Human lactoferrin activates NF-jB through the Toll-like receptor 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 Inoue4 and Makio Hayakawa1 School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Japan Department of Immunology, Juntendo University School of Medicine, Japan Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan 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 (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 (TRAF2) and TRAF5 are deficient, hLF causes NF-jB activation at a level comparable to that seen in wild-type MEFs, whereas TRAF6deficient MEFs show significantly impaired NF-jB activation in response to hLF TRAF6 is known to be indispensable in leading to NF-jB activation 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 lacking Toll-like receptor (TLR4), a unique TLR group member that triggers both MyD88-depependent and MyD88-independent signalings Importantly, 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 concerning 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-dependent 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 Biological action of hLF is mediated through TLR4 K Ando et al 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 function is not limited to the regulation of iron metabolism; 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 lipopolysaccharide (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 protein 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, myeloid 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 participate in the LF-dependent modulation of the inflammatory 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 inhibition of signal transduction mediated by the CD14–LPS complex In contrast to the anti-inflammatory roles noted above, LF is known to activate immune cells to produce 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 production 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 candidate 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 nucleolin directly transduces the intracellular signals in response to LF, because nucleolin lacks the membrane-spanning region and the cytoplasmic domain responsible for signal transduction Another candidate 2052 LF receptor has been described, namely the low-density 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 identified by Suzuki et al [18] They indicate that this receptor 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 various 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 inflammatory 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 heterodimers of these proteins are active forms of NF-jB FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS K Ando et al with DNA-binding activity The most intensively studied NF-jB dimer is RelA:p50 and the activating process 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 concentrations (12.5–100 lgỈmL)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 experiments 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–agarose column effectively absorbed LPS After passing through this column, the level of endotoxin detected in the hLF preparation was usually < 0.6 Emg)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 NFjB 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 carbohydrate chains of hLF play an important role in the recognition of hLF by THP-1 macrophages [22] In Biological action of hLF is mediated through TLR4 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 antiRelA was added to the electrophoretic mobility shift assay (EMSA) reaction mixture, the bands were supershifted to the top of the gel, confirming that the classical 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 activation Furthermore, the observation that NF-jB activation induced by hLF is ‘endo-b-galactosidase sensitive’, whereas activation induced by LPS is ‘endo-b-galactosidase 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 intermediates, especially those just upstream of the IKK complex, 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 regulators 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 FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2053 Biological action of hLF is mediated through TLR4 K Ando et al Fig Human LF induces canonical NF-jB activation (A) THP-1 cells were treated with TNF (3 ngỈmL)1) or endotoxin-depleted hLF (500 lgỈmL)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 Separately, cells were stimulated with TNF (3 ngỈmL)1) for 20 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 ngỈmL)1) or endotoxin-depleted hLF (500 lgỈmL)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 mgỈmL)1 of BSA was loaded or not loaded onto a PMB–agarose column After determining the protein concentration, the eluate was used to treat MEFs for 60 Separately, MEFs were stimulated with IL-1b (3 ngỈmL)1) for 20 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 ⁄ Pi 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 Separately, MEFs were stimulated with IL-1b (3 ngỈmL)1) for 20 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 (F) MEFs were pretreated