Published Ahead of Print 20 August 2007.
2007, 75(11):5415. DOI: 10.1128/IAI.00601-07.
Jack Silver and Sanna M. Goyert
Sik Kim, Sophie C. Gangloff, Saul Teichberg, Alain Haziot,
Shalaka Metkar, Shanjana Awasthi, Erick Denamur, Kwang
: Effects of K1
Role of CD14 in Responses to Clinical
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INFECTION AND IMMUNITY, Nov. 2007, p. 5415–5424
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 11
Role of CD14 in Responses to Clinical Isolates of Escherichia coli:
Effects of K1 Capsule Expression?
Shalaka Metkar,1† Shanjana Awasthi,2†‡ Erick Denamur,3Kwang Sik Kim,4Sophie C. Gangloff,2§
Saul Teichberg,2Alain Haziot,2¶ Jack Silver,1and Sanna M. Goyert1*
Department of Microbiology and Immunology, CUNY Medical School, and Sophie Davis School for Biomedical Sciences, City
College of New York, New York, New York1; Center of Immunology and Inflammation, Laboratory of Innate Immunity, The
Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health Systems, Manhasset, New York2; Ecologie
et e ´volution des micro-organismes, INSERM U722, Faculte ´ de Me ´decine Xavier Bichat, Paris, France3; and Division of
Infectious Diseases, Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, Maryland4
Received 26 April 2007/Returned for modification 8 June 2007/Accepted 8 August 2007
Severe bacterial infections leading to sepsis or septic shock can be induced by bacteria that utilize different
factors to drive pathogenicity and/or virulence, leading to disease in the host. One major factor expressed by
all clinical isolates of gram-negative bacteria is lipopolysaccharide (LPS); a second factor expressed by some
Escherichia coli strains is a K1 polysaccharide capsule. To determine the role of the CD14 LPS receptor in the
pathogenic effects of naturally occurring E. coli, the responses of CD14?/?and CD14?/?mice to three different
isolates of E. coli obtained from sepsis patients were compared; two isolates express both smooth LPS and the
K1 antigen, while the third isolate expresses only LPS and is negative for K1. An additional K1-positive isolate
obtained from a newborn with meningitis and a K1-negative isogenic mutant of this strain were also used for
these studies. CD14?/?mice were resistant to the lethal effects of the K1-negative isolates. This resistance was
accompanied by significantly lower levels of systemic tumor necrosis factor alpha (TNF-?) and interleukin-6
(IL-6) in these mice than in CD14?/?mice, enhanced clearance of the bacteria, and significantly fewer
additional gross symptoms. In contrast, CD14?/?mice were as sensitive as CD14?/?mice to the lethal effects
of the K1-positive isolates, even though they had significantly lower levels of TNF-? and IL-6 than CD14?/?
mice. These studies show that different bacterial isolates can use distinctly different mechanisms to cause
disease and suggest that new, nonantibiotic therapeutics need to be directed against multiple targets.
Sepsis, characterized by a systemic bacterial infection and
systemic inflammatory response syndrome, can lead to life-
threatening conditions, including severe sepsis (sepsis with
multiorgan failure) and septic shock (sepsis with persistent
arterial hypotension) (1, 4, 25). Although the gross symptoms
of sepsis and septic shock appear to be similar and indepen-
dent of the specific type of the invading bacterium, the bacteria
isolated from these patients can be highly varied in terms of the
properties that promote their pathogenicity and/or virulence.
Two widely studied bacterial factors that can influence patho-
genicity are lipopolysaccharide (LPS) (or endotoxin) (20, 29,
33, 34) and some of the capsular polysaccharide K antigens (3,
LPS resides in the outer membrane of all gram-negative
bacteria and can play a major role in pathophysiological re-
sponses by triggering production of large amounts of proin-
flammatory mediators, such as tumor necrosis factor alpha
(TNF-?) and interleukin-6 (IL-6). This induction occurs
through the interaction of LPS with its receptor, CD14 (13),
and its signaling complex, Toll-like receptor 4 (TLR4)/MD2
(for a review, see reference 11). CD14 is expressed strongly on
the surface of monocytes/macrophages (15) and weakly on
granulocytes (18) and some dendritic cells (27, 48). Previous
studies have shown that mice deficient in CD14 (CD14?/?)
produce little or no proinflammatory cytokines (TNF-?, IL-1,
IL-6) in response to LPS and Escherichia coli O111 and are
highly resistant to the lethal effects of both of these factors
(19). These observations indicate that CD14 can play a major
role in the pathophysiological responses caused by LPS and/or
live bacteria expressing LPS.
In addition to LPS, the surface of most E. coli strains is also
covered with a layer of polysaccharides that can be distin-
guished serologically as K antigens (28, 47). K antigens vary in
structure, but only a few of the more than 80 different sero-
types described play a role in enhancing the virulence of a
particular gram-negative bacterium. One example of a K anti-
gen that promotes virulence is the K1 antigen, most commonly
expressed by E. coli isolated from neonates suffering from
septic shock and/or meningitis (39, 42). The K1-polysaccharide
capsule masks surface structures of the bacterium, including
LPS, as well as sites for the binding of opsonins. Masking of the
latter enhances virulence in part by inhibiting the ability of the
host to phagocytose K1-positive E. coli (37). Although LPS is
also masked, it nevertheless has been suggested to play a role
in the pathogenicity of K1-positive E. coli (5); however, the
* Corresponding author. Mailing address: CUNY Medical School/
Sophie Davis School of Biomedical Education, City College of New
York, 160 Convent Ave., New York, NY 10031. Phone: (212) 650-
7773. Fax: (212) 650-7797. E-mail: firstname.lastname@example.org.
† S. Metkar and S. Awasthi contributed equally to this work.
