or GBS-induced
TNF production (Fig. 5). These data indicate that GBS is capable of
signaling TNF release from human monocytes through both CD14 and CD18,
whereas the LPS response in this case is mediated mainly by
CD14.
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B translocation
To evaluate further whether LPS and GBS use different receptor
molecules and/or intracellular signal transduction pathways, we
assessed their abilities to induce TNF release and NF-B activation
in peritoneal macrophages derived from LPS-responsive (C3H/OuJ) and
LPS-hyporesponsive (C3H/HeJ) mouse strains. Stimulation of C3H/OuJ
mouse macrophages with 1 µg/ml LPS resulted in the induction of
NF-B binding activity after 30 min of treatment, while GBS-mediated
NF-B translocation was evident only after 120 to 240 min (Fig. 6A). As little as 0.1
ng/ml LPS caused the appearance of active NF-B in nuclear extracts
from C3H/OuJ macrophages, and the response reached a plateau at 10
ng/ml LPS (Fig. 7). Macrophages derived
from C3H/HeJ mice showed very weak, if any, NF-B induction in
response to LPS stimulation, whereas GBS-induced NF-B responses
followed similar kinetic and dose dependence in both C3H/OuJ and
C3H/HeJ mouse strains (Figs. 6 and 7). Similarly, the highest
concentration of LPS used (1 µg/ml) caused very low TNF production
from C3H/HeJ macrophages (50 pg/ml), in contrast to the strong TNF
response (up to 165,000 pg/ml) exhibited by LPS-treated C3H/OuJ
macrophages (Table I). Similar to the
NF-B results, Table I shows that GBS induced marked TNF responses in
both C3H/OuJ and C3H/HeJ macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in response to LPS or GBS. Secondly,
antagonistic anti-CD14 Abs greatly inhibited the capacity of both
stimuli to induce TNF release in human monocytes and to activate
NF-B in CD14-expressing CHO cells. Third, LBP, a protein known to
catalyze the transfer of LPS to CD14 (5, 6), significantly enhanced the
TNF-inducing potency of both LPS and GBS in human monocytes as well as
their capacity to activate NF-B in CHO/CD14 cells. Fourth, sCD14, an
important mediator of LPS effects (4, 5, 14, 15, 16), increased the ability
of not only LPS, but also GBS, to cause TNF release from human
monocytes under serum-free conditions, and the combination of sCD14 and
LBP further potentiated these responses. Recently, LBP and sCD14 have
been found to transfer LPS to membranes consisting of certain classes
of lipoproteins (40), thereby enhancing and targeting cellular
responses to LPS. Hence, it is possible that sCD14 and LBP facilitate
the movement of not only LPS, but also GBS, into the cell membrane,
possibly to interact with signal-transducing molecules. Our data
combined with the results on CD14 dependency of several other bacterial
molecules (11, 20, 21, 22, 23) support the hypothesis that CD14-mediated
recognition of various bacterial products plays an important role in
cell activation (11).
A number of observations have shown that high doses of LPS can activate
cellular responses by a CD14-independent mechanism (17, 18, 24, 26).
This paper extends these findings and demonstrates that not only LPS,
but also GBS, when used at high concentrations activate TNF production
from human monocytes in a CD14-independent manner. These results
suggest the existence of receptor molecules distinct from CD14 that are
responsible for the signaling effects of LPS and Gram-positive
bacteria. In this respect, expression of the leukocyte integrins CR3 or
CR4 in CHO cells has been reported to enable NF-B translocation in
response to LPS (30, 31). In addition, CR3 binds unopsonized bacteria
and LPS to macrophages, monocytes, and granulocytes (41), and
associates with CD14 on the surface of human neutrophils after
stimulation with LPS (32). Thus, it is plausible that these leukocyte
integrins represent phagocyte receptors capable of mediating LPS
effects in the absence of CD14. To study whether CR3 and CR4 mediate
cellular responses elicited by Gram-positive bacteria, we first
compared the abilities of LPS and GBS to activate NF-B in CHO cells
engineered to express CR3 or CR4. In agreement with previously reported
data (30, 31), LPS induced NF-B in both CHO/CR3 and CHO/CR4 cell
transfectants. However, GBS failed to activate NF-B in either of the
cell lines, indicating that single expression of CR3 or CR4 is not
sufficient to enable GBS responsiveness. It could be argued that the
concentrations of GBS used to stimulate CHO/CR3 or CHO/CR4 cells were
not high enough to elicit NF-B activation. Based on the differences
in concentrations of GBS vs LPS required to induce comparable levels of
TNF in the monocyte experiments, one might expect that milligram per
milliliter concentrations of GBS would be necessary for activation of
NF-B in CHO/CR3 or CHO/CR4 transfectants. However, the feasibility
of this approach is limited because of practical and technical reasons.
