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ProductionαDown-Regulates TNF-
butStaphylococcus aureusby
CD14 Plays No Major Role in Shock Induced
Sophie C. Gangloff and Sanna M. Goyert
Alain Haziot, Naoki Hijiya, Karine Schultz, Fan Zhang,
http://www.jimmunol.org/content/162/8/4801
1999; 162:4801-4805; ;J Immunol
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 1999 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
by guest on February 25, 2013http://jimmunol.org/Downloaded from
CD14 Plays No Major Role in Shock Induced by
Staphylococcus aureus but Down-Regulates TNF-
a
Production
1
Alain Haziot, Naoki Hijiya, Karine Schultz, Fan Zhang, Sophie C. Gangloff, and
Sanna M. Goyert
2
Recent in vitro studies have suggested that CD14, a major receptor for LPS, may also be a receptor for cell wall components of
Gram-positive bacteria and thus play a role in Gram-positive shock. To analyze the in vivo role of CD14 in responses to Gram-
positive bacteria, CD14-deficient and control mice were injected with Staphylococcus aureus, and the effects on lethality, bacterial
clearance, and production of cytokines were analyzed. Survival of CD14-deficient and control mice did not differ significantly after
administration of various doses of either unencapsulated or encapsulated S. aureus; furthermore, mice in both groups displayed
similar symptoms of shock. In addition, inflammatory cytokines such as TNF-
a
and IL-6 were readily detectable in the serum of
CD14-deficient mice injected with live or antibiotic-killed S. aureus. Surprisingly, the serum concentration of TNF-
a
in CD14-
deficient mice was at least threefold higher than in control mice after injection of either unencapsulated or encapsulated S. aureus,
suggesting that CD14 down-regulates TNF-
a
. A similar increase in serum TNF-
a
occurred when CD14-deficient animals were
injected with gentamicin-killed bacteria even though no symptoms of shock were observed. These studies indicate that CD14, in
contrast to its key function in responses to the Gram-negative bacterium, Escherichia coli 0111, does not play a prominent role
in septic shock induced by S. aureus, and that the symptoms of S. aureus shock are not due solely to TNF-
a
. The Journal of
Immunology, 1999, 162: 4801–4805.
A
severe bacterial infection caused by either Gram-posi-
tive or Gram-negative organisms can lead to hemody-
namic shock accompanied by symptoms such as fever,
tachycardia, and hyperventilation, and possibly ending in multior-
gan failure (1). CD14, a GPI-anchored protein that is highly ex-
pressed on the surface of human monocytes and most macrophages
(2, 3), is a key mediator of septic shock induced by the Gram-
negative bacterium, Escherichia coli 0111. The symptoms of
shock can be reproduced with administration of purified LPS (en-
dotoxin), the major toxic component of the outer membrane of
Gram-negative bacteria (4–6). Mice deficient in CD14 show no
gross symptoms of shock (weakness, ruffled fur, labored breathing)
and are resistant to the lethal effects of both LPS and E. coli 0111
(4). Furthermore, they produce very low amounts of proinflamma-
tory cytokines (TNF-
a
, IL-6, IL-1) (4) when injected with either
LPS or live E. coli. It has been proposed that CD14 may also be an
important mediator of septic shock caused by Gram-positive bac-
teria based on the ability of anti-CD14 mAbs to inhibit the re-
sponses of monocytes/macrophages to Gram-positive cell wall
components (7–13). To address the role of CD14 in Gram-positive
septic shock, the responses of CD14-deficient mice to Staphylo-
coccus aureus were compared with normal mice by measuring
lethality, bacterial clearance, and cytokine production.
Materials and Methods
Mice
CD14-deficient mice (4) were backcrossed three to six times with
C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) or with
BALB/c (Harlan Sprague-Dawley, Madison, WI). Age- and sex-matched
CD14-deficient and control mice of the same strain (C57BL/6J or BALB/c)
were used as indicated in the text. All experiments were Institutional An-
imal Care and Use Committee approved and were performed in compliance
with the National Institute of Health and the New York State regulations on
animal handling and usage.
Bacteria
Ten different isolates of S. aureus (unencapsulated) were obtained from
the clinical microbiology laboratory, North Shore Health Systems
(a kind gift of Dr. Christine Ginocchio), from infected patients. S. au-
reus, strain M, was a gift of Dr. C. Y. Lee (University of Kansas), and
was previously shown to be a highly virulent encapsulated strain (14).