‡ Present address: Department of Pharmaceutical Sciences, Univer-
sity of Oklahoma Health Science Center, Oklahoma City, OK.
§ Present address: Laboratoire d’Immunologie et de Microbiologie,
EA 3796-IFR53, UFR Pharmacie, 1 Av. MalJuin, 51100 Reims,
¶ Present address: Institut National de la Sante ´ et de la Recherche
Me ´dicale Unite ´ 396, Paris, France.
?Published ahead of print on 20 August 2007.
on February 25, 2013 by guest
mechanism and exact role of LPS in the pathogenicity of K1-
positive E. coli have not been clearly documented. It is con-
ceivable that as the K1-positive bacteria divide, LPS and other
membrane components are released, making them available to
activate the innate immune system via CD14. In addition, since
it has been proposed that CD14 acts as a pattern recognition
receptor, direct interaction of the K1-polysaccharide capsule
with CD14 may also trigger host responses.
The studies described here were designed to determine the
role of CD14 in the pathogenic effects of three different iso-
lates of E. coli obtained from sepsis patients that differ in
several characteristics, including their O (LPS) antigen sero-
types and the presence of a K1 capsule; two isolates express
both smooth LPS and the K1 antigen, while the third isolate
expresses only LPS and is negative for K1. An additional K1-
positive E. coli strain isolated from a neonate with meningitis
and a K1-negative isogenic mutant of this strain were also
analyzed. Using CD14?/?and normal (CD14?/?) mice, the
role of CD14 in the host’s response to these five isolates was
examined with respect to TNF-? and IL-6 production, bacte-
rial clearance, and survival.
MATERIALS AND METHODS
Animals. CD14?/?mice (10th backcross on BALB/c) (13, 19) and age- and
weight-matched 6- to 8-week-old CD14?/?wild-type BALB/c mice (Harlan,
Indianapolis, IN) were housed in the animal facility of the City College of New
York, New York, NY, or the Feinstein Institute of Medical Research, Manhas-
set, NY, and were provided with nonsterile laboratory chow (Harlan Teklad,
Madison, WI) and water ad libitum. All animals were maintained and studied in
accordance with recommendations of the Institute of Laboratory Animal Re-
sources (National Academy of Sciences, Washington, DC) and the Institutional
Animal Care and Use Committees of the City College of New York, New York,
NY, and the Feinstein Institute for Medical Research, Manhasset, NY.
Bacteria. Three clinical E. coli isolates utilized in these studies (E. coli isolates
59, 69, and 61) were obtained from patients and were characterized to determine
various virulence factors as described by Picard et al. (31). The characteristics are
summarized in Table 1. These bacteria were shown to belong to the same
phylogenetic group (group 5), defined as nonhemolytic, carboxylase type B2 (Mf
62), and mannose-resistant hemagglutinin-positive organisms. These bacteria
also expressed pap and aer operons; they were negative for the sfa/foc, afa, and
hly operons and ibe-10 gene expression (31). In addition, E. coli isolates 69, 59,
and 61 were biotyped by the Gastroenteric Disease Center (Pennsylvania State
University, University Park) to determine specific O and H serotypes, fimbrial
antigens, and toxin production (Table 1). An additional K1-positive isolate,
RS218 (O18:K1:H7), obtained from a newborn with meningitis, and a K1-neg-
ative isogenic mutant of this isolate (O18:H7), have been described previously
Serum sensitivity. The sensitivity of each bacterial isolate to serum (comple-
ment)-mediated killing was tested as described by Russo et al. (38), with some
modifications. Briefly, fresh human serum was preadsorbed with log-phase E. coli
to remove isoagglutinins, centrifuged at 14,000 ? g for 5 min, filter sterilized, and
stored in aliquots at ?80°C. Each bacterial isolate was washed in Veronal buffer
and resuspended (1 ? 105CFU/ml) in preadsorbed serum (with and without heat
inactivation; final concentration, 80%) for 1 h at 37°C. Bacteria were then diluted
in Veronal buffer and plated on tryptic soy agar, and the number of surviving
bacteria was determined. All isolates were resistant to both normal and heat-
inactivated adsorbed serum. Known serum-resistant (E. coli CP9) and serum-
sensitive (E. coli CP923) strains (kind gifts of T. Russo ) were used as
Electron microscopy of bacterial isolates. The presence of a K1 capsule was
confirmed by electron microscopy of ruthenium red (RR)-stained E. coli using
the modified method of Luft (26). Briefly, fresh bacterial colonies from Trypti-
case soy agar plates (Difco, Detroit, MI) were suspended in 0.3 M sucrose,
pelleted, fixed by resuspension in 8 ml of 2.5% glutaraldehyde in 0.1 M cacody-
late buffer (pH 7.3) containing 0.15% (wt/vol) RR, and incubated overnight at
4°C. The fixed cells were rinsed in 0.1 M cacodylate buffer with RR, pelleted, and
resuspended in 3 ml of buffered 2% osmium tetroxide containing 0.15% RR for
1 h. The cells were then rinsed two times in buffer containing RR and dehydrated
in a graded ethanol series (25%, 50%, and finally 70% ethanol) containing RR
for 15-min periods. The cells were then resuspended in a small volume (400 ?l)
and centrifuged with a Beckman 152 Microfuge. The intact pellet was dehydrated
in the absence of RR with 95% ethanol followed by four changes of 100%
ethanol and then infiltrated with effapoxy resin (2:1, 1:1, and 1:2 ethanol-effapoxy
