A THP-1 cells were treated with TNF 3 ngÆmL1 or endotoxin-depleted hLF 500 lgÆmL1 for the indicated periods of time and then nuclear extracts were prepared.. The NF-jB DNA-binding activi
Trang 1receptor 4 pathway while it interferes with the
lipopolysaccharide-stimulated TLR4 signaling
Ken Ando1, Keiichi Hasegawa1, Ken-ichi Shindo1, Tomoyasu Furusawa1, Tomofumi Fujino1, Kiyomi Kikugawa1, Hiroyasu Nakano2, Osamu Takeuchi3, Shizuo Akira3, Taishin Akiyama4, Jin Gohda4, Jun-ichiro Inoue4and Makio Hayakawa1
1 School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Japan
2 Department of Immunology, Juntendo University School of Medicine, Japan
3 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan
4 Division of Cellular and Molecular Biology, Department of Cancer Biology, Institute of Medical Science, The University of Tokyo, Japan
Keywords
human lactoferrin; innate immunity;
lipopolysaccharide; nuclear factor-jB
(NF-jB); Toll-like receptor 4 (TLR4)
Correspondence
Makio Hayakawa, School of Pharmacy,
Tokyo University of Pharmacy and Life
Science, 1432-1 Horinouchi, Hachioji, Tokyo
192-0392, Japan
Fax: +81-42-676-4508
Tel: +81-42-676-4513
E-mail: hayakawa@ps.toyaku.ac.jp
(Received 26 August 2009, revised 15
February 2010, accepted 18 February 2010)
doi:10.1111/j.1742-4658.2010.07620.x
Lactoferrin (LF) has been implicated in innate immunity Here we reveal the signal transduction pathway responsible for human LF (hLF)-triggered nuclear factor-jB (NF-jB) activation Endotoxin-depleted hLF induces NF-jB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs) In MEFs, in which both tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-jB activation
at a level comparable to that seen in wild-type MEFs, whereas TRAF6-deficient MEFs show significantly impaired NF-jB activation in response
to hLF TRAF6 is known to be indispensable in leading to NF-jB activa-tion in myeloid differentiating factor 88 (MyD88)-dependent signaling 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.
Trang 2Lactoferrin (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 regulafunc-tion of iron
metabo-lism; it also plays multiple roles in host defense, and in
immune and inflammatory reactions [1–4]
LF shows antimicrobial activities against many
pathogens, including different types of bacteria [2] In
Gram-negative bacteria, it was observed that LF
specifically binds to porins present on the outer
membrane [5] and induces the rapid release of
lipo-polysaccharide (LPS), resulting in enhanced bacterial
susceptibility to osmotic shock, lysozyme, or other
antibacterial molecules [6] The LPS-binding activity of
LF may account for the other properties of this
pro-tein in the modulation of the inflammatory process [3]
The stimulation of mammalian cells by LPS occurs
through a series of interactions with several proteins,
including the LPS-binding protein (LBP), CD14,
mye-loid differentiation-2 (MD-2) and Toll-like receptor
(TLR)4 [7] Na et al [8] reported that macrophages
pretreated with the LF–LPS complex were rendered
tolerant to LPS challenge They suggested that the
down-regulation of TLR4 signaling is responsible for
this tolerance Alternatively, serum LBP may
partici-pate in the LF-dependent modulation of the
inflamma-tory response Elass-Rochard et al [9] showed that LF
prevented the LBP-mediated binding of LPS to CD14
In addition, Baveye et al [10] demonstrated that LF
interacted with soluble CD14, resulting in the
inhibi-tion of signal transducinhibi-tion mediated by the CD14–LPS
complex
In contrast to the anti-inflammatory roles noted
above, LF is known to activate immune cells to
pro-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 receptor that is involved in the LF-induced cytokine production is still obscure
In this study, we demonstrated that endotoxin-free human LF (hLF) directly activates nuclear factor-jB (NF-jB), which acts as the master regulator of immune and inflammatory responses, in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs) By characterizing vari-ous MEFs which lack adaptor or receptor molecules that trigger NF-jB activation, we found that TLR4 is responsible for hLF-induced NF-jB activation Furthermore, the carbohydrate chains of hLF were shown to play a crucial role in hLF-induced NF-jB activation Thus, we assume that the carbohydrate chains of hLF activate TLR4, which mediates the production of cytokines and chemokines Notably, when cells were simultaneously treated with LPS and endotoxin-depleted hLF, the levels of cytokines and chemokines produced were significantly lower than those of cells treated with LPS alone, suggesting that hLF may have a role as a moderate activator of the immune system while it can suppress the strong inflam-matory reactions induced by LPS
Results
LF has been shown to induce the expression of various cytokines such as TNF, IL-1 and IL-8 [11] Extensive research has established that NF-jB plays a critical role in the inducible expression of these cytokines involved in immune function and inflammation [20] Here we clearly demonstrated that hLF significantly 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
Trang 3with 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
It should be noted that the hLF used in our
experi-ments was prepared by passing it through a polymyxin
B (PMB)–agarose column, which is used to remove
contaminating endotoxin from the solution As shown
in Fig 1D, dose-dependent activation of NF-jB was
observed in MEFs treated with various concentrations
of LPS, whereas no activation of NF-jB was observed
in MEFs treated with fractions passed through a
PMB–agarose column, indicating that the
PMB–aga-rose column effectively absorbed LPS After passing
through this column, the level of endotoxin detected in
the hLF preparation was usually < 0.6 EUÆmg)1 To
prove that hLF-induced NF-jB activation is not
caused by traces of residual endotoxin, we compared
the levels of NF-jB activation in cells treated with
fractions of hLF passed through PMB–agarose versus
cells treated with fractions of hLF not passed through
PMB–agarose As shown in Fig 1E, NF-jB activation
occurred at a similar magnitude independently of
PMB–agarose purification of hLF, demonstrating that
hLF, but not the contaminating endotoxin, causes
NF-jB activation Furthermore, the treatment of cells with
actinomycin D or cycloheximide did not affect the
hLF-induced NF-jB activation (Fig 1F), indicating
that hLF directly triggers NF-jB activation without
requiring newly synthesized proteins such as TNF or
IL-1, well-known activators for NF-jB Thus, we have
demonstrated that hLF has the activity to stimulate
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 tion Furthermore, the observation that NF-jB activa-tion induced by hLF is ‘endo-b-galactosidase sensitive’, whereas activation induced by LPS is ‘endo-b-galacto-sidase resistant’, enables us to rule out the possibility that the trace amount of residual LPS in the post-PMB agarose fractions of hLF is responsible for NF-jB activation
We next focused on the molecular mechanism of how initial signaling that leads to the activation of IKK is triggered after hLF is recognized by cells Although a remarkable diversity of stimuli lead to the activation of NF-jB, many of the signaling intermedi-ates, especially those just upstream of the IKK com-plex, are thought to be shared [24] In particular, TNF receptor-associated factor (TRAF) families of proteins are key intermediates in nearly all NF-jB signaling pathways [24] Among seven TRAF proteins identified to date, TRAF2, TRAF5 and TRAF6 have been most extensively characterized as positive regula-tors of signaling to NF-jB From the study using TRAF2⁄ TRAF5 double-knockout mice, TRAF2 and TRAF5 were shown to be involved in TNF-induced NF-jB activation [25] However, TRAF6 has the most divergent TRAF-C domain, which mediates the interaction between TRAF proteins and the tails of cell-surface receptors, and is the only TRAF that
is involved in the signal from the members of the Toll⁄ IL-1 receptor [26] In order to verify the roles of
Trang 4TRAF proteins in hLF-induced NF-jB activation, we
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 activation in TRAF6-deficient MEFs However, by introducing a TRAF6 cDNA into those cells, NF-jB activation was restored (Fig 4C), suggesting that TRAF6 has an important role in hLF-induced NF-jB activation in MEFs
TRAF6 is known to be indispensable for NF-jB activation in the myeloid differentiating factor 88 (MyD88)-dependent signaling pathway; however, the role of TRAF6 in the MyD88-independent⁄ Toll ⁄ IL-1 receptor-domain-containing adaptor inducing inter-feron-b (TRIF)-dependent signaling pathway has not been clarified extensively [27] TRIF interacts directly with TRAF6 via its TRAF6-binding motifs in the N-terminal region [28,29] Jiang et al [29] showed that TRAF6-deficient MEFs that overexpressed TLR3 failed to activate NF-jB in response to poly(I:C), indicating that TRAF6 is critical in TRIF-dependent NF-jB activation downstream of TLR3 However, in our previous study, using macrophages isolated from TRAF6-deficient mice, TRAF6 was not required in the TRIF-dependent signaling including NF-jB acti-vation [30] This discrepancy concerning the require-ment of TRAF6 may reflect the cell-type-specific regulation of TRIF-signaling Indeed, in contrast to the results using TRAF6-deficient MEFs shown in Fig 4C and Fig S1, TRAF6-deficient macrophages clearly responded to hLF in terms of IKK activation and nuclear translocation of RelA, although the earlier responses observed in TRAF6+⁄)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)1of 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 i containing hLF was loaded or not loaded onto a PMB–agarose column After determining the protein concentration, 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 per-formed using densitometric analysis (Image Gauge 4.0) Similar results were obtained in three separate experiments (F) MEFs were pre-treated with actinomycin D (5 lgÆmL)1) or cycloheximide (5 lgÆmL)1) for 30 min and then treated with IL-1b (3 ngÆmL)1) or endotoxin-depleted hLF (500 lgÆmL)1) for the indicated periods of time Nuclear extracts were prepared and the RelA levels were determined using immunoblotting Using the same nuclear extracts, immunoblotting was carried out to detect the levels of histone H-1 as a loading control.
IB, immunoblotting.
Trang 5were weakened (Fig 4D) These results suggest that
hLF-induced NF-jB activation may involve the
TRIF-dependent pathway
TRIF-dependent NF-jB activation proceeds
down-stream of TLR3 or TLR4 However, TLR3-triggered
signaling is independent of MyD88, whereas TLR4 activates both MyD88-dependent and MyD88-indepen-dent⁄ TRIF-dependent signaling pathways [27] There-fore, we next examined the role of MyD88 in the hLF-stimulated signaling pathway leading to NF-jB
A
C B
D
E
F
Trang 6activation 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
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-depletrea-ted hLF (500 lgÆmL)1) containing 12.5 lgÆmL)1of oligosaccharides or with the endotoxin-depleted frac-tion of ActE-digested hLF (ActE–hLF) containing 12.5 lgÆmL)1of oligosaccharides Separately, cells were stimulated with TNF (3 ngÆmL)1) for 20 min Nuclear extracts were prepared and EMSA was performed in the presence or absence of anti-RelA, as described in the Materials and methods SS, supershifted band (C) THP-1 cells were treated for 90 min with endotoxin-depleted hLF (500 lgÆmL)1), ActE–hLF contain-ing 12.5 lgÆmL)1of oligosaccharides, or purified carbohydrate chains derived from endotoxin-depleted hLF (hLF–CC) containing 50 lgÆmL)1
of oligosaccharides Separately, cells were stimulated with TNF (3 ngÆmL)1) for 20 min Nuclear extracts were prepared and then subjected
to EMSA to analyze the NF-jB DNA-binding activities or to immunoblotting to detect the RelA levels (D) LPS or hLF were treated or left untreated with endo-b-galactosidase and then hLF was subjected to endotoxin depletion as described in the Materials and methods MEFs were stimulated with various concentrations of LPS or hLF for 60 min Nuclear and cytoplasmic extracts were prepared as described in the Materials and methods The IKK activities in the cytoplasmic extracts were determined using the IKK immunoprecipitation assay Using the same cytoplasmic extracts, immunoblotting was carried out to detect glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels as a load-ing control Nuclear extracts were analyzed by immunoblottload-ing to detect the RelA levels Usload-ing the same nuclear extracts, immunoblottload-ing 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 IB, immunoblotting.
Trang 7hLF, IKK activation was observed at 40 min,
but declined rapidly (Fig 5B) From these results,
hLF-induced NF-jB activation may occur through the
MyD88-dependent earlier step and through the
MyD88-independent⁄ TRIF-dependent later step Thus,
TLR4 could be a possible candidate as the receptor
responsible for hLF-stimulated signal transduction
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)1of 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)1of LPS Similarly to IP10, hLF stimu-lated the production of a significantly higher amount of TNF than present in the control but the levels were lower than in LPS-stimulated cells (Fig 7B)
LF has been described as the molecule that inter-feres with the biological actions of LPS [3,8–10] Indeed, the levels of IP10 and TNF produced by THP-1 cells simultaneously treated with LPS and hLF were significantly lower than those produced by cells treated with LPS alone (Fig 7A,B) When the NF-jB activities were examined using the 3x jB-Luc luciferase reporter vector, the action of LPS was also impaired in the presence of hLF, which alone induced
a lower, but significant, level of NF-jB activation (Fig 7C) By contrast, hLF failed to inhibit TNF-induced NF-jB activation (Fig S3), suggesting that hLF may specifically attenuate the LPS–TLR4 signal-ing pathway Interestsignal-ingly, ActE-digested hLF, in which the amount of oligosaccharide was equivalent
to that of intact hLF, failed to inhibit LPS-dependent NF-jB activation, while it induced NF-jB activation
at a level comparable to that induced by intact hLF (Fig 7C) Similarly, purified hLF carbohydrate chains that can stimulate NF-jB activation also failed to inhibit the action of LPS (Fig 7D) By contrast, endo-b-galactosidase treatment did not affect the inhibitory action of hLF on LPS-stimulated NF-jB activation, whereas it impaired hLF-dependent NF-jB activation (Fig 7E) These results suggest that the polypeptide moiety of hLF is required for inhibiting 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
NF-jB-dependent luciferase production was measured as described
in the Materials and methods Data are expressed as the mean ±
SD of triplicate determinations Bars represent fold induction
com-pared with the control (*P < 0.01) IB, immunoblotting.
