Download Involvement of CD14 and Complement Receptors CR3 and CR4 in

Involvement of CD14 and Complement Receptors CR3 and
CR4 in Nuclear Factor-kB Activation and TNF Production
Induced by Lipopolysaccharide and Group B Streptococcal
Cell Walls1
Andrei E. Medvedev,2* Trude Flo,† Robin R. Ingalls,‡ Douglas T. Golenbock,‡ Giuseppe Teti,§
Stefanie N. Vogel,* and Terje Espevik†
This study was undertaken to evaluate the role of CD14 and complement receptors type 3 (CR3) and 4 (CR4) in mediating TNF
release and NF-kB 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 b-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-kB translocation, which was inhibited by anti-CD14 mAb and enhanced by LPS binding protein. While LPS showed fast
kinetics of NF-kB activation in CHO/CD14 cells, a slower NF-kB response was induced by GBS. LPS also activated NF-kB 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-kB 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-kB translocation, whereas LPS was active only
in C3H/OuJ macrophages. Thus, LPS and GBS differentially involve CD14 and CR3 or CR4 for signaling NF-kB 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. The Journal of Immunology, 1998, 160: 4535– 4542.
L
ipopolysaccharide, a glycolipid of the outer membrane of
Gram-negative bacteria, is an important mediator of endotoxic shock, a syndrome that is associated with uncontrolled production of inflammatory cytokines upon interaction of
bacteria with immune cells (1, 2). CD14, a 55-kDa glycosylphosphatidylinositol-anchored protein expressed on the surface of
monocytes and neutrophils (3, 4), binds LPS with high affinity.
*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
Received for publication November 3, 1997. Accepted for publication December
30, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI18797 (to
S.N.V.), RO1GM54060, and STD CRC AI38515 (to D.T.G.); Ministero della Sanità
of Italy AIDS Grant 9305– 44 (to G.T.); and the Norwegian Cancer Society and the
Norwegian Research Council (to T.E). The opinions or assertions contained within
are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or the Department of Defense. Research was conducted according to the principles set forth in Guide for the Care and Use of Laboratory Animals, prepared by the
Institute of Laboratory Animal Resources, National Research Council, DHEW Publication (National Institute of Health) 85-23.
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:
[email protected]
Copyright © 1998 by The American Association of Immunologists
The serum LPS binding protein (LBP)3 significantly increases LPS
binding to CD14 (5, 6) and facilitates transfer of LPS monomers
into high density lipoproteins (7), thereby regulating its activities.
CD14 is appreciated as one of the components of an LPS receptor
complex due to the ability of anti-CD14 Abs to block LPS responses (5, 8) and because transfection of CD14 into CD14-negative cells greatly enhances LPS-induced activities (9 –11). CD14
also exists as a soluble protein, present in plasma in microgram
quantities (12), and complexes of sCD14 and LPS have been
shown to activate CD14-deficient cells (13–16). Moreover, low or
intermediate concentrations of LPS do not induce cellular responses in CD14 knockout mice (17, 18) or in cells lacking membrane CD14 (10, 11). Conversely, transgenic mice that overexpress
CD14 show hypersensitivity to LPS (19). Since not only LPS but
also bacterial compounds from several Gram-positive species
show CD14 dependence (11, 20 –23), CD14 was postulated to be
a pattern recognition receptor that recognizes a wide variety of
bacterial products, leading to cell activation (11). However, high
LPS concentrations induce cytokine production and gene expression via a CD14-independent pathway (17, 18), and lipid IVA, the
tetra-acyldisaccharide lipid A precursor, inhibits LPS responses in
human monocytes without affecting LPS-CD14 binding (24). Interestingly, in mouse macrophages, lipid IVA is an LPS mimetic,
as evidenced by its ability to stimulate arachidonic acid release and
TNF production (25). Furthermore, regardless of whether human
or murine CD14 is expressed in the given rodent or human cell
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.
0022-1767/98/$02.00
4536
UTILIZATION OF CD14, CR3, AND CR4 BY LPS AND GBS
lines, lipid IVA always is an antagonist in human cells and an
agonist in mouse cells (26). These findings question the role of
CD14 as a signal-transducing receptor and suggest that LPS may
interact with another, as yet unidentified molecule(s) capable of
propagating intracellular signals.