with actinomycin D (5 lgỈmL)1) or cycloheximide (5 lgỈmL)1) for 30 and then treated with IL-1b (3 ngỈmL)1) or endotoxindepleted hLF (500 lgỈmL)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 TRAF proteins in hLF-induced NF-jB activation, we first examined whether or not overexpression of dominant 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 activation, 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 control 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 activation did not occur in MEFs lacking both TRAF2 and TRAF5 (Fig 4A) By contrast, hLF and ActEtreated 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-deficient MEFs, neither hLF nor IL-1 induced NF-jB activation (Fig 4B and Fig S1) As shown in Fig 4C, 2054 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 interferon-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 activation [30] This discrepancy concerning the requirement 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 FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS K Ando et al A Biological action of hLF is mediated through TLR4 B C D E F were weakened (Fig 4D) These results suggest that hLF-induced NF-jB activation may involve the TRIF-dependent pathway TRIF-dependent NF-jB activation proceeds downstream of TLR3 or TLR4 However, TLR3-triggered signaling is independent of MyD88, whereas TLR4 activates both MyD88-dependent and MyD88-independent ⁄ TRIF-dependent signaling pathways [27] Therefore, we next examined the role of MyD88 in the hLF-stimulated signaling pathway leading to NF-jB FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2055 Biological action of hLF is mediated through TLR4 A K Ando et al B C D Fig 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 treated for 60 with endotoxin-depleted hLF (500 lgỈmL)1) containing 12.5 lgỈmL)1 of oligosaccharides or with the endotoxin-depleted fraction of ActE-digested hLF (ActE–hLF) containing 12.5 lgỈmL)1 of oligosaccharides Separately, cells were stimulated with TNF (3 ngỈmL)1) for 20 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 with endotoxin-depleted hLF (500 lgỈmL)1), ActE–hLF containing 12.5 lgỈmL)1 of oligosaccharides, or purified carbohydrate chains derived from endotoxin-depleted hLF (hLF–CC) containing 50 lgỈmL)1 of oligosaccharides Separately, cells were stimulated with TNF (3 ngỈmL)1) for 20 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 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 loading control Nuclear extracts were analyzed by 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 Quantification of the bands was carried out using densitometric analysis (Image Gauge 4.0) Similar results were obtained in three separate experiments IB, immunoblotting activation When MyD88-deficient MEFs were stimulated with hLF, the earlier activation observed at 40 was not obvious; however, significant IKK activation occurred 80 after stimulation with hLF 2056 (Fig 5A) Delayed, but significant, NF-jB DNA binding and nuclear translocation of RelA was also observed in MyD88-deficient MEFs (Fig 5A) By contrast, when TRIF-deficient MEFs were stimulated with FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS K Ando et al A B Fig 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 vector, 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 epitope 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 ngỈmL)1), IL-1b (6 ngỈmL)1), endotoxin-depleted hLF (500 lgỈmL)1) or endotoxin-depleted hLF-CC, containing 50 lgỈmL)1 of oligosaccharides, was added to the culture and incubated for a further 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 compared with the control (*P < 0.01) IB, immunoblotting hLF, 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- Biological action of hLF is mediated through TLR4 lated NF-jB activation was not impaired in MEFs lacking TLR2, which triggers only the MyD88-dependent signaling pathway (Fig 6B) These results demonstrated that TLR4 is responsible for hLF-evoked signal transduction TLR4 can activate two separate transcription factors: NF-jB and interferon regulatory factor (IRF3); the former is activated by the MyD88-dependent pathway and the TRIF-dependent pathway and the latter is activated 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 lgỈmL)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 EmL)1 of LPS Similarly to IP10, hLF stimulated 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 interferes 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 TNFinduced NF-jB activation (Fig S3), suggesting that hLF may specifically attenuate the LPS–TLR4 signaling pathway Interestingly, 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 FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2057 Biological action of hLF is mediated through TLR4 K Ando et al A C B D Fig 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 with endotoxin-depleted hLF (500 lgỈmL)1) or with endotoxin-depleted ActE–hLF containing 12.5 lgỈmL)1 of oligosaccharides Separately, cells were stimulated with TNF (3 ngỈmL)1) or IL-1b (3 ngỈmL)1) for 20 