Of importance, it should be noted that the same concentrations of GBS
that failed to activate NF-B in CHO/CR3 or CHO/CR4 cells induced
NF-B translocation in CHO/CD14 cells. In addition, recombinant LBP
significantly potentiated the ability of LPS to activate NF-B in
CHO/CR3 cells, yet in combination with GBS, it did not induce NF-B
(our unpublished observation). Of note, in CHO/CD14 cells, both LPS-
and GBS-induced NF-B responses were markedly enhanced by LBP. These
results seem to rule out the possibility that GBS fails to activate
NF-B in CHO cells transfected with CR3 or CR4 due to its
insufficient concentration.
As a second approach, we studied the involvement of CD14 and the common
ß-chain of the leukocyte integrins, CD18, in LPS- and GBS-induced TNF
production from human monocytes. Treatment of cells with both
anti-CD14 and anti-CD18 blocking mAb considerably decreased the
ability of GBS to induce TNF. In contrast, LPS was found to signal TNF
release mainly through CD14, as anti-CD14, but not anti-CD18,
mAb potently inhibited its activity. In line with our findings, LPS was
reported to induce cytokine production normally from PBMC derived from
patients with deficiency in CD18 expression (33). Predominant
expression of CD14 vs CD18 on the surface of human monocytes has been
reported (42), suggesting that compared with CD14, CD18 only weakly
contributes to LPS-mediated TNF release. However, blocking CD18 led to
an even greater suppression of GBS-mediated TNF release compared with
that caused by anti-CD14 mAb, indicating that the CD18 contribution
to the response is also important. Taken together, the results indicate
that GBS is incapable of signaling NF-B activation in CHO/CR3 or
CHO/CR4 cells, whereas transfection of CD14 renders CHO cells GBS
responsive. On the other hand, in human monocytes, GBS uses both CD14
and the leukocyte integrins for signaling TNF production, while LPS
acts predominantly through CD14.
Interestingly, CHO cells that express mutated CR3 molecules deficient
in the cytoplasmic domains exhibit impaired phagocytic activity, yet
respond to LPS by NF-B translocation (31). Furthermore, myeloid
cells derived from patients with deficient expression of CD18 bind and
respond to endotoxin normally (33). These findings argue against the
hypothesis that CR3 or CR4 can function as signal-transducing receptors
for LPS. Rather, they may act in a manner analogous to that proposed
for CD14, e.g., CR3 and CR4 bind LPS and transfer it to yet
unidentified protein, which, in turn, transmits a signal across the
plasma membrane. Of note, stimulation of human neutrophils with LPS was
found to lead to association of CD14 with CR3, followed by dissociation
of the CD14-CR3 complex in the course of cell attachment to substrates
(32). In addition, preliminary results show enhanced LPS-induced
NF-B activation in CHO cells cotransfected with CD14 and CR3
compared with the responses exhibited by either CHO/CD14 or CHO/CR3
single cell transfectants (data not presented). These results suggest
that LPS responses may require the formation of molecular complexes
among CD14 and CR3 or CR4 with other signal-transducing molecules,
possibly, by a mechanism similar to those used by the IL-6 and LIF
receptor systems (43).
It is possible that GBS binding to the leukocyte integrins triggers
intracellular pathways that signal TNF production, but not those
responsible for NF-B activation. Likewise, LPS interaction with CD18
might not recruit signaling molecules that mediate TNF release in human
monocytes, even though LPS can use CR3 or CR4 for triggering NF-B
activation in CHO cells. Different signaling molecules may be recruited
by these complexes, depending on whether the leukocyte integrins bind
LPS or GBS; in addition, these complexes may have various compositions
in different cell types. Thus, although CHO cells have integrin-coupled
intracellular pathways capable of LPS-mediated NF-B activation, they
could lack those necessary for GBS-induced NF-B activation through
the integrin receptors. In this respect, it is noteworthy that
LPS-responsive, CD14-transfected CHO cells fail to respond to
mycobacterial lipoarabinomannan, a molecule that otherwise activates
THP-1 monocytic cells through CD14 (22). Experiments are in progress to
distinguish between these possibilities.
