Human lactoferrin activates NF-jB through the Toll-like
receptor 4 pathway while it interferes with the
lipopolysaccharide-stimulated TLR4 signaling
Ken Ando
1
, Keiichi Hasegawa
1
, Ken-ichi Shindo
1
, Tomoyasu Furusawa
1
, Tomofumi Fujino
1
, Kiyomi
Kikugawa
1
, Hiroyasu Nakano
2
, Osamu Takeuchi
3
, Shizuo Akira
3
, Taishin Akiyama
4
, Jin Gohda
4
,
Jun-ichiro Inoue
4
and Makio Hayakawa
1

pathways, while the role of TRAF6 in the MyD88-independent signaling
pathway has not been clarified extensively. When we examined the
hLF-dependent NF-jB activation in MyD88-deficient MEFs, delayed, but
remarkable, NF-jB activation occurred as a result of the treatment of cells
with hLF, indicating that both MyD88-dependent and MyD88-independent
pathways are involved. Indeed, hLF fails to activate NF-jB in MEFs lack-
ing Toll-like receptor 4 (TLR4), a unique TLR group member that triggers
both MyD88-depependent and MyD88-independent signalings. Impor-
tantly, the carbohydrate chains from hLF are shown to be responsible for
TLR4 activation. Furthermore, we show that lipopolysaccharide-induced
cytokine and chemokine production is attenuated by intact hLF but not by
the carbohydrate chains from hLF. Thus, we present a novel model con-
cerning the biological function of hLF: hLF induces moderate activation of
TLR4-mediated innate immunity through its carbohydrate chains; however,
hLF suppresses endotoxemia by interfering with lipopolysaccharide-depen-
dent TLR4 activation, probably through its polypeptide moiety.
Abbreviations
ActE, actinase E; bLF, bovine lactoferrin; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
hLF, human lactoferrin; IKK, IjB kinase; IL, interleukin; IP10, interferon-c-inducible protein-10; IRF, interferon regulatory factor; JNK,
c-Jun N-terminal kinase; LBP, LPS-binding protein; LF, lactoferrin; LPS, lipopolysaccharide; LRP, low-density lipoprotein receptor-related
protein; MD-2, myeloid differentiation-2; MEF, mouse embryonic fibroblast; MyD88, myeloid differentiating factor 88; NF-jB, nuclear
factor-jB; PMB, polymyxin B; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; TRIF,
Toll ⁄ interleukin-1 receptor-domain-containing adaptor inducing interferon-b.
FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2051
Introduction
Lactoferrin (LF) is an iron-binding glycoprotein that is
abundant in exocrine secretions, including milk and
the fluids of the digestive tract [1]. Although LF
belongs to a family of transferrins, its biological func-
tion is not limited to the regulation of iron metabo-

duce several cytokines, such as tumor necrosis factor
(TNF), interleukin (IL)-8 and IL-12 [11–13]. However,
the molecular mechanism of how LF activates the
intracellular signaling pathway to induce the produc-
tion of these cytokines remains to be elucidated.
At the surface of cells, molecules should exist that
bind to LF and transduce intracellular signals to evoke
LF-dependent biological responses. One such candi-
date LF receptor is nucleolin [14], a 105 kDa nuclear
protein that has also been described as a cell-surface
receptor for several ligands, such as matrix laminin1
and midkine [15,16]. However, it is unlikely that nucle-
olin directly transduces the intracellular signals in
response to LF, because nucleolin lacks the mem-
brane-spanning region and the cytoplasmic domain
responsible for signal transduction. Another candidate
LF receptor has been described, namely the low-den-
sity lipoprotein receptor-related protein (LRP ⁄ LRP1)
[4]. LRP recognizes more than 30 different ligands,
including LF, and acts as a ‘cargo’ receptor, removing
such ligands from the cell surface [17]. However, the
involvement of LRP in the production of cytokines in
response to LF has not yet been described. A third
candidate molecule is the intestinal LF receptor identi-
fied by Suzuki et al. [18]. They indicate that this recep-
tor is responsible for taking up iron from LF into cells
in infants [19]. However, the intestinal LF receptor is
described as a GPI-anchored protein that lacks the
cytoplasmic domain responsible for signal transduction
[18]. Thus, the molecular nature of the cell-surface