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 lgỈmL)1) for 60 Separately, cells were stimulated for 20 with TNF (3 ngỈmL)1) or IL-1b (3 ngỈmL)1) 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, 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 with endotoxin-depleted ActE–hLF containing 12.5 lgỈmL)1 of oligosaccharides Separately, cells were stimulated with IL-1b (3 ngỈmL)1) for 20 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, 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 ngỈmL)1), IL-1b (3 ngỈmL)1), or endotoxin-depleted hLF (500 lgỈmL)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 described the species-specific differences in the structures of LF glycans (i.e all LFs contain biantennary glycans of the N-acetyllactosamine type; however, only 2058 hLF contains a-1,3-fucosylated N-acetyllactosamine residues within them) In addition, only hLF possesses poly-N-acetyllactosaminic glycans By contrast, bovine FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS K Ando et al Biological action of hLF is mediated through TLR4 A Fig NF-jB activation induced by hLF occurs through MyD88-dependent and MyD88-independent ⁄ TRIF-dependent pathways (A) Wild-type and MyD88) ⁄ ) MEFs were treated with endotoxin-depleted hLF (500 lgỈmL)1), TNF (3 ngỈmL)1) or IL-1b (3 ngỈmL)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 determined 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 EmL)1), TNF (3 ngỈmL)1), poly(I:C) (50 lgỈmL)1) or endotoxin-depleted hLF (500 lgỈmL)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 B A Fig TLR4 is responsible for hLF-induced NF-jB activation (A) Wild-type and TLR4) ⁄ ) MEFs were treated with LPS (1500 EmL)1), IL-1b (3 ngỈmL)1) or endotoxin-depleted hLF (500 lgỈmL)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, immunoblotting 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 treated with LPS (1500 EmL)1), peptidoglycan (PGN) (10 lgỈmL)1), or endotoxin-depleted hLF (500 lgỈmL)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 B FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2059 Biological action of hLF is mediated through TLR4 A B D E K Ando et al C F Fig 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 lgỈmL)1), LPS (75 EmL)1), or endotoxin-depleted hLF (500 lgỈmL)1) plus LPS (75 EmL)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 lgỈmL)1), LPS (75 EmL)1), or endotoxin-depleted hLF (500 lgỈmL)1) plus LPS (75 EmL)1) for 24 h The levels of TNF released in the media were determined 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 lgỈmL)1), endotoxin-depleted ActE–hLF containing 12.5 lgỈmL)1 of oligosaccharides, LPS (150 EmL)1), endotoxin-depleted hLF (500 lgỈmL)1) plus LPS (150 EmL)1), or endotoxin-depleted ActE–hLF containing 12.5 lgỈmL)1 of oligosaccharides plus LPS (150 EmL)1) was added to the culture, which was incubated for a further 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-transfected with the 3x jB-Luc luciferase reporter vector and the b-galactosidase expression vector After 24 h, endotoxin-depleted hLF-CC containing 50 lgỈmL)1 of oligosaccharides, LPS (150 EmL)1), or hLF-CC containing 50 lgỈmL)1 of oligosaccharides plus LPS (150 EmL)1) was added to the culture, which was incubated for a further 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 with endo-b-galactosidase-untreated ⁄ -treated hLF (300 lgỈmL)1), LPS (75 EmL)1), or endo-b-galactosidase-untreated ⁄ -treated hLF (300 lgỈmL)1) plus LPS (75 EmL)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 lgỈmL)1), endotoxin-depleted bLF (500 lgỈmL)1), LPS (150 EmL)1), endotoxin-depleted hLF plus LPS, or endotoxin-depleted bLF plus LPS was added to the culture, which was incubated for a further 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 Endo-b, endo-b-galactosidase; IB, immunoblotting 2060 FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS K Ando et al LF (bLF) contains glycans of the oligomannosidic type, representing the unique member of the transferrin family containing both N-acetyllactosamine and oligomannosidic-type glycans [31] As shown in Fig 7F, bLF did not induce NF-jB activation but strongly attenuated LPS-induced NF-jB activation, similarly to hLF These results clearly demonstrate that both hLF and bLF conserve the polypeptide structure that contributes to the impairment of LPS action, while only hLF contains ‘active carbohydrate chains’ to stimulate TLR4-mediated signaling pathways As the candidate of ‘active carbohydrate chains’, we focused on poly-N-acetyllactosaminic glycans Besides hLF, poly-N-acetyllactosaminic glycans are known to exist in human erythrocytes [32,33] Therefore, we examined whether or not carbohydrate chains isolated from human erythrocytes induce NF-jB activation When wild-type MEFs were treated with human erythrocyte-derived carbohydrate chains, nuclear translocation of RelA was clearly demonstrated, whereas the same treatment did not induce the nuclear translocation of RelA in TLR4-deficient MEFs at any time-point indicated (Fig S4A) Human erythrocyte-derived carbohydrate chains also induced