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 b2 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 b-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-kB 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-kB 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-kB-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 GBSinduced TNF production from human monocytes. The data suggest
that while LPS and GBS share CD14, they differentially use CR3
and CR4 for triggering NF-kB 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-kB translocation. This suggests
that, in contrast to LPS, GBS does not use the sphingomyelin pathway for signaling TNF release and NF-kB activation.
ville, 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 3 107 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 A1 buffy coats (The BloodBank,
Norwegian University of Science and Technology, Trondheim, Norway) as
previously described (35). Adherent cell monolayers were cultured in 24well plates (Costar, Cambridge, MA) in either AIM serum-free medium
(Life Technologies, Grand Island, NY) supplemented with 1% L-glutamine
and 40 mg/ml gentamicin or in AIM medium containing 25% human A1
serum. Monocytes were stimulated for 8 h at 37°C with the indicated preparations. Thereafter, supernatants were collected and stored at 280°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 mg/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 3 106 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 3 106 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 280°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 3 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 3 g, 10 min, 4°C) and
resuspended in 50 ml 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 3 g, 10 min, 4°C), and supernatants were
collected and stored at 280°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)
Materials and Methods
Reagents
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 3 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 (Rock-
The NF-kB-specific oligonucleotide probe 59-AGTTGAGGGGACTTTC
CCAGGC-39 (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 kB light chain gene
enhancer was 32P end labeled with T4 polynucleotide kinase (Promega).
Nuclear extracts (4 mg) were incubated with 0.2 ng of DNA probe in a
binding buffer containing 2 mg 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 (0.253 Tris borateEDTA, 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
The Journal of Immunology
previously (37). The lower limit of detection in this assay was 0.35
pg/ml TNF.
Results
4537
caused a moderate NF-kB 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
LPS and GBS induce TNF production from human monocytes in
a CD14-dependent manner, and human serum, LBP, and sCD14
enhance the response
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 50to 100-fold more potent than GBS in stimulating TNF release in
both the presence and the absence of normal human A1 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 CD14independent mechanism of cell stimulation. As shown in Figure 1,
A and B, human serum significantly potentiated not only LPSstimulated 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 6
274 to 1258 6 66 pg/ml. However, GBS-induced TNF secretion
(698 6 62 pg/ml) was not inhibited by BPI (744 6 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.
Transfection of CHO cells with CD14 cDNA renders them
responsive for induction of NF-kB 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-kB,
whereas no NF-kB translocation was seen in control CHO/NEO
transfectants. In CHO/CD14 cells, detectable NF-kB 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-kB 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
FIGURE 1. Effects of anti-CD14 mAb, sCD14, and LBP on LPS- and
GBS-stimulated TNF production from human monocytes. Human monocytes were stimulated with serial dilutions of ReLPS from S. minnesota (A)
or GBS (B) either in AIM containing 25% human A1 serum (open symbols) or in serum-free AIM medium (closed symbols) alone (circles), in the
presence of anti-CD14 mAb 3C10 (triangles) or with a control mAb 6H8
(squares). C, Cells were incubated with 20 ng/ml LPS from P. aeruginosa
or with 2 mg/ml GBS alone (blank bars) and in the presence of 100 ng/ml
rLBP (left-hatched bars), 100 ng/ml sCD14 (cross-hatched bars), and their
combination (black bars). Supernatants were harvested after 6 h and assayed for TNF. Shown are data from a representative experiment (mean 6
SD of triplicate wells). Similar results were obtained in two other independent experiments.
4538
UTILIZATION OF CD14, CR3, AND CR4 BY LPS AND GBS
FIGURE 2. LPS and GBS elicit NF-kB activation
in CHO/CD14, but not in CHO/NEO cell lines. CHO/
NEO (A) and CHO/CD14 (A and B) cells were incubated for the indicated time periods (minutes) with 10
mg/ml ReLPS from S. minnesota or with 50 mg/ml
GBS in AIM serum-free medium. Following stimulation, nuclear extracts were prepared, and EMSAs
were performed. Arrowheads indicate the main inducible NF-kB-specific bands. The results of a representative experiment (of three performed) are given.
(Fig. 1, A and B), 250- to 500-fold higher concentrations of GBS
were needed to observe NF-kB 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-kB (Fig.
3A). As was the case for human monocytes (Fig. 1C), recombinant
LBP markedly potentiated NF-kB 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.
LPS, but not GBS, activates NF-kB 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-kB 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-kB 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 mg/ml) was required to achieve strong NF-kB binding activity in both cell lines. One microgram per milliliter of LPS induced
moderate responses, and only a weak NF-kB translocation was in-
duced by 100 ng/ml LPS (Fig. 4, A and C). These concentrations of
LPS, however, did not induce NF-kB 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-kB in either CHO/
CR3 or CHO/CR4 cells, even when used at 200 mg/ml. These results
indicate that while LPS can use CR3 or CR4 for signaling NF-kB
activation in CHO cells, the expression of these leukocyte integrins
does not render CHO cells responsive to GBS.
Effects of mAb against CD14 and CD18 on LPS- and GBSinduced TNF production from human monocytes
In the next series of experiments, the involvement of CD14 and
CD18, the common b-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 antiCD14 mAb markedly decreased the capacities of both LPS and
GBS to stimulate TNF release. Interestingly, anti-CD18 mAb
FIGURE 3. Effects of anti-CD14 mAb 3C10 and recombinant LBP on the abilities of LPS and GBS to induce NF-kB in CHO/CD14 cells. A, Cells were
preincubated for 30 min on ice with 10 mg/ml 3C10 in AIM serum-free medium. Then, the indicated concentrations of ReLPS from S. minnesota or GBS
were added, and stimulation was performed for 60 min. B, Cells were stimulated for 60 min under serum-free conditions with the indicated concentrations
of ReLPS from S. minnesota or with GBS either alone or in the presence of 100 ng/ml recombinant LBP. Nuclear extracts were prepared, and EMSAs were
conducted as described in Materials and Methods. Arrowheads indicate the main NF-kB-inducible bands. Shown are the results of a representative
experiment. Similar data were obtained in two other independent experiments.
The Journal of Immunology
4539
FIGURE 4. LPS, but not GBS, induces NF-kB in CHO/CR3 and CHO/CR4 cells. A, CHO/CR3 cells were treated with 10 mg/ml ReLPS from S.
minnesota or with 200 mg/ml GBS for the indicated time periods (time-course experiment) or with serial dilutions of the stimuli for 180 min (dose-response
experiment). B, CHO/CR4 cells were stimulated with 10 mg/ml ReLPS from S. minnesota or with 100 mg/ml GBS for the indicated time periods. C, Serial
dilutions of the stimuli were added to CHO/CR4 cells, and incubation was continued for 180 min. All treatments were performed in AIM serum-free
medium. Following stimulations, nuclear extracts were prepared and analyzed for NF-kB binding activity as measured by EMSA. NF-kB-specific bands
are shown by arrowheads. The results of a representative experiment (of three performed) are depicted.
caused an even greater suppression of GBS-mediated TNF production than did the anti-CD14 mAb (Fig. 5). In contrast, LPSinduced 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.
C3H/OuJ macrophages (Table I). Similar to the NF-kB results,
Table I shows that GBS induced marked TNF responses in both
C3H/OuJ and C3H/HeJ macrophages.
Macrophages derived from both C3H/OuJ and C3H/HeJ mouse
strains respond to GBS with TNF production and NF-kB
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-kB 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 mg/ml LPS
resulted in the induction of NF-kB binding activity after 30 min of
treatment, while GBS-mediated NF-kB 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-kB 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-kB induction in response to LPS
stimulation, whereas GBS-induced NF-kB 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 mg/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
FIGURE 5. Effects of anti-CD14 and anti-CD18 mAb on LPS- and
GBS-induced TNF production from human monocytes. Cells were preincubated with anti-CD14 mAb, 3C10, anti-CD18 mAb, HB 203, or a control
mAb, 6H8, in AIM serum-free medium (all mAb were used at a concentration of 5 mg/ml) for 30 min followed by addition of 0.1 mg/ml LPS from
P. aeruginosa or 10 mg/ml GBS. Supernatants were collected after 8 h of
stimulation and assayed for TNF activity. Spontaneous TNF release from
monocytes incubated with medium alone was ,25 pg/ml. The results of a
representative experiment (mean 6 SD of triplicate determinations) are
shown. Similar data were obtained in three other independent experiments.
4540
UTILIZATION OF CD14, CR3, AND CR4 BY LPS AND GBS
Table I. Comparison of the TNF-inducing potencies of LPS and GBS
in C3H/OuJ and C3H/HeJ mouse peritoneal macrophages a
TNF (pg/ml)
Addition
Amount
(mg/ml)
C3H/OuJ
1.0
0.1
0.01
0.001
165,000 6 9354
72,500 6 1246
40,533 6 6534
6,080 6 320
LPS
GBS
50.0
10.0
2.0
0.4
0
20,334 6 1775
8,345 6 359
3,189 6 143
318 6 11
36 6 5
C3H/HeJ
50 6 8
12 6 6
10 6 3
12 6 4
16,955 6 998
7,318 6 371
2,932 6 274
247 6 37
9 6 0.7
a
Macrophages from C3H/OuJ (Lpsn) and C3H/HeJ (Lpsd) mouse strains were
incubated with the indicated concentrations of LPS from E. coli or GBS for 20 h at
37°C. Cell-free supernatants were harvested and assayed for TNF in the WEHI 164
clone 13 cell line as described in Materials and Methods. Results are shown as
mean 6 SD of a representative experiment. Similar data were obtained in two other
experiments.
FIGURE 6. Time course of NF-kB activation caused by LPS and GBS
in C3H/OuJ and C3H/HeJ mouse peritoneal macrophages. C3H/OuJ (A)
and C3H/HeJ (B) peritoneal macrophages were incubated with 1 mg/ml
K235 LPS from E. coli or with 50 mg/ml GBS for various periods of time
in RPMI/2% FCS medium followed by preparation of nuclear extracts and
NF-kB EMSAs. The main inducible NF-kB band is shown. The data from
a representative experiment are presented. Similar results were obtained in
four other independent experiments.
Discussion
In the present study, several lines of evidence indicate that LPS and
GBS use CD14 for signaling cell activation. First, transfection of
CD14 cDNA into CHO cells conferred upon them the ability to translocate NF-kB 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-kB in CD14expressing 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-kB in CHO/CD14 cells. Fourth, sCD14,
an important mediator of LPS effects (4, 5, 14 –16), increased the
ability of not only LPS, but also GBS, to cause TNF release from
FIGURE 7. Dose response of NF-kB stimulation in C3H/OuJ and C3H/HeJ mouse peritoneal
macrophages elicited by LPS and GBS. EMSAs
were performed on nuclear extracts from macrophages treated for 120 min with the indicated concentrations of LPS from E. coli K235 or with GBS
in RPMI/2% FCS. An arrowhead indicates the main
NF-kB-specific band. Shown are the results of a
representative (of three performed) experiment.
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, 20–23) 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-kB 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-kB in
The Journal of Immunology
CHO cells engineered to express CR3 or CR4. In agreement with
previously reported data (30, 31), LPS induced NF-kB in both
CHO/CR3 and CHO/CR4 cell transfectants. However, GBS failed
to activate NF-kB 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-kB 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-kB 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-kB in CHO/CR3
or CHO/CR4 cells induced NF-kB translocation in CHO/CD14
cells. In addition, recombinant LBP significantly potentiated the
ability of LPS to activate NF-kB in CHO/CR3 cells, yet in combination with GBS, it did not induce NF-kB (our unpublished observation). Of note, in CHO/CD14 cells, both LPS- and GBS-induced NF-kB responses were markedly enhanced by LBP. These
results seem to rule out the possibility that GBS fails to activate
NF-kB 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 b-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-kB 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-kB 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-kB 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 re-
4541
sults 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-kB 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-kB 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-kB activation, they
could lack those necessary for GBS-induced NF-kB 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-kB 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-kB 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-kB
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-kB
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-kB activation in CHO cells and TNF release from human monocytes. In addition, it demonstrates the ability of GBS to mediate NF-kB 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.
4542
UTILIZATION OF CD14, CR3, AND CR4 BY LPS AND GBS
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