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*
Lipid Metabolism Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114;
Maxwell Finland Laboratory for Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, MA 02118; and
Leukocyte Biology and Inflammation Program, Renal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-6 production in
macrophages taken from these animals, and this loss in responsiveness
is associated with impaired activation of both the NF-
B and the
c-Jun N-terminal mitogen-activated protein kinase pathways. The binding
and uptake of heat-killed Escherichia coli, measured by
FACS analysis, did not differ between CD14-null and wild-type
macrophages. However, in contrast to the findings with LPS, whole
E. coli stimulated similar levels of TNF- release
from CD14-null and wild-type macrophages at a dose of 10 bioparticles
per cell. This effect was dose dependent, and at lower bacterial
concentrations CD14-deficient macrophages produced significantly less
TNF- than wild type. Approximately half of this CD14-independent
response appeared to be mediated by CD11b/CD18, as demonstrated by
receptor blockade using neutrophil inhibitory factor. An inhibitor of
phagocytosis, cytochalasin B, abrogated the induction of TNF- in
CD14-deficient macrophages by E. coli. These data
indicate that CD14 is essential for macrophage responses to free LPS,
whereas other receptors, including CD11b/CD18, can compensate for the
loss of CD14 in response to whole bacteria. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as well as proinflammatory eicosanoids and NO
(1). Three cloned families of molecules on the surface of leukocytes are known to bind the toxic lipid A moiety of LPS. These include CD14, the macrophage scavenger receptors (SR-A family), and the ß2 or CD11/CD18 leukocyte integrins (4). LPS binding to CD14 is enhanced by serum factors; these serum components include the acute phase reactant LPS binding protein (LBP)4 and soluble CD14 (sCD14). When expressed on the surface of cells, CD14 has no intrinsic signaling capabilities, but is postulated to present LPS to its high affinity signal transducer, Toll-like receptor (TLR) 4 (5, 6, 7, 8). CD14 also enhances the responses of phagocytes to bacterial products that activate cells via TLR2 (e.g., peptidoglycan and lipopeptide), although the exact contribution of TLR2 to LPS responses remains to be clarified. Although all of the above receptors are components of the host response to endotoxin, the extraordinary sensitivity of the macrophage response to LPS appears to depend upon the presence of either membrane-bound or soluble CD14.
The presence of sCD14 in serum and the expression of membrane-bound CD14 on myeloid cells have complicated the analysis of alternative receptors involved in activation by bacterial products. This is particularly true of the ß2 integrins (CD11/CD18), which bind LPS, especially when LPS is presented to phagocytes as a component of a membrane (e.g., whole bacteria; Ref. 9). Although phagocytes bind the LPS moiety of Gram-negative bacteria via CD11/CD18, this interaction was felt initially to be biologically insignificant because children with congenital CD11/CD18 deficiency responded normally to LPS (10). In retrospect, this observation might have been predicted because the cells that were harvested from these children expressed CD14. When expressed in a cell line that does not coexpress CD14, the ß2 integrins function as low affinity LPS receptors (11, 12, 13). Although the ED50 for CD11/CD18 engagement is higher than that for CD14, few qualitative differences in LPS activation by these two distinct receptor systems have been described (14). Like CD14, CD11/CD18 can activate cells in the absence of cytoplasmic signaling residues, as demonstrated by experiments that showed equivalent LPS responses to a full length and "tailless" integrin (12). It seems likely that other, less well-characterized, cell surface proteins that bind bacteria may also activate phagocytes, but these will be challenging to identify as long as the highly sensitive CD14 activation pathway can be engaged.
To explore the contributions of both CD14-dependent and -independent
signaling mechanisms to LPS and bacterial stimulation, we chose to
generate mice lacking this receptor. Macrophages from our CD14-null
mice proved to be unresponsive to LPS stimulation over an LPS dosage
range of two log orders (1–100 ng/ml), confirming similar observations
Haziot et al. previously made in CD14-null mice generated with a
different gene-targeting strategy (15). In this work, we
further confirm that the lack of cytokine responsiveness in
LPS-stimulated CD14-null macrophages is associated with impaired
activation of both the NF-B and the c-Jun N-terminal
mitogen-activated protein kinase (MAPK) pathways (16, 17).
In contrast with their unresponsiveness to LPS, CD14-null macrophages
retained cytokine responses to whole bacterial particle stimulation. On
exposure to 10 heat-killed Escherichia coli, per cell or
greater, CD14-null and wild-type macrophages produced similar amounts
of TNF-. At 1 E. coli per cell, CD14-null macrophage
cytokine responses were definitely impaired but not lost. This
CD14-independent cytokine production was substantially reduced when
neutrophil inhibitory factor (NIF), a competitive inhibitor of the
ß2 integrin CD11B, was included in the
experiments. Inhibition of phagocytosis by cytochalasins completely
abolished CD14-independent responses. These data indicate that
CD14-null mice will be of considerable value in dissecting the
contributions of the multiple response pathways macrophages use to
combat bacterial infections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein-free LPS derived from E. coli K235 was provided by Dr. S. Vogel (Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD). Recombinant CD14 was purified from the conditioned medium of Chinese hamster ovary/CD14 cells as described elsewhere (18). The Ancyclostoma caninium (hookworm) protein, NIF, was provided by Dr. Matthew Moyle (Corvas International, San Diego, CA) (19). Endotoxin-free cell culture media and additives were purchased from Life Technologies (Grand Island, NY).
Generation of CD14-deficient mice
A P1 clone with 
60 kb of mouse genomic DNA, including the
gene encoding CD14, was obtained from Genome Systems (St. Louis, MO)
after screening a genomic library using a combination of PCR and
Southern hybridization as previously described (20). The
genomic structure of the CD14 locus was mapped by a
combination of restriction digest and Southern blotting. A targeting
vector, KO3CD14 (Fig. 1
A), was
generated that removed the entire coding sequence of the
CD14 gene. KO3CD14 contained the 4 kb of sequence
immediately upstream of the start site of translation of the CD14
coding sequence, an intervening neor cassette
derived from PGKneopA that replaced all CD14 coding sequences, and the
2.5 kb of sequence immediately downstream of the coding region. A
thymidine kinase gene was also incorporated into the plasmid as a
negative selectable marker to reduce the frequency of nonhomologous
recombinant transformants. The linearized vector was electroporated
into J1 embryonic stem (ES) cells derived from 129/SvEv mice, and 200
G418/gancyclovir resistant clones were selected. Genomic DNA from
selected colonies was digested with SacI and analyzed by
Southern blotting. Southern blots were hybridized with a
32P-labeled NheI-NcoI
fragment located outside of the targeting vector, washed in 2 x
SSC at 55°C and 65°C, and exposed to BioMax-MR film (Kodak,
Rochester, NY). The NheI-NcoI probe recognizes a
6-kb fragment in the endogenous CD14 locus and a 17-kb
fragment in the targeted locus.
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B). CD14-deficient
mice were obtained by intercross of mice hemizygous for CD14 with the
expected Mendelian ratio. Backcrossing of the CD14-deficient mice into
the C57BL/6J strain has been continued for four generations in a
pathogen-free facility, but the macrophages used for the experiments
described in this work were derived from animals that were backcrossed
for one or two generations. For experiments requiring autologous serum,
CD14 wild-type, -hemizygous, and -null mice were bled by retro-orbital
puncture using heparinized microcapillary tubes. Blood was pooled
within genotypes and centrifuged at 5000 rpm for 10 min, then serum was
isolated. Isolation of peritoneal macrophages
Peritoneal macrophages were isolated from 12-wk-old mice 4 days following i.p. injection of 1 ml of 3% thioglycollate broth (Difco, Detroit, MI) as we previously described (21). Unless otherwise stated, wild-type and hemizygous controls for CD14-null mice were F2 littermates. Mice were anesthetized using Metofane (Union, NJ) and sacrificed by cervical dislocation. Peritoneal lavage was performed with 6 ml of HBSS (Life Technologies) containing 0.5 mM EDTA. Collected peritoneal cells were treated with 0.17 M ammonium chloride to disrupt contaminating RBC, washed twice in HBSS, and resuspended in DMEM (Life Technologies) containing 100 U/ml penicillin and 100 µg/ml streptomycin and supplemented with 2.5% autologous mouse serum. Peritoneal cells were allowed to adhere to tissue culture plastic for 2 h, after which nonadherent cells were removed by rinsing the cell monolayer with media. Adherent cells were typically >97% positive for the macrophage-specific F4/80 Ag as determined by FACS analysis.
Western analysis
For Western analysis, peritoneal macrophages were seeded at 3 x 106 cells per well in six-well plates. Cells were lysed in TNET buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100) and centrifuged for 5 min at 15,000 rpm to pellet debris. To detect CD14 and phosphorylated or total c-Jun N-terminal kinase (JNK), equivalent amounts of protein were heat denatured in the presence of Laemmli sample buffer containing 5% 2-ME and electrophoresed on 10% SDS-PAGE gels. To detect F4/80, samples were run on 8% polyacrylamide gels under nonreducing conditions. Proteins were transferred to Immobilon-polyvinylidene difluoride membrane (Millipore, Bedford, MA) and blocked in TBS-T containing 5% nonfat milk for CD14 and F4/80, or 1% BSA in TBS-T for phosphoJNK. Primary Abs used were anti-murine CD14 rat monoclonal Ab (catalog no. 09471D; PharMingen, San Diego, CA), anti-F4/80 cell culture supernatant (1:5 dilution) from the F4/80 hybridoma cell line (American Type Culture Collection, Manassas, VA), rabbit anti-phosphoJNK Ab (Promega, Madison, WI), and rabbit antistress-activated protein kinase/JNK (New England Biolabs, Beverly, MA). Blots were incubated with HRP-conjugated secondary Ab (Sigma, St. Louis, MO) against the appropriate species and developed using an enhanced chemiluminescence detection system.
Detection of nuclear NF-B
EMSAs for nuclear NF-B were performed as previously described
(22). Briefly, 4 µg of crude nuclear protein was
incubated with a 32P-labeled oligonucleotide
containing the consensus sequence for NF-B binding from the murine
IgG light chain gene enhancer. The resulting complexes were analyzed by
nondenaturing PAGE. The gels were transferred to filter paper, dried,
and exposed to x-ray film. Scanning densitometry of the autoradiographs
was performed using Molecular Analyst software.
Cytokine ELISAs
Peritoneal macrophages were seeded in six-well plates at 5
x 105 cells per well in 0.6 ml of medium.
Peritoneal macrophages were stimulated with LPS from E. coli
K235 or heat-inactivated E. coli K12 bioparticles (Molecular
Probes), and cell supernatants were harvested at the indicated times.
Supernatants were centrifuged for 5 min to remove cellular debris,
transferred to new tubes, and stored at -70°C until analysis.
TNF- and IL-6 in cell supernatants were measured by ELISA using
matched pair Abs from Endogen (Cambridge, MA).
Binding assays
The binding of bacterial membrane particles to peritoneal macrophages was assessed as previously described (14). BODIPY-labeled E. coli K12 bioparticles (Molecular Probes) were added to CD14 wild-type or -null peritoneal macrophages (ratio of bacteria to cells was 100:1 or 10:1) in media containing 2.5% autologous serum for 5, 15, and 30 min at 37°C. To assess binding and uptake, cells were washed in PBS and assessed by FACS analysis. To assess uptake alone, cells were incubated in 2% trypan blue for 1 min to quench extracellular fluorescence, washed in PBS, and assessed by FACS analysis. The ability of trypan blue to quench BODIPY-labeled particles has been previously established in cells lines that cannot engage in CR3-mediated phagocytosis due to the expression of mutant "tailless" CD11/CD18 (12). Ten thousand gated events were recorded for each condition, excluding nonbound bacteria and cellular debris on the basis of forward and side light scatter. A second gate was established separating bright from nonfluorescent macrophages. Results are expressed as a percentage of cells that fall within this gate.
Blocking studies
Cells were preincubated with 4 µg of NIF protein/ml to inhibit CD11b/CD18 binding, or with 10 µM cytochalasin B to inhibit phagocytosis, for 15 min before stimulation with E. coli bioparticles. Inhibitors were maintained in culture media for the duration of the experiment (4 h).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Screening of ES cell clones by Southern blotting, as described in
Fig. 1A, revealed the correct disruption of the
CD14 allele in 2 of 100 clones. Although both ES cell clones
were injected into blastocysts, only one line produced high percentage
chimeras. Male chimeras from this line were backcrossed to C57BL6J
females and offspring were screened for germline transmission of the
targeted allele using tail DNA. F1 hemizygotes
were intercrossed, and screening revealed the expected Mendelian ratio
of CD14-null, -hemizygous, and wild-type offspring. A representative
Southern blot depicting the three genotypes is shown in Fig. 1B. The wild-type allele is 6 kb and the targeted allele is
17 kb. CD14-null mice appeared to be phenotypically normal. The
deletion had no obvious adverse effect on fertility or litter
size.
Western blot analysis of peritoneal macrophages from CD14-deficient
mice showed no expression of CD14 (Fig. 1C). As expected,
CD14 was detected in peritoneal macrophages from hemizygous and
wild-type mice. Western blot analysis of the same lysates for the
macrophage-specific Ag, F4/80, revealed similar levels of this protein
in CD14-null, -hemizygous, and wild-type macrophages, eliminating
nonspecific protein degradation as an explanation for the absence of
CD14 in the lysates of the null animals. These results established that
the CD14-null mice were homozygous for the targeted genomic allele and
that they were therefore unable to produce any CD14 protein.
Impaired responses of CD14-deficient macrophages to LPS
We evaluated pretranscriptional events, such as NF-B
translocation and JNK kinase activation, as well as cytokine
production, to determine the consequences of the absence of CD14 on
macrophage responses to endotoxin. A loss of NF-B activation in
response to LPS was observed in CD14-null peritoneal macrophages as
assessed by an EMSA (Fig. 2A).
Wild-type macrophages exhibited dose-dependent activation of NF-B in
response to as little as 1 ng/ml of LPS. In contrast, doses of up to
100 ng of LPS/ml did not induce NF-B activity in CD14-null
macrophages. However, IL-1ß, which acts independently of CD14,
induced the same level of NF-B translocation in CD14-null and
wild-type macrophages, indicating that the loss of LPS-induced NF-B
translocation in the null mice was a specific consequence of the CD14
deletion.
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B translocation, LPS induces the rapid activation
of several MAPKs, including the c-Jun N-terminal MAPK
(17). Western analysis revealed the accumulation of
phosphorylated JNK in wild-type macrophages within 30 min of LPS or
E. coli stimulation (Fig. 2B). In contrast, LPS
and E. coli activation of JNK was reduced in CD14-deficient
macrophages. However, IL-1ß, which stimulates JNK activation
independently of CD14, rapidly induced JNK phosphorylation in both
CD14-null and wild-type macrophages. These results indicate that
engagement of CD14 by either LPS or whole E. coli
contributes to JNK activation.
Corresponding with the loss of the early steps in LPS-induced signal
transduction, CD14-null macrophages exhibited a loss of cytokine
production in response to LPS (Fig. 3).
In wild-type macrophages, a 5-h incubation with LPS resulted in a
dose-dependent increase of both TNF- and IL-6 in culture
supernatants. However, no TNF- or IL-6 were detected in culture
supernatants from CD14-null macrophages stimulated with up to 100 ng/ml
of LPS from E. coli K235. Similar results were obtained
using LPS from Salmonella minnesota R595 (data not shown).
Although the precise level of CD14 expression required for sensitive
responses to LPS is unknown, an intermediate phenotype in cytokine
production was observed in LPS-stimulated hemizygous macrophages. The
reduction of TNF- (Fig. 3A) and IL-6 (Fig. 4A) production in
CD14-hemizygous macrophages, as compared with wild-type cells, clearly
demonstrates a CD14 gene dosage effect. Levels of TNF- and IL-6 were
undetectable in unstimulated macrophages from mice of all genotypes.
Taken together, these data demonstrate that CD14 appears to be critical
to the activation of several signal transduction pathways involved in
macrophage activation by LPS.
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sCD14 is believed to enhance delivery of LPS monomers to
membrane-bound CD14 at the cell surface, as well as to enable delivery
of LPS to additional components of the LPS signal transduction
apparatus in cells lacking surface CD14 (23). Therefore,
macrophages and sera from CD14-hemizygous and -null mice were used to
clarify the relative contributions of sCD14 and membrane CD14 to
cytokine responses to LPS. Both CD14-null and -hemizygous macrophage
cytokine production levels were restored to wild-type values by the
addition of sCD14 to the medium (Fig. 4A). These results are
similar to those found by Haziot et al. (15) despite our
use of concentrations of sCD14 that more closely mimic the physiologic
levels of that protein (2 µg/ml) as opposed to the 10-fold higher
concentration (20 µg/ml) used in the previous study. Using autologous
and CD14-null serum, we demonstrate the important contribution of serum
sCD14 in mediating signaling in response to low doses of LPS. At 1
ng/ml of LPS, serum sCD14 concentration significantly enhanced cytokine
production by membrane CD14-bearing cells (Fig. 4B).
Wild-type macrophages produced 10-fold more TNF- when cultured in
2.5% autologous serum than in 2.5% CD14-deficient serum. A similar
effect was observed when CD14-hemizygous macrophages were cultured in
autologous serum vs CD14-deficient serum. The degree of potency of
sCD14 proved to be unexpected; simply culturing cells from knockout
mice in FCS for several days was sufficient to restore near normal
LPS responses even after these cells were extensively washed in
serum-free medium (data not shown). This implies that residual levels
of sCD14, originally provided by culture in 5% FCS, were capable of
restoring LPS responsiveness to CD14-null macrophages. These data
confirm that circulating sCD14 contributes significantly to macrophage
responses to LPS, presumably by enhancing delivery of LPS to
membrane-bound CD14 or by directly enabling LPS delivery to the
TLRs.
E. coli binding and uptake is not altered in CD14-deficient macrophages
As CD14 can bind whole bacteria as well as its component LPS, we
examined the impact of CD14 deficiency on macrophage responses to
E. coli. CD14 has been reported to mediate the binding of
bacteria to macrophages as well as to function as a phagocytic receptor
for Gram-negative organisms, despite its lack of physical connections
to the cytoskeleton (24, 25). First, we used flow
cytometry to measure binding and uptake of BODIPY-labeled E.
coli bioparticles, a commercial preparation of fluorescently
labeled heat-killed bacteria. This assay takes advantage of the ability
of trypan blue to quench bound extracellular, but not intracellular,
fluorescent particles (14, 25). No significant differences
in E. coli binding and uptake were observed in
CD14-deficient macrophages, as compared with wild type. As shown in
Fig. 5, binding was evaluated using 100:1
E. coli per macrophage at 5-, 15-, and 30-min time points.
The percentage of binding was similar in CD14-deficient and wild-type
macrophages under all conditions. Similar results were obtained using
10:1 E. coli per macrophage (data not shown). No differences
were observed when the presence of extracellular organisms was quenched
with trypan blue (data not shown). In some experiments, a modest
reduction in binding was observed in CD14-null macrophages compared
with wild-type cells when bacteria were preincubated in
heat-inactivated (complement-deficient) serum (data not shown).
However, when data from five individual experiments were analyzed
together, this decrease was not statistically significant. Furthermore,
bacterial binding was not increased by sCD14 in either wild-type or
CD14-null macrophages. Incubation of CD14-null macrophages in medium
with 2% wild-type mouse serum containing endogenous sCD14 did not
increase bacterial binding, nor did the addition of recombinant sCD14
to either wild-type or null macrophages (data not shown). These
findings suggest that CD14 is not essential for bacterial uptake and
that, in the absence of CD14, other bacterial binding receptors (e.g.,
CD11b/CD18, SR-A, Fc
receptor, or mannose receptor) can
compensate.
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We next examined cytokine production from CD14-deficient
peritoneal macrophages stimulated with heat-killed E. coli
to determine the significance of the observation that bacterial
particles can bind and enter macrophages via alternate receptors to
CD14. In contrast to our findings with LPS, CD14-deficient macrophages
stimulated with 10 heat-killed E. coli per cell for 6 h
elaborated both TNF- (Fig. 6A) and IL-6 (data not shown)
in amounts equivalent to those produced by wild-type peritoneal
macrophages. This effect was dose dependent and at bacterial
concentrations <10 E. coli per cell, TNF- production by
CD14-null macrophages was significantly reduced compared with wild-type
macrophages (Fig. 6B). Although wild-type macrophages
produced TNF in response to as little as 0.1 E.
coli/macrophage, no cytokine production was observed at <1
E. coli/macrophage in CD14-null macrophages. This result
establishes that CD14 can also enhance the sensitivity of macrophage
responses to bacterial particles, but that this enhancement is observed
over a narrower range than that found with LPS. Although we did not
detect cytokine responses to LPS over a dosage range of 0.1–100 ng/ml
in our CD14-null animals, Haziot et al. did find some LPS
responsiveness in their animals at dosages of 1 µg/ml and higher. The
concentration of bacteria that gave half maximal stimulation in
CD14-null macrophages was 5 E. coli/macrophage. Using
current estimates of the amount of LPS present in the bacterial cell
wall of Gram-negative organisms (106 LPS
molecules/bacterium), this dose represents 40 ng/ml of LPS in our
experiments. This dose of LPS could not induce cytokine production in
CD14-null macrophages, suggesting that other factors, either intrinsic
to the bacterium or to the host cell, may contribute to the better
preserved response of CD14-null macrophages to whole organisms.
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produced in response to 10:1 heat-killed E. coli.
When CD14-null macrophages were taken from animals that were
129Sv/C57BL/6J hybrids and exposed to E. coli, their TNF-
production was intermediate between that produced by macrophages taken
from purebred mice of the two parental strains (Fig. 6A).
The CD14-null mice used in this experiment had been back-bred two
generations to C57BL/6J and thus were 75% C57BL/6J. These data
indicate that genetic variation at other murine loci contribute
to the macrophage cytokine response to bacterial stimulation and
that caution must be used in interpreting in vivo differences using
CD14-null mice that have not been back-bred to strain homogeneity. CD11b/CD18 mediates bacterial signaling in the absence of CD14
The CD11/CD18 family of leukocyte integrins can bind whole Gram-negative bacteria as a result of the recognition of the lipid A moiety in the outer membrane (9, 26). Like CD14, CD11/CD18 integrins have been reported to initiate signal transduction (11, 12, 13, 14) and are believed to enable LPS responses via the same downstream signaling molecules as CD14. Unlike CD14, CD11/CD18 integrins do not have a high affinity for monomeric LPS (27), but avidly interact with larger aggregates of LPS, including whole bacteria. In addition to its ability to bind directly to the lipid A moiety of LPS as a constituent of the bacterial membrane, CD11b/CD18 is also a receptor for complement (iC3b)- and LBP-coated bacteria (28).
Although all of the ß2 integrins appear to be
capable of mediating responses to LPS (13), mouse
peritoneal macrophages express CD11b/CD18 as the predominant
ß2 integrin (29). The hookworm
protein NIF has been demonstrated previously to inhibit CD11b/CD18
binding to its ligands by interfering with the function of the integrin
metal ion-dependent adhesion site domain (19). We used NIF
to block CD11b/CD18 binding to Gram-negative bacteria to quantitate
that portion of the E. coli-induced cytokine response that
occurred via this receptor in CD14-null macrophages. In a rosetting
assay, NIF pretreatment inhibited binding of iC3b-coated sheep RBC to
CD14-null peritoneal macrophages, confirming its ability to inhibit
binding to CD11b/CD18 in these cells (data not shown). Pretreatment of
CD14-null macrophages with NIF reduced TNF- production by 50% in
response to 5 E. coli per macrophage, a dose that was
determined to give half maximal TNF- production (Fig. 7). In contrast, no difference was
observed in TNF- production by wild-type macrophages treated with
NIF (Fig. 7, inset). These data imply that bacterial
internalization via CD11b/CD18 accounts for a substantial fraction of
the induction of cytokine signaling that occurs in the absence of
CD14.
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Because the concentration of LPS contained in the doses of
E. coli bioparticles used in our study is unable to initiate
signal transduction in CD14-null macrophages alone, we hypothesized
that the internalization of intact bioparticles was essential for
presentation of the inflammatory components of the bacterial membrane
to the signaling apparatus. To test whether phagocytosis of E.
coli was required for TNF- production, we pretreated CD14-null
and wild-type macrophages with cytochalasin B. Cytochalasin B inhibited
TNF- production by CD14-deficient, but not CD14-intact, macrophages
stimulated with 1–5 E. coli per cell for 4 h (Fig. 8). Inhibition of phagocytosis in both
wild-type and CD14-null macrophages was confirmed by examining the
uptake of fluorescently labeled E. coli bioparticles in
cytochalasin B-treated cells (data not shown). These data imply that
the initiation of cytokine signaling by alternative bacterial receptors
requires internalization of the whole bacterium in the absence of CD14
on the cell surface.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We generated mice in which the entire coding region of the
CD14 gene was deleted. This strategy, unlike the gene
interruption technique used in the generation of the previous CD14-null
mouse, ensures that no CD14 gene product could be made
(15). Despite this difference in our gene-targeting
strategy, like Haziot et al. (15), we found the phenotype
of our CD14-null mouse to be characterized by profound
hyporesponsiveness to purified endotoxin. No response of CD14-null
macrophages was observed at 100 ng of LPS per milliliter, whereas this
concentration gave a maximal response in wild-type cells. Addition of
physiologic amounts of sCD14 restored LPS responsiveness to the null
macrophages. Additionally, we confirm previous observations that two
major downstream effectors of LPS activation, NF-B translocation and
JNK phosphorylation, require upstream activation by CD14 (16, 17). Our data also confirm previous studies suggesting that CD14
sensitizes macrophages to the presence of purified LPS by greater than
two orders of magnitude. Thus, it is clear from our work and from that
of others, that the exquisite sensitivity of macrophage activation to
LPS stimulation requires CD14 (15, 16, 30). However, in
sharp contrast to this finding, we now demonstrate that CD14-null
macrophages can be activated by heat-killed whole E. coli in
a dose-dependent manner. At higher bacterial concentrations, the
stimulation of cytokines by wild-type and CD14-null macrophages is
indistinguishable. This previously unexplored finding demonstrates that
other signal transduction pathways can compensate for the loss in CD14
activity.
We found no difference in the binding and uptake of E. coli
in CD14-null and wild-type macrophages over a range of doses (10:1,
100:1) and conditions, including the presence of heat-inactivated or
normal serum and wild-type vs CD14-null serum. In the absence of CD14,
bacterial internalization via alternate phagocytic receptors such as
CD11b/CD18 may be a critical feature of proinflammatory responses to
Gram-negative bacteria. We demonstrate that 5 E. coli per
macrophage is sufficient to induce half maximal TNF- production in
CD14-null macrophages. Using the estimate that a single bacterium
contains 106 LPS molecules (31),
this E. coli concentration represents 40 ng of LPS per
milliliter. This concentration of purified LPS is unable to stimulate
CD14-null macrophages, yet when presented to macrophages in the context
of whole bacteria, a robust inflammatory response resulted. We
hypothesize that in the absence of CD14, alternative receptors
contribute to the internalization of the bacteria and allow effective
presentation of bacterial LPS to the downstream signaling apparatus
(e.g., TLRs). Underhill and colleagues recently described the
localization of TLR2 to the phagolysosome following phagocytosis of
zymosan, implying that signal transduction can occur after particle
internalization via intracellular TLR activation (32).
Thus, internalized Gram-negative bacilli may engage TLRs within the
phagolysosome, in the absence of CD14. However, the preservation of
CD14-null macrophage cytokine stimulation in response to whole E.
coli, despite the failure to phosphorylate JNK, suggests that
JNK-independent signal transduction pathways are activated by bacteria
that are not engaged by LPS alone. Alternatively, other proinflammatory
molecules in E. coli membranes, such as lipoproteins and
peptidoglycan, may account for part of the residual responses observed
in the CD14-null mice. It is well established that these molecules have
immunostimulatory capabilities, (33, 34, 35, 36, 37, 38, 39, 40); however, in
vitro studies have indicated that, like LPS, activation by these
alternative bacterial products is also enabled by CD14 (36, 40, 41, 42, 43).
Our hypothesis that E. coli internalized via alternate
receptors engage a signaling pathway within the phagolysosome is
supported by our finding that inhibition of bacterial phagocytosis in
CD14-null macrophages completely abrogates cytokine production. One
receptor that mediates bacterial signaling in the absence of CD14
appears to be CD11b/CD18. Using NIF to block this receptor, we show
that CD11b/CD18 accounts for approximately half of the CD14-independent
TNF- production. CD11b/CD18 has been shown to selectively modulate
LPS-induced gene expression, qualitatively influencing inflammatory
responses to bacterial endotoxin (29). As a number of
studies have provided evidence for direct interactions between
GPI-linked proteins, Fc receptors, and the integrins
(44, 45, 46, 47, 48, 49), it seems likely that the relationships between
phagocytosis receptors and downstream signaling pathways will be
complex.
The studies presented in this report suggest that macrophages rely heavily on CD14 to activate their arsenal of host defenses when confronting an early infection characterized by low pathogen number. We demonstrate that at low bacterial inocula (1 E. coli per cell), the inflammatory response is severely compromised in the absence of CD14. One prediction of this work is that animals inoculated with low dosages of pathogens might be especially susceptible to infection in the absence of CD14. For example, Jack et al. observed that LBP-null mice, when infected with small numbers of Salmonella typhimurium, were hypersusceptible to infection and thus succumbed to sepsis (50). In an animal model of human infection where a high inoculum is used, the outcome might be expected to more closely resemble endotoxin challenge. This might explain the contradictory findings of Haziot et al., who observed that CD14 knockout mice were resistant to Gram-negative infection (15) when they used very large numbers of bacteria to infect their CD14 knockout mice.
It is our expectation that the widespread availability of CD14-null mice should facilitate a detailed understanding of bacterial activation of host defenses in vivo and that this work may lead to improved therapies for septic shock. Perhaps just as importantly, there is a growing number of diseases that are thought to use the signal activation pathways used by LPS, including asthma, atherosclerosis, arthritis, and inflammatory bowel disease. These diseases should also benefit from the opportunity to use genetically engineered mice that serve as models of those diseases and that can now be rendered null for a critical component of the LPS signal transduction pathway.
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Footnotes |
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2 K.J.M. and L.P.A. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Mason W. Freeman, Massachusetts General Hospital, 55 Fruit Street, GRJ1328, Boston, MA 02114.
4 Abbreviations used in this paper: LBP, LPS binding protein; TLR, Toll-like receptor; NIF, neutrophil inhibitory factor; ES, embryonic stem; JNK, c-Jun N-terminal kinase; sCD14, soluble CD14.
Received for publication March 22, 2000. Accepted for publication July 24, 2000.
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B induced by LPS does not require tyrosine kinase activity. J. Biol. Chem. 269:22252.
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