Analysis using india ink staining confirmed that the strain M isolate
contained its capsule while all of the other clinical isolates lacked a
capsule (data not shown).
Preparation of S. aureus and injections. S. aureus was cultured in tryptic
soy broth (Difco, Detroit, MI) to midlogarithmic phase, harvested by cen-
trifugation (2500 3 g, 30 min, 4°C), washed twice with pyrogen-free saline
(Baxter Healthcare, Deerfield, IL), and resuspended in saline. The concen-
tration of bacteria was estimated from the absorbance at 530 nm or by
fluorescence (LIVE/DEAD kit; Molecular Probes, Eugene, OR) and con-
firmed by viable CFU counts on agar plates. Antibiotic-killed bacteria were
prepared by incubating the washed bacteria in saline containing 800
m
g/ml
gentamicin (Life Technologies, Gaithersburg, MD) for2hat37°C, fol-
lowed by two washes in saline and plating on agar plates to confirm the
efficiency of the treatment. No viable bacteria were detected in neat sam-
ples of gentamicin-treated S. aureus. Mice were injected with bacteria and
monitored for their physical signs and death for up to 3 wk.
Determination of the number of bacteria and cytokines in blood and
organs. Blood samples and organs were collected and processed as de-
scribed (4). Bacterial CFU were determined by plating serial 10-fold di-
lutions on agar. The concentration of TNF-
a
in mouse serum was measured
by cytotoxicity using WEHI-2F cells, as previously described (15). The
concentration of IL-6 in mouse serum was measured by ELISA (Endogen,
Boston, MA).
North Shore University Hospital/New York University School of Medicine, Manhas-
set, NY 11030
Received for publication August 26, 1998. Accepted for publication January 19, 1999.
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 Grant AI23859, Council
for Tobacco Research Grant 2218 (to S.M.G.), and the American Heart Association
New York State Affiliate Grant-In-Aid 960159 (to A.H.).
2
Address correspondence and reprint requests to Dr. Sanna M. Goyert, Division of
Molecular Medicine, North Shore University Hospital/NYU School of Medicine, 350
Community Drive, Manhasset, NY 11030. E-mail address: sannag@aol.com
Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00
by guest on February 25, 2013http://jimmunol.org/Downloaded from
Cell wall preparation
The cell walls of the encapsulated S. aureus strain M were prepared as
described by De Jonge et al. (16). Briefly, bacteria were grown in1Lof
tryptic soy broth, chilled, and harvested by centrifugation. The bacteria
were boiled for 30 min in a 4% SDS solution in saline. The cells, recovered
by centrifugation and washed seven times in water, were mixed with
baked, acid-washed glass beads and disrupted by vigorous agitation (15
min vortex and 15 min shaking at 540 rpm/min (G24 shaker; New Bruns-
wick Scientific, Edison, NJ)). After an initial centrifugation (10 min at
2000 3 g), the supernatant was collected and centrifuged at 27,500 3 g for
30 min and the pellet was resuspended in a 100 mM Tris-HCl, pH 7.5,
solution supplemented with 10
m
g/ml DNase I (Sigma, St. Louis, MO),
50
m
g/ml RNase A (Boehringer Mannheim, Indianapolis, IN), and 20 mM
MgSO
4
. The resuspended pellet was then incubated for 90 min at 37°C;
CaCl2 (10 mM final concentration) and trypsin (100
m
g/ml; EM Labora-
tories, Elmsford, NY) were added, and the suspension was further incu-
bated for 24 h at 37°C. The enzymes were then inactivated by boiling 15
min in the presence of 1% SDS. This preparation was then washed five
times with water, once with 8 M LiCl, once with 100 mM EDTA, twice
with water, once with acetone, and six times with water before
lyophilization.
Stimulation of peritoneal macrophages
Thioglycolate-elicited peritoneal cells (2 3 10
6
/ml) were incubated for 3 h
at 37°C under 5% CO
2
in RPMI-HEPES supplemented with 1% autolo-
gous serum in 24-well plates. After removal of nonadherent cells, macro-
phages were incubated for3hinthepresence of increasing concentrations
of cell walls from S. aureus strain M or soluble peptidoglycan from S.
aureus (a gift of R. Dziarski Indiana University School of Medicine) in 0.5
ml RPMI-HEPES/1% autologous serum with or without polymyxin B (5
m
g/ml; Sigma). The supernatants were then collected and assayed for
TNF-
a
by ELISA, as described below.