nuclear translocation of RelA in THP-1 cells (Fig S4B, lane versus lane 2) However, simultaneous addition of the carbohydrate chains did not affect LPS-induced RelA nuclear translocation (Fig S4B, lane versus lane 4) From these results, we postulate that the polyN-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 concerns immuno-activating (or pro-inflammatory) properties and the other concerns immunosuppressive (or anti-inflammatory) properties that are based on attenuation of the LPS action [1,4] It is necessary to determine 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) function of LF represents the pro-inflammatory cytokine production induced by LF In this study, we demonstrated that hLF induced significant activation of NF-jB, a master regulator of immune reactions that plays a critical role in pro-inflammatory cytokine pro- Biological action of hLF is mediated through TLR4 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 NF-jB activation, and reached the conclusion that TLR4 is indispensable TLR4 is an essential receptor for LPS recognition [34,35] In addition, TLR4 is implicated in the recognition of taxol, a diterpene purified from the bark of the western yew (Taxus brevifolia) [36,37] Furthermore, TLR4 has been shown to be involved in the recognition of endogenous ligands, such as heat shock proteins (Hsp60 and Hsp70), the extra domain A of fibronectins, oligosaccharides of hyaluronic acid, heparan sulfate and fibrinogen However, very high concentration of these endogenous ligands are required to activate TLR4 [38] In addition, it has been shown that contamination of the Hsp70 preparation with LPS confers the ability to activate TLR4 [39] In this study, we used hLF passed through a PMB–agarose column to minimize contamination with endotoxin It is of note that the magnitude of NF-jB activation induced by a pre-PMB–agarose fraction of hLF (500 lgỈmL)1), which contained 13 EmL)1 of endotoxin, was comparable to that induced by a postPMB–agarose fraction of hLF (500 lgỈmL)1), which contained < 0.3 EmL)1 of endotoxin (Fig 1E) This result indicates that the effect of endotoxin (13 EmL)1) on NF-jB activation is negligible in the presence of hLF (500 lgỈmL)1) By contrast, the action of hLF (500 lgỈmL)1) is not influenced by endotoxin (13 EmL)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, particularly, in milk [1] In human milk, the hLF concentration may vary from mgỈmL)1 (mature milk) to mgỈmL)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 lgỈmL)1) than found in human milk, and therefore it is likely that hLF acts as the endogenous activator for TLR4 in the intestines of the breast-fed infants while concomitantly acting as a competitor of LPS Interestingly, ActE-digested hLF does not inhibit LPS and it potently activates NF-jB, similarly to intact hLF (Fig 7C) By contrast, endob-galactosidase-treated hLF retained the inhibitory property towards LPS, whereas its activity, in terms of NF-jB activation, was impaired compared with that of intact hLF (Fig 7E) These results suggest that hLF may have divergent intramolecular modules in its carbohydrate chains and its polypeptide moiety: the former FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2061 Biological action of hLF is mediated through TLR4 K Ando et al with an immuno-activating property and the latter with an anti-inflammatory property The current view on the TLR4 activation induced by LPS is as follows: LBP (a serum protein or a cell-associated protein) binds to the LPS aggregates An LPS monomer is then transferred to CD14 CD14 is required for the efficient transfer of the LPS monomer to MD-2, which is either soluble form or associated with the ectodomain of TLR4 The formation of a trimeric LPS–MD-2–TLR4 complex is thought to be the final event of the recognition of extracellular LPS [41] In the present study, we were unable to reveal the molecular mechanism of how hLF triggers TLR4 activation Interestingly, Baveye et al [10] reported that hLF interacts with soluble CD14 and inhibits the expression of E-selectin and intercellular adhesion molecule (ICAM) induced by the CD14–LPS complex Therefore, we can postulate that the CD14–hLF complex, instead of the CD14–LPS complex, may be presented to TLR4, thereby triggering TLR4 activation through the carbohydrate 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-Tlr4lps-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 discrepancy 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 glycanenriched fraction derived from human erythrocytes activated NF-jB, but did not affect the LPS-induced activation of NF-jB (Fig S4A, B) There is intense interest in the effects of breast-feeding on infants and the mechanisms behind these effects Compared with formula-fed infants, breast-fed infants are known to have a high level of resistance to infectious diseases [43] In particular, several lines of evidence suggest that breast-feeding provides strong protection against diarrheal disease [44] Human milk contains numerous components that support the infant’s host 2062 defense [44] Among them, hLF may play an important role in stimulating the infant’s own immune system by activating TLR4-mediated signaling while preventing the high levels of inflammation induced by LPS Thus, we should highlight TLR4 as the key molecule required for the biological activity of hLF Materials and methods Reagents LF from human milk, LF from bovine milk, LPS from Escherichia coli serotype O111:B4 and anti-FLAG M2 were purchased from Sigma Chemical Co (St Louis, MO, USA) Peptidoglycan from Staphylococcus aureus was obtained from Fluka The human IP10 ELISA kit was obtained from Invitrogen Corporation (Carlsbad, CA, USA) Recombinant human IL-1b and recombinant human TNF-a were purchased from R&D Systems, Inc (Minneapolis, MN, USA) Antibodies specific for NF-jB p65 (C20), histone H-1 (AE-4) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 6C5) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Anti-IKKc (C73-764) was from BD Biosciences Pharmingen (San Diego, CA, USA) Antibodies against JNK and phospho-JNK (Thr183 ⁄ Tyr185) were obtained from Cell Signaling Technology (Danvers, MA, USA) The TNF-a Human Biotrak Easy ELISA kit was obtained from GE Healthcare BioSciences KK, Japan Actinase E was obtained from KakenSeiyaku Co., Japan Endo-b-galactosidase (EC 3.2.1.103) from Escherichia freundii and the EndospecyÒ ES-50M kit were purchased from Seikagaku Co., Japan The PMB–agarose 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 commercially obtained hLF using the EndospecyÒ ES-50M kit While the endotoxin activity of E coli LPS was approximately 510 000 Emg)1, hLF usually contained endotoxin activity of 15–26 Emg)1 of protein The NaCl ⁄ Pi (PBS) solution containing 20 mg of hLF was loaded onto the PMB–agarose column (1 mL of gel) The protein concentration of the eluate was then determined according to the method of Bradford [45] After passing through the column, the endotoxin activity in hLF was < 0.6 Emg)1 (i.e more than 96% of LPS was removed from hLF) ActE digestion of hLF NaCl ⁄ Pi containing hLF (15 mgỈmL)1) was subjected to digestion with ActE (5 mgỈmL)1) for 48 h at 37 °C After inactivating the enzyme by boiling for 10 min, the resultant FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS K Ando et al reaction mixture was centrifuged for 20 at 3000 g The supernatant was filtered through a 0.22-lm filer membrane (MILLEX-GV; Millipore, Billerica, MA, USA), the filtrate was loaded onto the PMB–agarose column and the eluate was collected Then, the concentration of the saccharides was determined using a phenol ⁄ sulfuric acid method [46] and expressed as the equivalent concentration of glucose Isolation of the carbohydrate chains from hLF In order to obtain peptide ⁄ amino acid-free N-linked oligosaccharides from hLF, hLF was subjected to hydrazinolysis and purified as described previously [22] After confirming that amino acids were not detectable in the purified fraction, the concentration of the oligosaccharides was determined using a phenol ⁄ sulfuric acid method [46] and expressed as the equivalent concentration of glucose Endo-b-galactosidase treatment of hLF and LPS hLF (4 mgỈmL)1) or LPS (15–450 EmL)1) was treated with endo-b-galactosidase (0.1 mL)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 ⁄ Pi 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 maintained in RPMI-1640 supplemented with 10% fetal bovine and streptomycin serum, penicillin (50 mL)1) )1 (50 lgỈmL ) in a humidified atmosphere of 5% CO2 at 37 °C MEFs were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin (50 mL)1), streptomycin (50 lgỈmL)1) and 50 lm b-mercaptoethanol in a humidified atmosphere of 10% CO2 at 37 °C The 3x jB-Luc luciferase reporter vector (0.5 lg) and the b-galactosidase expression vector pAct ⁄ b-gal (0.1 lg) were co-transfected into THP-1 cells using the FuGENE6 transfection reagent (Roche Diagnostics, IN, USA) After 24 h of incubation, cells were treated with TNF, IL-1b, hLF or purified carbohydrate chains from hLF (hLF-CC) and then incubated for a further h The cells were then harvested and the cellular luciferase activities were measured using a chemiluminescence photometer and normalized to the b-galactosidase expression Data were analyzed using the Student’s t-test To evaluate the effect of dominant negative forms of TRAF2 or TRAF6 on NF-jB activation, cells were transfected with pME-FLAG-TRAF2DN or pME-FLAG-TRAF6DN during transfection with the reporter plasmids, as described previously [47] Biological action of hLF is mediated through TLR4 Differentiation of splenocytes from TRAF6-deficient mice into macrophages Spleen cells from 2-week-old TRAF6+ ⁄ ) and TRAF6) ⁄ ) mice [30] were incubated for h in MEMa (Gibco) containing 10% fetal bovine serum, and nonadherent cells were further cultured with 10 ngỈmL)1 of macrophage colonystimulating factor Adherent cells obtained after 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 ⁄ Pi on ice and then suspended in 800 lL of buffer A (10 mm Hepes ⁄ KOH, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, mm dithiothreitol, 0.2 mm phenylmethanesulfonyl fluoride, lgