INFECTION AND IMMUNITY, Sept. 2002, p. 4892–4896 Vol. 70, No. 9
0019-9567/02/$04.00
0 DOI: 10.1128/IAI.70.9.4892–4896.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Toll-Like Receptor 4 (TLR4)-Deficient Murine Macrophage Cell Line
as an In Vitro Assay System To Show TLR4-Independent Signaling
of Bacteroides fragilis Lipopolysaccharide
Eva Lorenz,1* Dhavalkumar D. Patel,2 Thomas Hartung,3 and David A. Schwartz4
Department of Internal Medicine, Section of Infectious Diseases, Wake Forest University Medical Center, Winston-Salem,1 and
Department of Medicine, Section of Allergy and Clinical Immunology,2 and Department of Medicine, Division of
Pulmonary and Critical Care Medicine,4 Duke University Medical Center, Durham, North Carolina, and
Department of Pharmacology, University of Konstanz, Konstanz, Germany3
Received 22 February 2002/Returned for modification 15 May 2002/Accepted 18 June 2002
Bacterial lipopolysaccharides (LPS) activate cells of innate immunity, such as macrophages, by stimulating
signaling through toll-like receptor 4 (TLR4). We and others have hypothesized that LPS derived from different
bacterial species may function through TLR4-independent mechanisms. To test this hypothesis, we have
generated using a nonviral transformation procedure a bone marrow-derived macrophage cell line called
10ScNCr/23 from mouse strain C57BL/10ScNCr. This mouse strain has a deletion of the TLR4 locus, causing
the mouse strain to be nonresponsive to stimulation by LPS from Escherichia coli while responding normally
to other bacterial substrates, such as lipoteichoic acid (LTA) from Staphylococcus aureus, which signal TLR4
independently. Stimulation with LTA induces five- and sixfold increases in 10ScNCr/23 cell line tumor necrosis
factor alpha and macrophage inflammatory protein-2 (MIP-2) secretion, but no increases in either cytokine
were found when cells were stimulated with E. coli LPS. Bacteroides fragilis-derived LPS, however, can effectively
stimulate MIP-2 expression in the absence of functional TLR4 in the 10ScNCr/23 cell line. This gives rise to
the notion that LPS from some bacterial species will utilize alternative receptors to stimulate the innate
immune response.
Macrophages and the macrophage-mediated innate immune
response have received increased attention recently through
the cloning and functional characterization of the Toll protein
family of transmembrane receptors (18). Macrophages are
considered the first line of defense against bacterial pathogens,
where the toll-like receptors (TLR) have been implicated as
the major receptors for bacterial pathogen binding on the cell
membrane (23). The Toll family consists of a minimum of 10
different members (21), each binding to a specific group of
pathogens. TLR4 is the best characterized receptor of the
family and was shown to be the signaling receptor for endo-
toxically active lipopolysaccharide (LPS) of gram-negative bac-
teria (15), while TLR5 binds bacteria expressing the flagellin
protein (8). TLR2, in combination with TLR6, is thought to
bind gram-positive bacteria, yeast, mycoplasma, and viruses
(7). TLR9 more recently has been shown to be involved in
initiating an immune response in response to CpG DNA (3),
while TLR3 has been identified as the receptor for double-
stranded RNA (1). Recent evidence has shown that the lines
defining the specificity of the Toll receptor-pathogen interac-
tion are not as clear as originally thought. Certain bacteria,
such as spirochetes, can signal through TLR2 as well as TLR4
(19), and heat shock protein was shown to signal through both
TLR2 and TLR4 (2), which can synergize (5). These findings
generate the need for a simple assay to determine whether
ligands signal through TLRs and whether signaling can occur
through more than one TLR.
In addition, recent evidence has indicated that even though
all TLRs are believed to signal through an MyD88-initiated
signal transduction cascade, different downstream proteins,
such as kinases, are involved in the signal transduction events
initiated by TLR2 and TLR4 in monocytes. Even though this is
the first evidence of TLR-specific signaling and has only been
shown in one cell type so far, it is very likely that different
TLRs initiate different signal transduction cascades. Most
likely this differential signaling is true for additional cell types
besides monocytes.
The cell line described in this paper is derived from mouse
strain C57BL/10ScNCr. This mouse strain has a deletion of the
TLR4 gene (17) but is wild type for the interleukin-12 (IL-12)
gene, in contrast to its close relative C57BL/10ScCr (16). The
macrophages from this mouse strain are nonresponsive to
stimulation by LPS. This makes the novel cell line 10ScNCr/23
an important tool to define the TLR specificity of novel bac-
terial substrates as well as a useful tool to dissect the differen-
tial cytokine expression pattern in response to TLR-mediated
signal transduction.
MATERIALS AND METHODS
Generation of cell lines. The cell line 10ScNCr/23 was generated using the
previously described methods of Monner and Walker with minor modifications
(12, 22). The cell line was propagated using microbeads until a sufficient number
and density were reached to cryopreserve the clonal isolates. The PCR analysis
(carried out by M. A. Freudenberg, Max-Planck Institute for Immunobiology,
Freiburg, Germany) revealed that the cell line 10ScNCr/23 carried a deletion of
the TLR4 locus, identical to that observed in C57BL/10ScCr mice, but the point
* Corresponding author. Mailing address: Section of Molecular
Medicine and Infectious Diseases, Wake Forest University School of
Medicine, Medical Center Boulevard, Winston-Salem NC 27157-1042,
Phone: (336) 716-4322. Fax: (336) 716-1214. E-mail: elorenz@wfubmc
.edu.
4892

mutation in the IL-12R2 gene, characteristic for C57BL/10ScCr mice, was
absent (16).
Cell culture medium and maintenance. Once immortalized, cells were grown
in a conditioned medium, prepared fresh on a weekly basis. The conditioned
medium contained 10% heat-inactivated fetal calf serum, 1% L-glutamine, and
20% LADMAC (catalog no. CRL 2420; American Type Culture Collection)
supernatant in minimal essential medium (22). This medium provides the iso-
lated macrophages with colony-stimulating factor-1. To eliminate the potential
for contamination as a source for unusual cytokine secretion profiles, it was
verified that the cell lines tested negative for mycoplasma.
Immune stimuli. Lipoteichoic acid (LTA) was prepared from Staphylococcus
aureus by butanol extraction as described elsewhere (13). LPS from Escherichia
coli 0111:B4 and LPS from E. coli 026:B6 were purchased as lyophilized powder
from Sigma, solubilized in Hank’s buffer, and tested for endotoxin content as
described previously (10). LPS from E. coli 0111:B4 does not signal through
TLR2, as determined previously (11). The Bacteroides fragilis LPS was a gift of
Ribi, Inc. The endotoxin preparations for in vitro testing using the cell line were
selected based on their failure to induce IL-1 production from whole human
blood at concentrations comparable to that of E. coli-derived LPS (T. Hartung,
unpublished data).
ELISAs. All cytokine assays were done in duplicate based on 50 l of cell
culture supernatant, including a standard curve for normalization in between
different experiments. Enzyme-linked immunosorbent assay (ELISA) kits were
purchased from R&D Biosystems and used according to the manufacturer’s
instructions. ELISA plates were read on a microplate reader (Powerwave; Biotek
Systems) with attached software (KCjunior; Biotek Systems) that allowed a
transformation of the absorbance reading into concentration curves (in pico-
grams per milliliter). The lowest limits of detection for the cytokines when using
this method were 5.1 pg/ml for tumor necrosis factor alpha (TNF-) and 7.8
pg/ml for macrophage inflammatory protein-2 (MIP-2).
Flow cytometry. Cells were labeled with fluorescein-conjugated antibodies to
F4/80 (mature macrophages), CD11b (bone marrow-derived macrophages), and
C11a (a general macrophage marker) (12). Isotype control antibodies were rat
anti-mouse immunoglobulin G2a (IgG2a) for CD11a and rat anti-mouse IgG2b
for F4/80 as well as CD11b. All antibodies were purchased from CalTag Labo-
ratories (Burlingame, Calif.) and were used according to the manufacturer’s
specifications. Analysis was performed on a Coulter Epics XL flow cytometer
(Beckman-Coulter, Fullerton, Calif.), and data were analyzed using CellQuest
software (Becton-Dickinson, Mountain View, Calif.).
Statistics. All statistical analyses were performed using the SPSS version 9.0
software package. Statistical significance was determined using a cutoff of
0.05.
RESULTS
The C57BL/10ScNCr-derived cell line exhibits a macro-
phage-like phenotype. To determine whether the resulting cell
line, 10ScNCr/23, was a clonal derivative of the primary
C57BL/10ScNCr-derived macrophages, we determined the ex-
pression level of macrophage-specific surface markers by flow
cytometry. Light microscopy had shown that the cell line 10Sc-
NCr/23 exhibited macrophage-like morphology. The resulting
cells were rounded in shape, adherent to tissue culture plates,
and sensitive to trypsin detachment (Fig. 1A). Flow cytometric
analysis of the cell line isolated in this study showed that all
three surface markers were expressed at robust levels in cell
line 10ScNCr/23, verifying the bone marrow-derived macro-
phage phenotype of the cells (Fig. 1B).
The murine macrophage cell line is unresponsive to LPS but
responsive to LTA. Mouse strain C57BL/10ScNCr was orig-
inally identified because of its hyporesponsive phenotype to
LPS stimulation. Since LTA signals through the TLR2 re-
ceptor (14) rather than the TLR4 receptor, which is deleted
FIG. 1. The cell line 10ScNCr/23 is derived from mouse bone mar-
row macrophages. (A) Light micrograph of cells derived from the cell
line 10ScNCr/23, indicating a macrophage-like appearance and adher-
ent phenotype. (B) Flow cytometry analysis to determine expression
levels of surface markers CD11a, CD11b, and F4/80, indicating the
bone marrow macrophage origin of 10ScNCr/23.
VOL. 70, 2002 C57BL/10ScNCr-DERIVED MACROPHAGE CELL LINE 4893