Trang 8described the species-specific differences in the
struc-tures of LF glycans (i.e all LFs contain biantennary
glycans of the N-acetyllactosamine type; however, only
hLF contains a-1,3-fucosylated N-acetyllactosamine residues within them) In addition, only hLF possesses poly-N-acetyllactosaminic glycans By contrast, bovine
Fig 4 TRAF6 is indispensable in hLF-induced NF-jB activation in MEFs, whereas TRAF6-independent NF-jB activation occurs in mouse macrophages stimulated with hLF (A) Wild-type and TRAF2 ⁄ 5) ⁄ )MEFs were treated for 60 min with endotoxin-depleted hLF (500 lgÆmL)1)
or with endotoxin-depleted ActE–hLF containing 12.5 lgÆmL)1of oligosaccharides Separately, cells were stimulated with TNF (3 ngÆmL)1) or IL-1b (3 ngÆmL)1) for 20 min Nuclear extracts were prepared and then subjected to EMSA to analyze NF-jB DNA-binding activities or to immunoblotting to detect the RelA levels Using the same nuclear extracts, immunoblotting was carried out to detect histone H-1 levels as
a loading control (B) Wild-type and TRAF6) ⁄ )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)1of 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)1), or endotoxin-depleted hLF (500 lgÆmL)1) for the indicated periods of time, and then nuclear and cytoplasmic extracts were prepared as described in the Materials and methods The IKK activities were determined in the cytoplasmic extracts using the IKK immunoprecipitation assay and nuclear extracts were used for 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.
Trang 9B
Fig 5 NF-jB activation induced by hLF
occurs through MyD88-dependent and
MyD88-independent ⁄ TRIF-dependent
path-ways (A) Wild-type and MyD88) ⁄ )MEFs
were treated with endotoxin-depleted hLF
(500 lgÆmL)1), TNF (3 ngÆmL)1) or IL-1b
(3 ngÆmL)1) for the indicated periods of time
and then cytoplasmic and nuclear extracts
were prepared as described in the Materials
and methods The IKK activities were
deter-mined in the cytoplasmic extracts using the
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
NF-jB activation (A) Wild-type and
TLR4) ⁄ )MEFs were treated with LPS
(1500 EUÆmL)1), IL-1b (3 ngÆmL)1) or
endo-toxin-depleted hLF (500 lgÆmL)1) for the
indicated periods of time Nuclear extracts
were prepared and then subjected to EMSA
to analyze NF-jB DNA-binding activities or
to immunoblotting to detect RelA levels.
Using the same nuclear extracts,
immuno-blotting was carried out to detect histone
H-1 levels as a loading control Cytoplasmic
extracts from TLR4) ⁄ )MEFs were prepared
and the IKK activities were determined.
(B) Wild-type and TLR2) ⁄ )MEFs were
trea-ted with LPS (1500 EUÆmL)1), peptidoglycan
(PGN) (10 lgÆmL)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.
Trang 10Fig 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 (75 EUÆmL)1), or endotoxin-depleted hLF (500 lgÆmL)1) plus LPS (75 EUÆmL)1) for 24 h The levels of TNF released in the media were deter-mined by ELISA Data are expressed as the mean ± SD of triplicate determinations (C) 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 ActE–hLF containing 12.5 lgÆmL)1of oligosaccharides, LPS (150 EUÆmL)1), endotoxin-depleted hLF (500 lgÆmL)1) plus LPS (150 EUÆmL)1),
or endotoxin-depleted ActE–hLF containing 12.5 lgÆmL)1of 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 Fig 3B Data are expressed as the mean ± SD of triplicate determinations Bars represent fold induction compared with the unstimulated control (D) THP-1 cells were co-trans-fected with the 3x jB-Luc luciferase reporter vector and the b-galactosidase expression vector After 24 h, endotoxin-depleted hLF-CC con-taining 50 lgÆmL)1of oligosaccharides, LPS (150 EUÆmL)1), or hLF-CC containing 50 lgÆmL)1of 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), endotoxin-depleted hLF plus LPS, or endotoxin-depleted bLF plus LPS was added to the culture, which was incubated for a further 5 h NF-jB-dependent luciferase production was measured as described in Fig 3B Data are expressed as the mean ± SD of tripli-cate determinations Bars represent fold induction compared with the unstimulated control Endo-b, endo-b-galactosidase; IB, immunoblotting.