stimulates NF-jB DNA binding in THP-1 cells
(Fig. 1A). In mammals, the family of NF-jB proteins
comprises five members: RelA ⁄ p65, RelB, c-Rel,
p50 ⁄ NF-jB1 and p52 ⁄ NF-jB2. Homodimers or hetero-
dimers of these proteins are active forms of NF-jB
Biological action of hLF is mediated through TLR4 K. Ando et al.
2052 FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS
with DNA-binding activity. The most intensively stud-
ied NF-jB dimer is RelA:p50 and the activating pro-
cess of this classical NF-jB dimer is described as the
‘canonical pathway’ that is initiated by the activation
of the IjB kinase (IKK) complex that is composed of
two catalytic subunits – IKKa (also known as IKK1)
and IKKb (also known as IKK2) – and a regulatory
subunit, IKKc (also known as NEMO) [21]. As shown
in the bottom panel of Fig. 1A, nuclear extract from
cells treated with hLF contained RelA, suggesting that
hLF activates NF-jB through the ‘canonical pathway’.
Figure 1B shows that significant NF-jB activation was
induced by hLF at physiologically relevant concentra-
tions (12.5–100 lgÆmL
)1
). Significant activation of
NF-jB was also observed in hLF-treated MEFs
(Fig. 1C). Furthermore, IKK, which is responsible for
the phosphorylation-induced degradation of IjB, was
activated in response to hLF (Fig. 1C, top panel). These
results suggest that hLF can stimulate the ‘canonical
pathway’, leading to the activation of the classical
NF-jB dimer, RelA:p50, in various types of cells.

the canonical NF-jB-activating pathway directly.
In our previous study, we indicated that the carbo-
hydrate chains of hLF play an important role in the
recognition of hLF by THP-1 macrophages [22]. In
order to evaluate the role of carbohydrate chains of
hLF, hLF was treated with actinase E (ActE) (which is
a nonspecific protease derived from Streptomyces
griseus and is also known as Pronase E [23]) in order
to digest the polypeptide region of hLF while the
carbohydrate chains of hLF remain intact (Fig. 2A).
As shown in Fig. 2B, ActE-digested hLF significantly
stimulated NF-jB DNA binding in MEFs. When anti-
RelA was added to the electrophoretic mobility shift
assay (EMSA) reaction mixture, the bands were super-
shifted to the top of the gel, confirming that the classi-
cal NF-jB dimer, RelA:p50, was activated (Fig. 2B). It
should be noted that ActE alone did not stimulate
NF-jB activation (data not shown). Furthermore, the
purified hLF carbohydrate chain fraction, in which
hLF-derived oligopeptides or amino acids were not
detectable, induced marked NF-jB activation in
THP-1 cells (Fig. 2C). By contrast, when hLF was
treated with endo-b-galactosidase, which is known to
cleave the carbohydrate chains at the internal
Galb1-4GlcNAc position, IKK activation and nuclear
translocation of RelA were significantly impaired,
while the same treatment did not affect LPS-induced
activation (Fig. 2D), suggesting that the carbohydrate
chains of hLF are critical for inducing NF-jB activa-
tion. Furthermore, the observation that NF-jB activa-

first examined whether or not overexpression of domi-
nant negative forms of TRAF2 or TRAF6, which lack
RING finger and zinc finger domains, impaired the
NF-jB activation in THP-1 cells in response to hLF.
As shown in Fig. 3A, similar amounts of dominant
negative forms of TRAF2 or TRAF6 were expressed
in THP-1 cells. While dominant negative TRAF2
effectively suppressed the TNF-induced NF-jB activa-
tion, no inhibition was observed in cells treated with
hLF or hLF-derived carbohydrate chains, as in the
case of IL-1-stimulated cells (Fig. 3B). By contrast,
THP-1 cells overexpressing dominant negative TRAF6
did not show NF-jB activation in response to IL-1,
hLF or hLF-derived carbohydrate chains, whereas
their response to TNF was comparable to that of con-
trol cells (Fig. 3B). These results suggest that TRAF6,
but not TRAF2, is involved in hLF-triggered NF- jB
activating signals. Then, we further investigated the
role of TRAFs in hLF-triggered NF-jB activation by
characterizing cells lacking TRAF isoforms. As
reported previously [25], TNF-stimulated NF-jB acti-
vation did not occur in MEFs lacking both TRAF2
and TRAF5 (Fig. 4A). By contrast, hLF and ActE-
treated hLF significantly stimulated NF-jB DNA
binding and nuclear translocation of RelA in
TRAF2 ⁄ TRAF5-deficient MEFs at levels comparable
to those in wild-type MEFs (Fig. 4A). In TRAF6-defi-
cient MEFs, neither hLF nor IL-1 induced NF-jB
activation (Fig. 4B and Fig. S1). As shown in Fig. 4C,
ActE-digested hLF also failed to induce NF-jB

macrophages
Fig. 1. Human LF induces canonical NF-jB activation. (A) THP-1 cells were treated with TNF (3 ngÆmL
)1
) or endotoxin-depleted hLF
(500 lgÆmL
)1
) for the indicated periods of time and then nuclear extracts were prepared. The NF-jB DNA-binding activities in the nuclear
extracts were determined using EMSA and the RelA levels in the nuclear extracts were determined using immunoblotting. Using the same
nuclear extracts, EMSA was used to determine the activity of the constitutively produced DNA-binding protein, Oct-1, as a loading control.
(B) THP-1 cells were treated with endotoxin-depleted hLF, at the indicated concentrations, for 90 min. Separately, cells were stimulated with
TNF (3 ngÆmL
)1
) for 20 min. Nuclear extracts were prepared and analyzed by immunoblotting for the presence of RelA. Using the same
nuclear extracts, immunoblotting was carried out to detect histone H-1 as a loading control. (C) MEFs were treated with TNF (3 ngÆmL
)1
)or
endotoxin-depleted hLF (500 lgÆmL
)1
) for the indicated periods of time, and nuclear and cytoplasmic extracts were prepared as described in
the Materials and Methods. The IKK activities in the cytoplasmic extracts were determined. The NF-jB and Oct-1 DNA-binding activities of
the nuclear extracts were measured using EMSA and the RelA levels of the nuclear extracts were determined by immunoblotting. (D) An
LPS solution containing 0.1 mgÆmL
)1
of BSA was loaded or not loaded onto a PMB–agarose column. After determining the protein concen-
tration, the eluate was used to treat MEFs for 60 min. Separately, MEFs were stimulated with IL-1b (3 ngÆmL
)1
) for 20 min. Nuclear extracts
were prepared and analyzed by immunoblotting to detect the levels of RelA. Using the same nuclear extracts, immunoblotting was carried
out to detect histone H-1 levels as a loading control. Quantification of the bands was performed using densitometric analysis (Image Gauge
4.0). Similar results were obtained in three separate experiments. (E) NaCl ⁄ P

fore, we next examined the role of MyD88 in the
hLF-stimulated signaling pathway leading to NF-jB
A
C
B
D
E
F
K. Ando et al. Biological action of hLF is mediated through TLR4
FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2055
activation. When MyD88-deficient MEFs were stimu-
lated with hLF, the earlier activation observed at
40 min was not obvious; however, significant IKK
activation occurred 80 min after stimulation with hLF
(Fig. 5A). Delayed, but significant, NF-jB DNA bind-
ing and nuclear translocation of RelA was also
observed in MyD88-deficient MEFs (Fig. 5A). By con-
trast, when TRIF-deficient MEFs were stimulated with
AB C
D
Fig. 2. Carbohydrate chains of hLF are responsible for NF- jB activation. (A) hLF was digested with actinase E as described in the Materials
and methods. The resultant sample was subjected to SDS ⁄ PAGE followed by Coomassie Brilliant Blue (CBB) staining. (B) MEFs were trea-
ted for 60 min with endotoxin-depleted hLF (500 lgÆmL
)1
) containing 12.5 lgÆmL
)1
of oligosaccharides or with the endotoxin-depleted frac-
tion of ActE-digested hLF (ActE–hLF) containing 12.5 lgÆmL
)1
of oligosaccharides. Separately, cells were stimulated with TNF (3 ngÆmL

Figure 6A clearly shows that hLF did not induce
IKK activation, NF-jB DNA binding or nuclear
translocation of RelA in TLR4-deficient MEFs at any
time-point studied. In addition, hLF failed to activate
c-Jun N-terminal kinase (JNK) in TLR4-deficient
MEFs, whereas it significantly induced JNK activation
in wild-type MEFs (Fig. S2). By contrast, hLF-stimu-
lated NF-jB activation was not impaired in MEFs
lacking TLR2, which triggers only the MyD88-depen-
dent signaling pathway (Fig. 6B). These results demon-
strated that TLR4 is responsible for hLF-evoked
signal transduction.
TLR4 can activate two separate transcription factors:
NF-jB and interferon regulatory factor 3 (IRF3); the
former is activated by the MyD88-dependent pathway
and the TRIF-dependent pathway and the latter is acti-
vated by the TRIF-dependent pathway [27]. NF-jB
induces several pro-inflammatory cytokines, such as
TNF or IL-1b, whereas IRF3 induces interferon-b,
thereby leading to the induction of interferon-inducible
genes such as interferon-c-inducible protein-10 (IP10).
Therefore, we next examined whether or not hLF indeed
induced TNF and IP10 production. As shown in
Fig. 7A, 500 lgÆmL
)1
of hLF induced the production of
a large amount of IP10 in THP-1 cells, although the
amount produced was lower than that in cells treated
with 75 EUÆmL
)1

LPS action, whereas the carbohydrate chains of hLF
act to stimulate TLR4.
Spik et al. [31] reported the primary structures of
LF glycans from humans, mice, cows and goats. They
A
B
Fig. 3. TRAF6, but not TRAF2, is involved in hLF-triggered NF-jB
activation. (A) THP-1 cells were co-transfected with the 3x jB-Luc
luciferase reporter vector and the b-galactosidase expression vec-
tor, together with the expression vector encoding the FLAG-tagged
dominant negative form of TRAF2 (TRAF2DN) or TRAF6
(TRAF6DN), as described in the Materials and methods. After 24 h,
cells were lysed in SDS ⁄ PAGE sample buffer, and the resultant cell
lysates were subjected to immunoblotting to detect the FLAG epi-
tope or to detect b-actin levels as a loading control. (B) THP-1 cells
were co-transfected with the 3x jB-Luc luciferase reporter vector
and the b-galactosidase expression vector, together with the
expression vector encoding TRAF2DN or TRAF6DN, as described
for Fig. 3A. After 24 h, TNF (10 ngÆmL
)1
), IL-1b (6 ngÆmL
)1
), endo-
toxin-depleted hLF (500 lgÆmL
)1
) or endotoxin-depleted hLF-CC,
containing 50 lgÆmL
)1
of oligosaccharides, was added to the
culture and incubated for a further 5 h. Cells were harvested and

) ⁄ )
MEFs were treated with endotoxin-depleted hLF (500 lgÆmL
)1
) for 60 min. Separately, cells
were stimulated for 20 min with TNF (3 ngÆmL
)1
) or IL-1b (3 ngÆmL
)1
). Nuclear extracts were prepared and then subjected to EMSA to ana-
lyze NF-jB DNA-binding activities or to immunoblotting to detect RelA levels. Using the same nuclear extracts, immunoblotting was carried
out to detect histone H-1 levels as a loading control. (C) Wild-type MEFs, TRAF6
) ⁄ )
MEFs and TRAF6
) ⁄ )
MEFs ectopically overexpressing
TRAF6 were treated for 60 min with endotoxin-depleted ActE–hLF containing 12.5 lgÆmL
)1
of oligosaccharides. Separately, cells were stimu-
lated with IL-1b (3 ngÆmL
)1
) for 20 min. Nuclear extracts were prepared and then subjected to EMSA to analyze NF-jB DNA-binding activi-
ties or to immunoblotting to detect RelA levels. Using the same nuclear extracts, immunoblotting was carried out to detect histone H-1
levels as a loading control. (D) Spleen macrophages, differentiated from the splenocytes of TRAF6
+ ⁄ )
and TRAF6
) ⁄ )
mice, were treated
with TNF (3 ngÆmL
)1
), IL-1b (3 ngÆmL

IKK immunoprecipitation assay. The NF-jB
DNA-binding activities in the nuclear
extracts were determined using EMSA and
the RelA levels in the nuclear extracts were
determined using immunoblotting. Using
the same nuclear extracts, immunoblotting
was carried out to detect histone H-1 levels
as a loading control. (B) TRIF
) ⁄ )
MEFs were
treated with LPS (75 EUÆmL
)1
), TNF
(3 ngÆmL
)1
), poly(I:C) (50 lgÆmL
)1
)or
endotoxin-depleted hLF (500 lgÆmL
)1
) for
the indicated periods of time. Cytoplasmic
extracts were prepared and the IKK
activities were determined. Using the same
cytoplasmic extracts, immunoblotting was
carried out to detect GAPDH levels as a
loading control. IB, immunoblotting.
A
B
Fig. 6. TLR4 is responsible for hLF-induced

)1
), or endotoxin-depleted
hLF (500 lgÆmL
)1
) for the indicated periods
of time. Nuclear extracts were prepared and
analyzed by immunoblotting to detect RelA
levels. Using the same nuclear extracts,
immunoblotting was carried out to detect
histone H-1 levels as a loading control. IB,
immunoblotting.
K. Ando et al. Biological action of hLF is mediated through TLR4
FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2059
A
DE F
BC
Fig. 7. Human LF moderately activates TLR4 via its carbohydrate chains whereas it attenuates LPS-triggered TLR4 activation independently
of the carbohydrate chains. (A) THP-1 cells were treated for 24 h with endotoxin-depleted hLF (500 lgÆmL
)1
), LPS (75 EUÆmL
)1
), or endo-
toxin-depleted hLF (500 lgÆmL
)1
) plus LPS (75 EUÆmL
)1
). The levels of IP10 released in the media were determined by ELISA. Data are
expressed as the mean ± SD of triplicate determinations. (B) THP-1 cells were treated with endotoxin-depleted hLF (500 lgÆmL
)1
), LPS

of oligosaccharides, LPS (150 EUÆmL
)1
), or hLF-CC containing 50 lgÆmL
)1
of oligosaccharides plus LPS (150 EUÆmL
)1
)
was added to the culture, which was incubated for a further 5 h. NF-jB-dependent luciferase production was measured as described in the
legend to Fig. 7C. Data are expressed as the mean ± SD of triplicate determinations. Bars represent fold induction compared with the
unstimulated control. (E) Human LF was treated or left untreated with endo-b-galactosidase and subjected to endotoxin depletion as
described in the Materials and methods. THP-1 cells were then stimulated for 90 min with endo-b-galactosidase-untreated ⁄ -treated hLF
(300 lgÆmL
)1
), LPS (75 EUÆmL
)1
), or endo-b-galactosidase-untreated ⁄ -treated hLF (300 lgÆmL
)1
) plus LPS (75 EUÆmL
)1
). Nuclear extracts
were prepared, and the levels of RelA were analyzed using immunoblotting. Using the same nuclear extracts, immunoblotting was carried
out to detect histone H-1 levels as a loading control. Quantification of the bands was carried out using densitometric analysis (Image Gauge
4.0). Similar results were obtained in three separate experiments. (F) THP-1 cells were co-transfected with 3x jB-Luc luciferase reporter
vector and b-galactosidase expression vector. After 24 h, endotoxin-depleted hLF (500 lgÆmL
)1
), endotoxin-depleted bLF (500 lgÆmL
)1
), LPS
(150 EUÆmL
)1

lane 1 versus lane 2). However, simultaneous addition
of the carbohydrate chains did not affect LPS-induced
RelA nuclear translocation (Fig. S4B, lane 3 versus
lane 4). From these results, we postulate that the poly-
N-acetyllactosaminic carbohydrate moiety may act as a
moderate activator of TLR4.
Discussion
LF has been described as the molecule that modulates
our immune system in vivo. However, two contrasting
descriptions for this have been put forward: one con-
cerns immuno-activating (or pro-inflammatory) prop-
erties and the other concerns immunosuppressive (or
anti-inflammatory) properties that are based on attenu-
ation of the LPS action [1,4]. It is necessary to deter-
mine the precise molecular mechanism of how these
contrasting functions are exerted. Most importantly,
the cell-surface receptor that recognizes LF and then
triggers intracellular signals to induce immunological
reactions must be identified. However, this has not yet
been fully elucidated.
The immuno-activating (or pro-inflammatory) func-
tion of LF represents the pro-inflammatory cytokine
production induced by LF. In this study, we demon-
strated that hLF induced significant activation of
NF-jB, a master regulator of immune reactions that
plays a critical role in pro-inflammatory cytokine pro-
duction. By characterizing MEFs in which the adaptor
or receptor molecules involved in NF-jB activation
are genetically deficient, we were able to narrow down
the signaling process responsible for hLF-induced

of endotoxin (Fig. 1E). This
result indicates that the effect of endotoxin
(13 EUÆmL
)1
) on NF-jB activation is negligible in the
presence of hLF (500 lgÆmL
)1
). By contrast, the action
of hLF (500 lgÆmL
)1
) is not influenced by endotoxin
(13 EUÆmL
)1
). Indeed, endotoxin-depleted hLF reduced
the levels of TNF and IP10 production induced by LPS
(Fig. 7, A and B). LF is secreted in the apo-form from
epithelial cells in most exocrine fluids, such as saliva,
bile, pancreatic and gastric fluids, tears and, particu-
larly, in milk [1]. In human milk, the hLF concentration
may vary from 1 mgÆmL
)1
(mature milk) to 7 mgÆmL
)1
(colostrum) [40]. We have shown, in the present study,
that hLF can induce NF-jB activation at much lower
concentrations (i.e. 25–500 lgÆmL
)1
) than found in
human milk, and therefore it is likely that hLF acts as
the endogenous activator for TLR4 in the intestines of

CD14–LPS complex, may be presented to TLR4,
thereby triggering TLR4 activation through the carbo-
hydrate chains of hLF. However, hLF may interfere
with the formation of the CD14–LPS complex, resulting
in the attenuation of LPS signaling. As LF is known to
interact with LPS, the binding of LPS with LBP may
also be interfered with in the presence of hLF [9].
Although the involvement of TLR4 in LF signaling
has already been reported by Curran et al. [42], their
conclusion was different from ours. They examined the
bLF-activated signal transduction, including NF-jB
signaling, using macrophages from congenic TLR4
) ⁄ )
C.C3-Tlr4
lps-d
mice. In contrast to our results, they
showed that bLF-stimulated IjB degradation was
rather enhanced in TLR4
) ⁄ )
macrophages, suggesting
that TLR4 is not required for bLF-induced NF-jB
activation [42]. At present, we cannot explain this dis-
crepancy. However, we should emphasize the role of
the carbohydrate chains of LF in TLR4 activation.
Species-specific differences exist in the structure of the
carbohydrate chains of LF [31]. As shown in Fig. 7F,
bLF, lacking poly-N-acetyllactosaminic glycans, fails
to activate NF-jB and attenuates the action of LPS.
By contrast, the poly-N-acetyllactosaminic glycan-
enriched fraction derived from human erythrocytes

was from BD Biosciences Pharmingen (San Diego, CA,
USA). Antibodies against JNK and phospho-JNK
(Thr183 ⁄ Tyr185) were obtained from Cell Signaling Tech-
nology (Danvers, MA, USA). The TNF-a Human Biotrak
Easy ELISA kit was obtained from GE Healthcare Bio-
Sciences KK, Japan. Actinase E was obtained from Kaken-
Seiyaku Co., Japan. Endo-b-galactosidase (EC 3.2.1.103)
from Escherichia freundii and the Endospecy
Ò
ES-50M kit
were purchased from Seikagaku Co., Japan. The PMB–aga-
rose column (Detoxi-GelÔ Endotoxin Removing Gel) was
from Pierce Biotechnology Inc. (Rockford, IL, USA).
Removal of contaminating endotoxins from hLF
First of all, we examined the endotoxin content in commer-
cially obtained hLF using the Endospecy
Ò
ES-50M kit.
While the endotoxin activity of E. coli LPS was approxi-
mately 1 510 000 EUÆmg
)1
, hLF usually contained endo-
toxin activity of 15–26 EUÆmg
)1
of protein. The NaCl ⁄ P
i
(PBS) solution containing 20 mg of hLF was loaded onto
the PMB–agarose column (1 mL of gel). The protein con-
centration of the eluate was then determined according to
the method of Bradford [45]. After passing through the col-

Endo-b-galactosidase treatment of hLF and LPS
hLF (4 mgÆmL
)1
) or LPS (15–450 EUÆmL
)1
) was treated
with endo-b-galactosidase (0.1 UÆmL
)1
) in 0.1 m sodium
acetate buffer (pH 5.8), containing 0.07 m NaCl, for 48 h at
37 °C. The resultant reaction mixtures were then dialyzed
against NaCl ⁄ P
i
. The dialyzed endo-b-galactosidase-treated
hLF was passed through the PMB–agarose column, as
described above, to remove contaminating endotoxin.
Cell culture, transient transfection and reporter
gene assay
The human monocytic leukemia cell line, THP-1, was main-
tained in RPMI-1640 supplemented with 10% fetal bovine
serum, penicillin (50 UÆmL
)1
) and streptomycin
(50 lgÆmL
)1
) in a humidified atmosphere of 5% CO
2
at
37 °C. MEFs were maintained in DMEM supplemented
with 10% fetal bovine serum, penicillin (50 UÆmL

taining 10% fetal bovine serum, and nonadherent cells were
further cultured with 10 ngÆmL
)1
of macrophage colony-
stimulating factor. Adherent cells obtained after 6 days of
culture were used as macrophages. The protocol of animal
preparations for the experiment was approved by the Ethics
Committee of our institute.
Preparation of the nuclear and cytoplasmic
extracts
Nuclear extracts were prepared according to the method of
Kida et al. [48], with slight modifications. Cells treated with
various agents were washed with NaCl ⁄ P
i
on ice and then
suspended in 800 lL of buffer A (10 mm Hepes ⁄ KOH, pH
7.9, 10 mm KCl, 1.5 mm MgCl
2
,1mm dithiothreitol, 0.2 mm
phenylmethanesulfonyl fluoride, 1 lgÆmL
)1
of aprotinin,
1 lgÆmL
)1
of leupeptin and 1 lgÆmL
)1
of pepstatin). After
incubation for 35 min on ice, Nonidet P-40 was added to the
samples to a concentration of 0.1%, then the samples were
left on ice for 5 min. After centrifugation (5 min, 2000 g,

After normalization of protein content according to the
protein assay, samples were resolved by SDS ⁄ PAGE and
K. Ando et al. Biological action of hLF is mediated through TLR4
FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2063
subjected to immunoblotting analyses. The immunocom-
plexes on the poly(vinylidene difluoride) membranes were
visualized using enhanced chemiluminescence detection.
Quantification of the bands was performed using densito-
metric analysis (Image Gauge 4.0).
IKK assay
Cells extracts were prepared using immunoprecipitation
buffer [50] with a slight modification (the Nonidet P-40
concentration was increased to 1.0%). After normalization
of the protein content according to the protein assay, endog-
enous IKK was immunoprecipitated with anti-IKKc, and
the in vitro kinase assay was performed as described
previously, using glutathione S-transferase (GST)–IjBa,
a member of IjB family of proteins, as the substrate [50].
Isolation of the carbohydrate chains from human
erythrocyte membrane proteins
The erythrocyte-rich fraction of healthy human blood
(blood group O) was obtained from the Japanese Red
Cross Tokyo Metropolitan Blood Center. Erythrocyte
membranes were then isolated and delipidated. In order to
obtain peptide ⁄ amino acid-free oligosaccharides, the delipi-
dated membranes were subjected to hydrazinolysis after
protease digestion. After confirming that amino acids were
not detectable in the purified fraction, the concentration of
the oligosaccharides was determined.
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Supporting information
The following supplementary material is available:
Fig. S1. Kinetics of hLF-induced NF-jB activation in
wild type and TRAF6
) ⁄ )
MEFs.
Fig. S2. Kinetics of hLF-induced JNK activation in
wild type and TLR4
) ⁄ )
MEFs.
Fig. S3. Human LF does not attenuate TNF-induced
NF-jB activation.
Fig. S4. Carbohydrate chains from human erythrocytes