for 30-min periods and finally 100% effapoxy overnight at 4°C), and this was
followed by embedding. Thin sections were stained with uranyl acetate and lead
citrate and examined with a JEOL JEM100 CXII transmission electron micro-
Bacterial culture. Isolates 69, 59, and 61 were grown in Trypticase soy broth or
agar (Difco, Detroit, MI). RS218 and an isogenic mutant of this strain were
grown in tryptic soy broth or agar supplemented with 50 ?g/ml streptomycin (for
the K1-positive isolate) or 50 ?g/ml streptomycin and 40 ?g/ml chloramphenicol
(for the K1-negative isolate). Individual isolates were grown in the appropriate
broth media (5 ml) after inoculation of a single colony and incubated at 37°C
overnight in an orbital shaker. The next day, 2.5 ml of each overnight culture was
used to inoculate 47.5 ml of fresh broth and incubated for 2 h at 37°C with
shaking. Bacterial growth was determined by measuring the optical density at 600
nm, and the number of bacteria (CFU/ml) was determined using a previously
determined growth curve. The number and viability of bacteria were confirmed
using a LIVE/DEAD Baclight kit (Molecular Probes, Eugene, OR) and by
individual plating on agar following injection. For all experiments, bacteria in log
phase were used. Pyrogen-free normal saline (Baxter Healthcare Corporation,
Deerfield, IL) was used to prepare bacterial suspensions for injection.
Survival studies and determination of sublethal doses of bacteria. Each isolate
of E. coli was grown to log phase, concentrated by centrifugation, and diluted in
saline, and various doses (0.2 ml) of each isolate were injected intraperitoneally
(i.p.) into CD14?/?BALB/c mice. The bacterial dose yielding close to 50%
survival in this small-scale experiment (four mice per dose and four different
doses for each bacterial isolate) was used for a larger-scale experiment to de-
termine the sensitivity of CD14?/?and CD14?/?mice to the different isolates of
bacteria over a 7-day period. Following i.p. injection, the mice were observed for
endotoxin shock-like symptoms, including ruffled fur, eye exudate, diarrhea,
prostration, lack of reactivity, and death, for 7 days. The individual symptoms
were each scored using a severity range of 0 to 3, the scores were combined to
obtain a single score for each mouse ranging from 0 to 15, and the mean scores
for all surviving mice were compared daily (Mann-Whitney test); the overall
results shown by the two curves were compared by two-way analysis of variance
TABLE 1. Characteristics of E. coli strains studied
E. coli isolate
Serum (80%) sensitivity
Heat toxin (a and b)b
Shiga-like toxin types 1 and 2b
Cytotoxic necrotizing factors 1 and 2b
Fimbrial antigens (K88, K99, 987P,
CS31A, F1845, F107)b
Mannose-resistant hemagglutinin antigen
Carboxylase B2 polymorphism type
a?, positive; ?, negative.
bTyping was performed by Pennsylvania State University (see Materials and
cM, multiple (bacteria cross-reacted with H4, H23, H37, and H44 H antigens).
5416METKAR ET AL.INFECT. IMMUN.
on February 25, 2013 by guest
(ANOVA). At the end of the observation period surviving mice were sacrificed
with an inhalation overdose of CO2.
Preparation of tissue samples. Mice were anesthetized with isofluorane, in-
jected (i.p.) with an individual isolate (?50% lethal dose), and at the appropriate
time point euthanized with CO2and exsanguinated. A sample (100 ?l) of hep-
arinized blood was taken for determination of the number of bacteria that it
contained; the remainder of the blood was centrifuged to obtain plasma, which
was sterile filtered, aliquoted, and stored at ?80°C for cytokine analyses. The
peritoneal lavage fluid was collected using 5 ml sterile normal saline; a portion
(100 ?l) was used for determination of the number of bacteria that it contained,
and the remainder was sterile filtered, aliquoted, and stored at ?80°C for cyto-
kine analyses. A laparotomy was performed under aseptic conditions, and the
lungs, liver, and spleen were collected in sterile saline, weighed, and homoge-
nized using sterile tissue homogenizers (Tekmar, Cincinnati, OH).
Determination of the number of CFU. Blood, peritoneal lavage fluid, and
organ homogenates, all stored on ice, were serially diluted and plated on the
appropriate agar plates. After overnight incubation at 37°C, bacterial colonies
were counted and the number of live bacteria was normalized to the total volume
of fluid or organ weight.
Activation of thioglycolate-elicited peritoneal macrophages with live bacteria.
Thioglycolate-elicited peritoneal macrophages were obtained and stimulated
with live bacteria as previously described (14, 19). Briefly, CD14?/?or CD14?/?
BALB/c mice were injected with 3 ml of 4% thioglycolate broth (Difco, Detroit,
MI) i.p. After 3 days, the mice were sacrificed, and the peritoneal lavage fluid was
collected using RPMI 1640 medium (Gibco BRL, Grand Island, NY). The lavage
fluid was centrifuged at 200 ? g for 20 min at room temperature, and the cell
pellet was washed once and resuspended in RPMI medium containing 1%
autologous serum. The cells were seeded into 24-well plates (Becton Dickinson,
Franklin Lakes, NJ) at a density of 2 ? 106cells per well, and the plates were
incubated at 37°C in 5% CO2for 3 h to allow the macrophages to adhere; the
nonadherent cells were removed by washing twice with RPMI 1640 medium.
Log-phase live bacteria (3 ? 105CFU) were then added to each well, and the
plates were incubated for 3 h at 37°C. The supernatants were then collected,
centrifuged, and filter sterilized, and the cell-free supernatants were stored at
?80°C for further analysis.
Cytokine assays. The amount of TNF-? in plasma or cell-free supernatant was
measured using a double antibody sandwich enzyme-linked immunosorbent as-
say (ELISA) (R&D Systems, Minneapolis, MN) according to the manufacturer’s
instructions. The optical density at 450 nm was read using a Thermomax ELISA
plate reader (Molecular Devices, Menlo Park, CA). The lower limit of detection
of TNF-? was 10 pg/ml. The assays were linear up to a concentration of 1,500
pg/ml for TNF-?. Samples with levels above the upper limit were diluted and
assayed again; samples with levels below the lower limit were assayed again using
a lower dilution. IL-6 expression was measured by an ELISA (Biosource, Car-
arillo, CA) performed according to the manufacturer’s instructions.
Statistics. Results of cytokine production and bacterial clearance experiments
were compared using the Mann-Whitney test, performed using GraphPad Prism
version 4.0b for Macintosh (GraphPad Software, San Diego, CA) (www
.graphpad.com). A P value of ?0.05 was considered significant, and P values of
?0.1 were also noted. Percent survival was calculated using the product limit
(Kaplan-Meier) method, and curves were compared using the log rank test; all
analyses were performed using GraphPad Prism software. Comparisons of
symptoms of infected CD14?/?and CD14?/?mice over time and clearance of
bacteria by the two strains over time were performed by two-way ANOVA
Characteristics of E. coli isolates. The characteristics of
three of the clinical isolates of E. coli used in these studies,
designated strains 59, 61, and 69, are shown in Table 1. As
previously described (31), these E. coli strains were isolated
from sepsis patients and are identical in most characteristics.
All three isolates express mannose-resistant hemagglutinin and
carboxylase B (Mf 62), as well as the aer and pap operons; the
aer operon encodes a molecule that competes with transferrin
for iron uptake, and the pap operon encodes an extraintestinal
attachment factor. Furthermore, all three isolates are negative
for hemolysin, sfa/foc, afa, hly, and the ibe-10 operon and do
not express toxins or fimbrial antigens (Table 1). However, the
three isolates also differ from each other in at least one of three
additional characteristics. E. coli isolates 59 and 61 differ in
both O-antigen (LPS) and H-antigen (flagellum) serotypes;
isolate 61 is O-antigen type 1 and H-antigen type 7, whereas
isolate 59 is O-antigen type 2 and H-antigen type M. In addi-
tion, although both of these isolates express the capsular K1
antigen, as determined serologically using anti-Neisseria men-
ingitidis group B antiserum (31), they differ from isolate 69,
which does not express the K1 antigen. This characteristic,
shown to be important in our subsequent studies, was con-
firmed by electron microscopy (Fig. 1). K1-positive E. coli
isolates 59 and 61 show a heavy, electron-dense layer of acidic
polysaccharides when they are stained with RR, which com-
pletely surrounds the bacterial cells on their outer wall surface.
The K1-negative isolate 69 and our previously studied E. coli
O111 isolate (also K1 negative) lack such staining (Fig. 1).
Thus, of the 20 different characteristics compared, E. coli
isolates 59 and 61 differ in their O- and H-antigen serotypes
FIG. 1. Transmission electron micrographs of K1-positive and K1-negative E. coli isolates. The arrows indicate the K1 capsular structures
stained with RR.
VOL. 75, 2007 CD14 AND RESPONSES TO K1-POSITIVE E. COLI5417
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but are identical in all other respects, including expression of
the K1 capsule. In contrast, E. coli isolate 69 lacks a K1 capsule
but has the same O- and H-antigen types as isolate 59, which
is K1 negative (Table 1).
CD14?/?mice show different levels of resistance to geneti-
cally distinct E. coli isolates. We previously showed that CD14-
deficient mice, in contrast to normal mice, are highly resistant
to E. coli O111 (19); this strain types as O antigen 1 and is
negative for the H (flagellum) antigen, as well as the K1 cap-
sular antigen. In order to extend these observations to clinical
isolates of E. coli, CD14?/?and CD14?/?BALB/c mice were
injected with isolate 59, 61, or 69 at a dose that was sublethal
to CD14?/?BALB/c mice (determined in a small-scale exper-
iment as described in Materials and Methods). Whereas 50%
of the CD14?/?mice injected with a 50% lethal dose (2 ? 106
CFU/g of body weight) of E. coli K1-negative isolate 69 died,
all CD14?/?mice injected with isolate 69 survived (P ? 0.005)
(Fig. 2C). In contrast, both CD14?/?and CD14?/?mice were
sensitive to sublethal doses of K1-positive E. coli isolates 59
(8 ? 104CFU/g of body weight) and 61 (5 ? 105CFU/g of
body weight) and displayed no significant differences in sur-
vival (Fig. 2A and B). Similar results (i.e., significant differ-
ences in survival for normal and CD14-deficient mice with
isolate 69 but not with isolate 59 or 61) were obtained in three
independent experiments (data not shown). Nevertheless, to
confirm these differences between CD14?/?and CD14?/?
mice exposed to K1-negative isolate 69, a dose-response anal-
ysis was performed. CD14?/?mice showed a clear survival
advantage over normal CD14?/?mice (P ? 0.0001, two-way
ANOVA), all of which died at every bacterial dose tested
(Table 2). It should be noted that this pattern of survival of
CD14?/?mice but not normal CD14?/?mice was previously
observed for the other K1-negative strain, E. coli O111, that
was previously studied (19).
In addition to survival, mice were also examined daily for
other gross symptoms of responsiveness often seen in endo-
toxemia, including ruffled fur, eye exudates, diarrhea, prostra-
tion, and lack of reactivity, and these symptoms were scored
daily on the basis of severity from 0 to 3 (Fig. 3). In our
previous studies (19), CD14-deficient mice showed few symp-
toms of shock, whereas normal mice quickly succumbed to E.
coli O111. When the symptoms of CD14?/?mice infected with
the K1-negative isolate, strain 69, were compared to those of
normal CD14?/?mice (Fig. 3C), it was observed that CD14?/?
mice displayed significantly less severe symptoms than
CD14?/?mice (P ? 0.0367), especially on day 1 (P ? 0.03). In
contrast, there were no differences in symptoms between
CD14?/?and CD14?/?mice infected with the K1-positive
isolates, strains 59 and 61 (Fig. 3A and 3B), except on day 1,
although the differences were less pronounced for mice in-
jected with the K1-positive isolate 61 (Fig. 3B). These differ-
ences in severity of symptoms over an extended period of time
correlated well with the differences in survival (Fig. 2 and 3).
However, there was no correlation between symptoms on day
1 and survival; rather, the differences in symptoms observed at
day 1 seemed to correlate well with levels of TNF-? expression
CD14?/?mice are resistant to a lethal dose of K1-negative
E. coli but not to K1-positive E. coli. To determine whether the
differences in survival of CD14?/?mice infected with the
FIG. 2. Survival of CD14?/?and CD14?/?mice after i.p. injection
of sublethal doses of K1-positive isolates 59 (A) and 61 (B) and
K1-negative isolate 69 (C). The data in panels A and B are the means
for 12 mice per group, and the data in panel C are the means of four
pooled experiments (24 mice per group). The statistical significance of
differences in survival curves was assessed by the log rank test using
GraphPad Prism software.
TABLE 2. Survival of mice injected with different amounts of
K1-negative isolate 69a
No. alive/total no.
Day 1 Day 2Day 3
3 ? 106
4.5 ? 106
6.75 ? 106
10 ? 106
aMice were injected i.p. with the doses of E. coli indicated and were observed
for 7 days; no additional deaths occurred after 3 days. The P value was 0.0001 as
determined by two-way ANOVA.
5418METKAR ET AL.INFECT. IMMUN.
on February 25, 2013 by guest
nonisogenic K1-positive (isolates 59 and 61) and K1-negative
(isolate 69) clinical isolates were due to the K1 capsule itself
and not to one of the other factors that distinguish these
isolates (Table 1), CD14?/?mice were injected with sublethal
doses of one of two additional E. coli isolates, either RS218, a
K1-positive E. coli obtained from a newborn with meningitis
(O18:K1:H7) (22), or an isogenic mutant of RS218 that lacks
the K1 capsule (O18:H7) (22). As was observed with the K1-
negative nonisogenic isolate 69, CD14?/?survived a dose of
the isogenic K1-negative mutant that was lethal for CD14?/?
mice (Fig. 4B), while both strains of mice were sensitive to the
parental K1-positive E. coli RS218 strain (Fig. 4A). Thus, the
presence of the K1 capsule alone was sufficient to eliminate
the resistance of CD14?/?mice to E. coli.
CD14?/?mice produce low levels of TNF-? in response to
both K1-positive and K1-negative E. coli. To determine
whether survival correlates with high or low levels of the proin-
flammatory cytokine TNF-?, the levels of in vivo production of
TNF-? were compared in mice injected with the nonisogenic
K1-positive and K1-negative E. coli strains. All three isolates
induced comparable levels of circulating TNF-? in normal
CD14?/?mice, with values ranging from a mean of 1.877 ng/ml
(K1-positive isolate 59) to 3.322 ng/ml (K1-negative isolate 69).
However, the level of circulating TNF-? was significantly re-
duced in CD14?/?mice with all three isolates (Fig. 5). The
decrease was most pronounced for the K1-negative isolate 69
(mean, 0.1409 ng/ml) and least pronounced for the K1-positive
isolate 61 (mean, 0.7403 ng/ml). The difference in TNF-? in-
duction between normal and CD14?/?mice was also observed
with both the K-1 positive RS218 isolate and its K1-negative
isogenic mutant and was maintained for up to 12 h (data not
To examine whether the differences in TNF-? levels be-
tween CD14?/?and CD14?/?mice in response to both K1-
positive and K1-negative isolates reflected differences in the
ability of CD14?/?and CD14?/?macrophages to respond to
these isolates, the abilities of all three nonisogenic isolates of
E. coli to stimulate production of TNF-? from peritoneal mac-
rophages isolated from both CD14?/?and CD14?/?mice
were determined. In these studies, a constant amount (3 ? 105
CFU) of the isolates was used. All three isolates induced pro-
duction of high levels of TNF-? from CD14?/?macrophages,
with values ranging from means of 6.649 ng/ml (K1-negative
FIG. 3. Symptoms of CD14?/?and CD14?/?mice after injection of sublethal doses of K1-positive isolates 59 (A) and 61 (B) and K1-negative
isolate 69 (C). The symptom index was calculated as described in Materials and Methods. Results at individual points were compared using the
Mann-Whitney t test. The statistical significance of differences in symptoms over time was assessed by two-way ANOVA.
FIG. 4. Survival of CD14?/?and CD14?/?mice after i.p. injection
of K1-positive RS218 E. coli (A) or its K1-negative isogenic mutant
(B). The statistical significance of differences in survival curves was
assessed by the log rank test using GraphPad Prism software.
FIG. 5. TNF-? levels in blood of CD14?/?and CD14?/?mice 1.5 h
following i.p. injection of K1-positive (isolates 59 and 61) or K1-
negative (isolate 69) E. coli. The results are means ? standard errors
of the means for six mice per group. All measurements were made in
triplicate. Results were compared using the Mann-Whitney t test.
VOL. 75, 2007 CD14 AND RESPONSES TO K1-POSITIVE E. COLI5419
on February 25, 2013 by guest
strain 69) to 13.91 ng/ml (K1-positive strain 61). However, the
amount of TNF-? induced was significantly less for CD14?/?
macrophages, irrespective of which isolate was used for stim-
ulation (Fig. 6). Thus, for all three isolates, the ability to induce
production of TNF-? was dependent on the presence of CD14.
However, although the levels of TNF-? correlated with sur-
vival for mice injected with K1-negative isolate 69, there was
no such correlation for mice injected with K1-positive isolates
59 and 61.
CD14?/?mice produce low levels of IL-6 in response to both
K1-positive and K1-negative E. coli. Studies by other workers
have suggested that low levels of IL-6 at 6 h after infection
correlate with survival in a polymicrobial model of infection
(35, 44, 50). To determine whether survival after infection with
K1-positive or K1-negative E. coli correlates with high or low
levels of the proinflammatory cytokine IL-6, the levels of in
vivo production of IL-6 at 6 h were compared in normal and
CD14?/?mice injected with K1-positive and K1-negative bac-
teria. Both of the K1-positive isolates, isolates 59 and RS218,
as well as the K1-negative isolates, isolate 69 and the isogenic
mutant of RS218, induced production of significantly more
IL-6 in normal mice than in CD14?/?mice (Fig. 7). However,
in spite of the presence of low levels of IL-6, there was no
survival advantage for CD14?/?mice infected with the K1-
positive isolates as there was with CD14?/?mice infected with
the K1-negative isolates.
Survival correlates with enhanced clearance of bacteria. To
further examine the basis for the resistance of CD14?/?mice
to K1-negative E. coli but not to K1-positive E. coli, the de-
grees of bacterial clearance of all three nonisogenic isolates
were initially compared. At the time that the samples were
analyzed (3 h after i.p. injection) all mice were bacteremic;
however, the degree of bacteremia varied. The CD14?/?mice
cleared the K1-negative E. coli isolate 69 much more efficiently
than the two K1-positive isolates (Fig. 8). As shown in Fig. 8C,
the bacterial counts of E. coli 69 in organs, the peritoneal
cavity, and the blood were significantly reduced in CD14?/?
mice compared to CD14?/?mice. The bacterial counts in the
blood and peritoneal lavage fluid of CD14?/?mice were 1/20
and 1/15 of the counts in their control counterparts, respec-
tively. However, CD14?/?and CD14?/?mice showed no dif-
ference in the bacterial counts in organs or fluids when they
were injected with the two K1-positive E. coli isolates, strains
59 and 61 (Fig. 8A and B). Similarly, a pattern of enhanced
FIG. 6. In vitro production of TNF-? by CD14?/?and CD14?/?
thioglycolate-elicited peritoneal macrophages after incubation with
log-phase live K1-positive (isolates 59 and 61) or K1-negative (isolate
69) E. coli. The results are means ? standard errors of the means of
two independent experiments. All measurements were made in tripli-
cate. Where there are no error bars, they fall within the bar. Results
were compared using the Mann-Whitney t test.
FIG. 7. IL-6 levels in blood of CD14?/?and CD14?/?mice 6 h
following i.p. injection of (A) nonisogenic K1-positive isolate 59 and
K1-negative isolate 69 and (B) K1-positive RS218 and its K1-negative
isogenic mutant. Results were compared using the Mann-Whitney t test.
FIG. 8. Bacterial counts (CFU/g of organ or CFU/ml of fluid) re-
covered 3 h following i.p. injection of K1-positive isolate 59 (A),
K1-positive isolate 61 (B), or K1-negative isolate 69 (C). The data are
expressed as means ? standard errors of the means on a log scale and
include data for blood (CFU/ml), total peritoneal lavage fluid (Perit.
fluid) (CFU) and the liver, lung, and spleen (CFU/g of organ). P values
were determined by a Mann-Whitney t test.
5420 METKAR ET AL.INFECT. IMMUN.
on February 25, 2013 by guest
bacterial clearance was observed at 3 h in CD14?/?mice com-
pared to CD14?/?mice when they were injected with the
K1-negative isogenic mutant but not when they were injected
with the parental K1-positive RS218 isolate (data not shown).
Thus, the ability to clear bacteria, rather than TNF-? or IL-6
levels, correlated with survival.