Preparation of spleen cells and stimulation with S. aureus
Spleen cells were washed in RPMI supplemented with HEPES (10 mM)
(Life Technologies). Adherent cells were prepared by incubation of washed
spleen cells in tissue culture dishes for4hat37°C in 5% CO
2
and removal
of nonadherent cells by gently washing the dishes three times with warm
RPMI-HEPES. Whole spleen cells and adherent cells were cultured in
RPMI-HEPES supplemented with 1% autologous serum. For stimulation,
increasing numbers of washed S. aureus were added to 5 3 10
6
spleen
cells, followed 30 min later with the addition of gentamicin (100
m
g/ml)
and further incubation for3hat37°C in 5% CO
2
. TNF-
a
was measured
in cell-free supernatants by ELISA using the mAb TN3 19.12 (17) and a
polyclonal goat anti-recombinant murine TNF-
a
(gifts of Dr. Schreiber,
Washington University, St. Louis, MO).
Statistical analyses
Statistical comparisons were made using the Fisher “exact” test (for sur-
vival studies) or the two-tailed Mann-Whitney test (all others).
Results
The role of CD14 in S. aureus-induced shock
Previous studies indicated that cell wall products from Gram-pos-
itive bacteria could stimulate the activation of human and murine
macrophages via CD14. These observations suggested that, as for
E. coli 0111 (4), some of the deleterious effects of Gram-positive
infection leading to shock might be CD14 dependent (7–13). To
test this hypothesis, CD14-deficient and control mice were injected
with various doses of S. aureus and monitored for lethality. A
panel of 11 different isolates of S. aureus was used. These con-
sisted of 10 unencapsulated isolates of S. aureus obtained from
infected patients, and one highly virulent encapsulated isolate,
strain M (14). Approximately 12 h following the injection of a
lethal dose of any of the isolates of S. aureus, both CD14-deficient
and control mice showed similar shocklike symptoms, including
ruffled hair, eye exudate, and diarrhea. A summary of the survival
data for CD14-deficient and control mice injected i.v. with an un-
encapsulated isolate of S. aureus (isolate 10) at doses ranging from
2.5 3 10
6
to 1.5 3 10
7
CFU/gram body weight is shown in Table
I. There is no increase in survival rate among the CD14-deficient
mice compared with the control mice. When the encapsulated iso-
late of S. aureus, strain M, was similarly tested, there was again no
statistically significant difference in survival between CD14-defi-
cient and control mice, although the CD14-deficient mice showed
a slight trend toward lower survival. Similar results were obtained
when S. aureus was administered i.p. instead of i.v. (data not
shown). These results indicate that there is no remarkable differ-
ence in the survival of CD14-deficient and normal mice after i.v.
or i.p. injection with either encapsulated or unencapsulated
S. aureus.
Clearance of S. aureus in CD14-deficient and control mice
CD14-deficient mice have previously been shown to more rapidly
clear the Gram-negative organism, E. coli 0111, than control mice,
resulting in reduced bacterial dissemination (4). Although lack of
CD14 has no effect on survival to a lethal S. aureus infection, it
might, nevertheless, play a role in the clearance of S. aureus. Ac-
cordingly, the number of bacteria in the blood and tissues of
CD14-deficient and control mice infected with encapsulated or un-
encapsulated S. aureus was examined. Mice were injected i.v. with
9 3 10
6
CFU/g of the unencapsulated isolate of S. aureus (number
10), and the number of bacteria present in blood and organs after
Table I. Data on unencapsulated and encapsulated S. aureus
Dose
(CFU/gbw)
No. of
Mice/Group
Survival
a
(control),
Days After Infection
%
Survival
b
Survival
a
(CD14-deficient),
Days After Infection
%
Survival
b
247 247
Unencapsulated
c
1.5 3 10
7
7 774 57 3 0 0 0
7.5 3 10
6
6 100 0 2 1 0 0
4.0 3 10
6
6 6 1 1 17 6 2 1 17
2.5 3 10
6
7 320 0 1 1 1 24
Encapsulated
d
2.9 3 10
7
4 111 25 0 0 0 0
2.5 3 10
7
3 000 0 0 0 0 0
1.75 3 10
7
10 000 0 1 0 0 0
1.4 3 10
7
7 433 43 2 0 0 0
0.7 3 10
7
12 12 8 7 58 9 9 7 58
a
Number of mice surviving.
b
Percent survival after 7 days.
c
Dose calculated per weight at each individual mouse.
d
Dose calculated as approximate based on total amount injected per mouse per weight range.
4802 CD14 AND GRAM-POSITIVE SHOCK
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6 h of infection was determined. As shown in Fig. 1, CD14-defi-
cient and control mice had very similar numbers of S. aureus in
their blood, liver, and spleen. A similar result was obtained with
the encapsulated strain of S. aureus (data not shown). These results
indicate that, in contrast to the effects observed after infection with
E. coli 0111, CD14 does not play a role in the clearance of
S. aureus.
The role of CD14 in the production of cytokines induced by S.
aureus
Previous in vitro studies have shown that Abs to CD14 can inhibit
the release of inflammatory mediators induced by cell wall com-
ponents from Gram-positive bacteria (7–13). To determine
whether CD14, despite no effect on survival or bacterial clearance,
might nevertheless play a role in the in vivo induction of inflam-
matory cytokines during infection with a live Gram-positive bac-
terium, the serum concentrations of TNF-
a
and IL-6 were mea-
sured in CD14-deficient and control mice after infection with the
encapsulated S. aureus strain M or the unencapsulated S. aureus
isolate 10 using the C57BL/6 or BALB/c background mice, re-
spectively. In keeping with previous reports (18, 19), the concen-
tration of TNF-
a
in the serum of control animals was very low or
absent (Fig. 2, a and c). Surprisingly, CD14-deficient animals pro-
duced substantially more TNF-
a
than control mice; animals in-
jected with either encapsulated or unencapsulated S. aureus pro-
duced at least three times as much TNF-
a
as control animals. In
contrast to the results with TNF-
a
, IL-6 was only slightly elevated
(maximum twofold) in CD14-deficient animals injected with this
dose of strain M (Fig. 2b) as compared with controls; at lower
doses of strain M, there was virtually no difference in IL-6 pro-
duction (data not shown), indicating that there is no CD14-depen-
dent IL-6 response to S. aureus in vivo. Furthermore, no differ-
ences in IL-6 expression were detected in animals injected with the
high dose of unencapsulated S. aureus (Fig. 2d).
The dramatic differences in TNF-
a
production by CD14-defi-
cient and control mice in response to S. aureus were consistently
observed in multiple experiments. Furthermore, the enhanced
TNF-
a
production by CD14-deficient mice was also observed in
vitro; when spleen cells from CD14-deficient and control mice
(C57BL/6) were incubated with live S. aureus (strain M), culture
supernatants from cells from CD14-deficient mice showed greater
levels of TNF-
a
than culture supernatants from cells from control
mice (Fig. 3). Similar results were obtained after a 6-h incubation
or when nonadherent spleen cells were removed before activation
(not shown). In contrast, when peritoneal macrophages from
CD14-deficient and control mice were exposed to either purified
cell walls or peptidoglycan from S. aureus, there was a clear de-
pendence on the presence of CD14 for the production of TNF-
a
(Fig. 4).
Antibiotic-treated S. aureus induce TNF-
a
, but not septic shock
To examine whether cell wall components of Gram-positive or-
ganisms are sufficient to cause shock, CD14-deficient and control
mice were injected with gentamicin-killed unencapsulated (isolate
10) S. aureus (Fig. 5a) and monitored for cytokine production,
symptoms of septic shock, and lethality. Gentamicin is a bacteri-
cidal antibiotic that acts by inhibiting protein synthesis without
FIGURE 1. Blood and organ counts (CFU) of S. aureus (unencapsulat-
ed) in CD14-deficient and control mice. CD14-deficient mice (backcrossed
to BALB/c six times) or control BALB/c were injected i.v. with 9 3 10
6
CFU/g of S. aureus isolate 10 in 0.2 ml nonpyrogenic saline. The blood and
the organs were collected 7 h later, and bacterial counts were determined
by serial dilution plating on agar. Results are represented as the mean (n 5
3) number of CFU in each organ 6 SEM.
FIGURE 2. Production of TNF-
a
and IL-6 in CD14-deficient and con-
trol mice after infection with S. aureus. CD14-deficient mice (backcrossed
to C57BL/6 mice three times) and control C57BL/6 were used in a and b,
and CD14-deficient mice (backcrossed to BALB/c six times) and control
BALB/c were used in c and d. Mice were injected i.v. with 3 3 10
8
CFU
(approximately 1.5 3 10
8
CFU/g) of encapsulated S. aureus (strain M) (a
and b)or2.53 10
7
CFU/g of unencapsulated S. aureus isolate 10 (c and
d) in 0.2 ml of nonpyrogenic saline. Mice were bled at the indicated times,
and TNF-
a
(a and c) and IL-6 (b and d) were measured in the serum. Open
squares, CD14 deficient; filled circles, controls. Results are represented as
the mean (n 5 3) 6 SEM.
FIGURE 3. Secretion of TNF-
a
by spleen cells in the presence of S.
aureus. Spleen cells (5 3 10
6
) from CD14-deficient and control mice were
incubated with increasing numbers of S. aureus (strain M). Thirty minutes
later, gentamicin (100
m
g/ml) was added to the samples; after further in-
cubation (total, 3 h), TNF-
a
was measured in the cell-free supernatant by
ELISA. The results are representative of three independent experiments,
and are shown as the mean of duplicate determinations 6 SD; lack of error
bars indicates that the error falls within the symbol. p, p , 0.02 (Mann-
Whitney test).
4803The Journal of Immunology
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directly altering the bacterial membrane and its components, leav-
ing an intact cell wall and capsule. This preparation differs from
cell walls prepared by the classical method particularly in that
proteins are not denatured. As can be seen in Fig. 5a, CD14-de-
ficient animals again produced substantially more TNF-
a
than
control animals, yet in neither case were gross symptoms of shock
(ruffled fur, labored breathing, eye exudate, diarrhea) or lethality
observed, even though the amounts of TNF-
a
produced were sim-
ilar to those in the serum of animals injected with live bacteria.
Furthermore, mice injected with the encapsulated S. aureus
showed a response similar to those injected with the unencapsu-
lated organism (Fig. 5b).
Discussion
The studies described were designed to analyze the role of CD14
in septic shock induced by the Gram-positive bacterium, S. aureus.
Previous studies have shown that CD14 plays a major role in the
response to the Gram-negative bacterium, E. coli 0111-B4 (4).
Since the symptoms seen in Gram-positive shock are very similar
to those seen in Gram-negative shock, it has been proposed that a
LPS analogue may reside in the cell wall of Gram-positive bacteria
that can similarly induce Gram-positive shock (1, 20). Indeed,
studies have shown that a combination of lipoteichoic acid and
peptidoglycan from S. aureus can induce shock in rats (21). Fur-
thermore, it has been proposed that CD14 serves as the receptor on
monocytes/macrophages for mediating this response (7–11).
To examine whether CD14 might play a role in the in vivo
response to Gram-positive bacteria, CD14-deficient and control
mice were injected with a lethal dose of S. aureus, and the effects
on lethality, bacterial clearance, and cytokine production were de-
termined. Surprisingly, no differences were observed between
CD14-deficient and control mice. Similar results were obtained
with the two different types of S. aureus utilized in these studies,
a highly virulent encapsulated form, strain M, which has a very
large polysaccharide capsule (type 1) (14), and the more common
unencapsulated strain of S. aureus, which is sensitive to phagocy-
tosis. The inability to demonstrate any differences in the gross
symptoms of septic shock or death rate in CD14-deficient and
control mice is quite distinct from what was observed with the
Gram-negative organism, E. coli 0111; there, CD14-deficient mice
showed no symptoms of shock or death when exposed to a lethal
dose of E. coli 0111.
In addition to the lack of a clear difference in symptoms and
death between CD14-deficient and control mice, there was no in-
dication of reduced cytokine production by CD14-deficient mice;
in fact, CD14-deficient mice produced more TNF-
a
than control
mice when exposed to live S. aureus (Fig. 2). This induction of
TNF-
a
could be reproduced by gentamicin-killed organisms (Fig.
5), suggesting that cell wall components may be responsible for
this response. However, the presence of a capsule (strain M),
which has been shown to inhibit some cell wall-mediated events
(22–24), did not influence TNF-
a
production, suggesting that the
bacterial component inducing TNF-
a
may not be masked by the
capsule. Alternatively, the TNF-
a
-inducing component may be a
non-cell wall component that does not require new protein
synthesis.
FIGURE 4. TNF-
a
production by peritoneal macrophages stimulated
with cell walls or soluble peptidoglycan (sPGN) from S. aureus.
Thioglycolate-elicited peritoneal macrophages from control and CD14-de-
ficient mice (C57BL/6 background) were incubated for3hat37°C with the
indicated concentrations of either S. aureus cell walls (a) or sPGN (b)in
RPMI-HEPES supplemented with 1% autologous serum. The cell-free su-
pernatants were assayed for TNF-
a
by ELISA and plotted as the mean of
duplicate determinations 6 SD; lack of error bars indicates that the error
falls within the symbol. Results are representative of two independent
experiments.