Another model of LPS signal transduction postulates that CD14 and other
LPS-binding molecules facilitate the transfer of LPS into the cell
membrane where it can activate the sphingomyelin pathway directly due
to a structural similarity between LPS and ceramide (44). Stimulation
of the sphingomyelin pathway with exogenous ceramide analogues or
bacterial sphingomyelinase mimics a number of LPS responses in C3H/OuJ
(Lpsn), but not in C3H/HeJ
(Lpsd), macrophages (45, 46). Furthermore,
mice genetically hyporesponsive to LPS have recently been found to
exhibit a defect in endocytic uptake and intracellular localization of
both LPS and ceramide (47). In addition, LPS has been shown to activate
ceramide-activated protein kinase in myeloid cells (48), supporting the
role of the sphingomyelin pathway in LPS signal transduction. This
paper demonstrates that GBS exhibited similar kinetic and dose
responses of NF-B translocation regardless of whether C3H/OuJ or
C3H/HeJ macrophages were used, whereas LPS was active only in C3H/OuJ
macrophages. Similar to CHO/CD14 cells, GBS induced NF-B activation
in mouse macrophages more slowly than LPS, and higher concentrations of
GBS compared with LPS were required to elicit the response. Consistent
with the NF-B results, GBS exhibited a marked ability to cause TNF
release from both C3H/OuJ and C3H/HeJ mouse macrophages, while LPS
induced TNF only in C3H/OuJ macrophages. Our findings extend earlier
reported observations showing the ability of cell walls from
Gram-positive Bacillus subtilis and Staphylococcus
aureus to induce nitrate production in C3H/HeJ macrophages (11).
Thus, in contrast to LPS signaling, a number of cellular responses
induced by Gram-positive cell walls, including nitrite production,
NF-B activation, and TNF release, are not influenced by an apparent
defect in the sphingomyelin pathway that is observed in macrophages
from C3H/HeJ mice.
In summary, this study shows that LPS and GBS share CD14, but
differentially use the leukocyte integrins for signaling NF-B
activation in CHO cells and TNF release from human monocytes. In
addition, it demonstrates the ability of GBS to mediate NF-B
activation and TNF release in LPS-hyporesponsive C3H/HeJ
(LPSd) mouse macrophages that also appear to
express a defect in the sphingomyelin signaling pathway. Identification
of structural GBS moieties, responsible for its biologic activity, will
help bring a better understanding of the differences between LPS and
GBS signaling as well as the mechanisms behind GBS signal
transduction.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrei E. Medvedev, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814–4788. E-mail address:
3 Abbreviations used in this paper: LBP, lipopolysaccharide binding protein; sCD14, soluble CD14; BPI, bactericidal permeability-increasing protein; GBS, group B streptococci type III; ReLPS, rough lipopolysaccharide; EMSA, electrophoretic mobility shift assay; CHO, Chinese hamster ovary.
Received for publication November 3, 1997. Accepted for publication December 30, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to LPS. J. Immunol. 150:5556.[Abstract]
by Mycobacterium tuberculosis components. J. Clin. Invest. 91:2076.
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C. W. Lee, S. Bennouna, and E. Y. Denkers Screening for Toxoplasma gondii-Regulated Transcriptional Responses in Lipopolysaccharide-Activated Macrophages Infect. Immun., March 1, 2006; 74(3): 1916 - 1923. [Abstract] [Full Text] [PDF]
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S. Kenzel, G. Mancuso, R. Malley, G. Teti, D. T. Golenbock, and P. Henneke c-Jun Kinase Is a Critical Signaling Molecule in a Neonatal Model of Group B Streptococcal Sepsis. J. Immunol., March 1, 2006; 176(5): 3181 - 3188. [Abstract] [Full Text] [PDF]
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