To confirm that differences in bacterial clearance persisted
over time, normal and CD14?/?mice were injected with a
sublethal dose of either the K1-positive RS218 isolate or its
K1-negative isogenic mutant and bacterial counts in peritoneal
fluid and blood were determined at various times (1 to 12 h)
after injection. As was observed with the nonisogenic isolates,
the K1-negative isogenic mutant was rapidly cleared in
CD14?/?mice but not in normal mice (Fig. 9B and D). In con-
time in both normal and CD14?/?mice, although the increase
was more pronounced in the blood than in the peritoneal cavity
(Fig. 9A and C). A pattern of enhanced bacterial clearance
similar to that shown in Fig. 8 was observed in the livers and
spleens of CD14?/?mice injected with the K1-negative iso-
genic mutant but not in the livers and spleens of CD14?/?mice
injected with the parental K1-positive RS218 isolate (data not
The present study was initiated to study the role of CD14 in
the response of the host to different clinical isolates of E. coli
obtained from sepsis patients. We used three extensively typed
clinical isolates (designated strains 59, 61, and 69) that share
the majority of genotypic and phenotypic characteristics but
differ in at least one of three characteristics, the O-antigen
(LPS) and/or H-antigen (flagellum) antigen serotype or the
presence of a K1 capsule (Table 1). Both LPS and the K1
capsule are known to be important virulence factors for E. coli.
LPS is a potent activator of innate immune responses, resulting
in the release of pro- and anti-inflammatory mediators from
hematopoetic and nonhematopoetic cells (16, 45). The smooth
form of LPS, found in most strains of gram-negative bacteria,
consists of a serologically defined O-antigen polysaccharide
linked to core polysaccharides and a lipid A moiety; the lipid A
moiety is embedded in the outer bacterial membrane. The
primary receptor for LPS, CD14, is expressed on the surface of
monocytes, macrophages, neutrophils, and some dendritic cells
(15, 18, 27, 48). It functions in the activation of cellular re-
sponses, including release of proinflammatory cytokines and
induction of endotoxemia (9, 10, 13, 16, 17, 19, 23, 24, 40, 41,
49), playing a major role in the symptoms leading to severe
sepsis and/or septic shock (4, 20, 29, 33). In general, it is widely
accepted that low-level expression of proinflammatory cyto-
kines and other inflammatory mediators is beneficial to the
host’s ability to eliminate the infecting bacteria, while high
levels of these mediators lead potentially to death.
In addition to LPS, the presence of a polysaccharide capsule
loosely associated with the outer surface of gram-negative bac-
teria may be associated with increased virulence; the polysac-
charides show much greater structural variability than LPS.
Capsular polysaccharides (serologically typed as K antigens)
represent a major surface antigen of E. coli, and more than 80
distinct K serotypes have been described (47). However, only a
few K-antigen serotypes have been associated with virulence;
these include K1, K5, K10, and K54 (47).
Structurally, the K1 capsule is a homopolymer of ?2,8-linked
N-acetylneuraminic acid. Unlike the toxic properties of LPS
FIG. 9. Time course of bacterial counts in the blood (A and B) and the peritoneal cavity (C and D) of CD14?/?and CD14?/?mice following
injection of K1-positive RS218 (top panels) or its K1-negative isogenic mutant (bottom panels). The statistical significance of differences in the
number of live bacteria recovered over time was assessed by two-way ANOVA.
VOL. 75, 2007 CD14 AND RESPONSES TO K1-POSITIVE E. COLI5421
on February 25, 2013 by guest
that cause activation of host cells, the virulence of bacteria
expressing such a capsule appears to be associated with evad-
ing the host response. Several mechanisms by which the cap-
sular polysaccharide aids the bacteria in eluding host defenses
have been postulated. Such mechanisms include masking of
LPS and thus preventing activation of the innate immune sys-
tem, as well as masking sites for deposition of opsonins and
thus inhibiting phagocytosis and sites required for comple-
ment-mediated killing (37). Thus, the available literature sug-
gests that the K1 capsule may be capable of inhibiting some
aspects of the early innate immune response that may normally
be beneficial early in infection.
In the course of performing these studies we observed major
differences in the responses of CD14-deficient and normal
mice to three different E. coli isolates (isolates 59, 61, and 69)
that seemed to correlate with the presence of a K1 capsule.
Accordingly, we used a K1-positive isolate, RS218, obtained
from a newborn with meningitis, and a K1-negative isogenic
mutant of this isolate to confirm observations made with the
Our results demonstrate that CD14?/?mice, but not
CD14?/?mice, are resistant to K1-negative E. coli, similar to
what we previously found using an E. coli O111 strain (19).
Furthermore, as was previously observed with E. coli O111, the
K1-negative organisms are rapidly cleared and induce very
little TNF-? or IL-6 production in CD14?/?mice. In contrast,
although the levels of TNF-? and IL-6 induced by the K1-
positive isolates in CD14?/?mice are dramatically reduced
compared to the levels in CD14?/?mice, there is no increase
in survival; both CD14?/?and CD14?/?mice die within 48 h.
Furthermore, there is no accelerated clearance of the K1-
positive bacteria, as was observed for the two K1-negative
isolates, in CD14?/?mice; thus, CD14?/?and CD14?/?mice
showed no difference in bacterial counts when they were in-
jected with any of the K1-positive isolates.
In contrast to our observation that CD14?/?mice are resis-
tant to a lethal dose of K1-negative E. coli, Cross et al. (6)
found that C3H/HeJ mice that are deficient in a functional
TLR4 molecule required for LPS-CD14 signaling (21, 32) are
as sensitive as normal mice (C3H/HeN) to a lethal dose of
K1-negative E. coli, as measured by survival and bacterial
clearance (5, 6). Furthermore, although CD14?/?mice show
sensitivity to K1-positive E. coli similar to that of CD14?/?
mice (Fig. 2), Cross et al. (6) found that C3H/HeJ mice lacking
TLR4 were ?1,000-fold more sensitive to K1-positive E. coli
than their parental control. The differences in the resistance of
CD14-deficient and TLR4-deficient mice to both K1-negative
and K1-positive E. coli suggest that TLR4 functions indepen-
dent of CD14 in response to some bacterial components.
Our studies also contrast with those of Bernheiden et al. (2),
who showed that CD14?/?and TLR4?/?mice are more sus-
ceptible to Salmonella enterica serovar Typhimurium than con-
trols. This difference between our observations and those of
Bernheiden and colleagues may be due to the fact that S.
enterica serovar Typhimurium is an intracellular pathogen,
while the E. coli isolates used in these studies are extracellular
The resistance of CD14?/?mice to the K1-negative isolates
but not to the K1-positive isolates indicates that entirely dif-
ferent mechanisms are responsible for the symptoms and death
induced by K1-positive E. coli and by K1-negative E. coli.
Production of large amounts of proinflammatory cytokines like
TNF-? is induced during gram-negative bacterial infections,
which are thought to be critical for inducing mortality during
septic shock (8, 43). For the K1-negative isolates, survival cor-
relates with the decrease in TNF-? production and rapid clear-
ance of the organism. In contrast, although there is a signifi-
cant decrease in TNF-? production in CD14?/?mice injected
with the K1-positive organisms, there is no enhanced survival,
suggesting that TNF-? is not responsible for death caused by
K1-positive E. coli. Furthermore, there is no beneficial effect
from the low levels of TNF-?, in contrast to the studies of
Cross et al. (5), who found that injection of a combination of
TNF-? and IL-1 reduced the sensitivity of C3H/HeJ (TLR4-
deficient) mice to K1-positive bacteria. It should be noted that
the small amount of TNF-? induced by the K1-positive iso-
lates, especially isolate 61, in CD14?/?mice indicates that
there is a bacterial component capable of inducing TNF-?
production via a non-CD14 mechanism.
The role of IL-6 in sepsis-induced morbidity and mortality
has been controversial. In previous studies, individual variation
in IL-6 expression was used to predict the survival of mice in
the early (acute) phase of cecal ligation and puncture-induced
sepsis (35, 44, 50). However, studies with IL-6-deficient mice
(36), as well as antibody therapy targeted to IL-6 (46), clearly
show that IL-6 is an indicator of the severity of disease rather
than a cause of disease. As was the case with IL-6-deficient
mice in a cecal ligation and puncture model of sepsis (36), the
low levels of IL-6 expression in CD14-deficient mice compared
to normal mice did not prevent mortality when mice were
infected with the K1-positive E. coli isolates, even though
CD14?/?mice displayed reduced symptoms of sepsis on day 1
compared to normal mice (Fig. 3).
Further evidence that the mechanisms resulting in death
induced by the K1-negative and K1-positive isolates are dis-
tinct is based on the pattern of symptoms observed after in-
jection. Initially (within 24 h), mice injected with the K1-pos-
itive organisms show significantly greater gross symptoms of a
response than those injected with the K1-negative bacteria
(Fig. 3). Furthermore, whereas these symptoms diminish sig-
nificantly after day 1 in surviving mice injected with the K1-
positive isolates, a similar significant decrease is not seen in
mice injected with K1-negative bacteria.
The lack of a major role for CD14 in the response to K1-
positive E. coli, coupled with the host’s inability to phagocytose
and kill K1-positive E. coli, may explain the results of other
workers studying the therapeutic effects of anti-CD14 mono-
clonal antibodies in models of septic shock or pneumonia in-
duced by K1-positive E. coli. In these studies, anti-CD14 re-
duced symptoms of endotoxemia but had no effect on bacterial
clearance (12, 30). The importance of bacterial clearance in
ameliorating the effects of septic shock is further supported by
studies in mouse models of polymicrobial infection which show
that survival during the chronic phase of infection correlates
best with the ability to control bacterial overgrowth and not
with proinflammatory cytokine levels (50).
These studies suggest that there is significant diversity in the
mechanisms and/or molecules associated with symptoms oc-
curring in response to bacteria expressing different virulence
factors. Indeed, although some bacteria may use specific cell
5422METKAR ET AL.INFECT. IMMUN.
on February 25, 2013 by guest
receptors, such as CD14, to mediate their effects, other bacte-
ria may use other receptors to induce severe sepsis or septic
shock. Accordingly, it may be difficult to treat all patients with
gram-negative septicemia with a single drug, as has been tried
previously with anti-TNF antibodies. Only a thorough under-
standing of the various mechanisms employed by different or-
ganisms to induce sepsis/septic shock will make it possible to
treat these syndromes effectively.
This work was supported in part by National Institutes of Health
grant RO1 AI23859 (to S.M.G.) and by a grant from la Fondation pour
la Recherche Me ´dicale, Paris (to S.C.G.).
We thank Ana Pino (Laboratory of Innate Immunity, CUNY Med-
ical School/Sophie Davis School of Biomedical Research) and Jeffrey
Moyse (Electron Microscopy Laboratory, Manhasset, NY) for their
excellent technical assistance.
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Editor: J. B. Bliska
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