FIGURE 5. Production of TNF-
a
in CD14-deficient and control mice
after injection of antibiotic-killed S. aureus. CD14-deficient mice (back-
crossed to BALB/c 10 times) and control BALB/c were used in a, and
CD14-deficient mice (backcrossed to C57BL/6 mice three times) and con-
trol C57BL/6 were used in b. Mice were injected i.v. with 5 3 10
7
CFU/
gram body weight of gentamicin-killed S. aureus isolate 10 (a)or13 10
9
CFU of gentamicin-killed S. aureus (strain M) (b) in 0.2 ml of nonpyro-
genic saline. Mice were bled at the indicated times, and TNF-
a
was mea-
sured in the serum. Open squares, CD14 deficient; filled circles, controls.
Results are represented as the mean (n 5 3) 6 SEM.
4804 CD14 AND GRAM-POSITIVE SHOCK
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Although TNF-
a
is produced, its induction is not sufficient to
produce death or symptoms of shock; mice exposed to gentamicin-
treated bacteria produce a similar amount of TNF-
a
as those ex-
posed to live S. aureus, and yet no death or symptoms of shock
were observed. The inability to observe differences in TNF-
a
pro-
duction in response to either live or antibiotic-killed S. aureus
brings into question the physiologic relevance of in vitro studies
that examine the induction of TNF-
a
by Gram-positive cell walls
or products.
The lack of a deleterious role for TNF-
a
in the septic shock
response to Gram-positive organisms may not be surprising in
view of the observation that the introduction of anti-TNF Ab into
mice after infection by live S. aureus actually increases the death
rate (19). Based on these studies, the authors have proposed that
low levels of TNF-
a
may actually be protective against Gram-
positive infections. Indeed, it should be noted that the total amount
of circulating TNF-
a
produced by control or CD14-deficient mice
after infection with S. aureus is 7 to 18 times lower than the
amount produced after infection with E. coli 0111 (Fig. 2a) (4), in
which TNF-
a
presumably has a deleterious effect (25). Whether
the protective effects referenced above are due to such reduced
levels of TNF-
a
remain to be determined. Similarly, the observa-
tion by others that heat-killed S. aureus can kill mice (26) does not
contradict our data in which we fail to observe death with gen-
tamicin-killed S. aureus, since the former experiments were per-
formed after sensitization with
D-galactosamine, an agent that
makes mice hypersensitive to even very small amounts of TNF-
a
.
The ability of CD14-deficient mice to produce TNF-
a
in re-
sponse to live or gentamicin-killed organisms is quite surprising in
view of the proposed role for CD14 in cytokine induction by
Gram-positive cell wall products. It indicates the presence of a
non-CD14 receptor that can respond to Gram-positive compo-
nents. This receptor is present on splenic macrophages since such
cells from CD14-deficient mice produce large quantities of TNF-
a
in response to S. aureus (Fig. 3). A similar CD14 independence
was seen in the in vitro response of human monocytes to heat-
killed unencapsulated pneumococcus (12).
The increased expression of TNF-
a
in CD14-deficient mice is
also surprising, and suggests a role for CD14 in down-regulating
TNF-
a
responses to Gram-positive organisms. Presumably, a cell
wall component of Gram-positive organisms that is not masked by
a capsule is responsible for this down-regulation, since gentami-
cin-killed unencapsulated or encapsulated bacteria show the same
effect. Whether this Gram-positive component is one of the so far
proposed ligands of CD14 (10, 27) or an unrelated component of
the bacterium remains to be determined. Such a mechanism of
negative signaling has recently been described with receptors such
as Fc
g
RII and CD22 (28–30), and requires phosphorylation by the
protein-tyrosine kinase Lyn (31). Although the molecular mecha-
nisms used by CD14, a GPI-anchored glycoprotein (3), to transmit
signals into the cell are still unknown, it is intriguing to note that
CD14 has also been shown to coprecipitate with Lyn (32); how-
ever, the molecular mechanisms regulating the generation of a neg-
ative signal by CD14 or an associated signaling molecule remain
to be determined.
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2. Goyert, S. M., E. Ferrero, W. J. Rettig, A. K. Yenamandra, F. Obata, and
M. M. Le Beau. 1988. The CD14 monocyte differentiation antigen maps to a
region encoding growth factors and receptors. Science 239:497.
3. Haziot, A., S. Chen, E. Ferrero, M. G. Low, R. Silber, and S. M. Goyert. 1988.
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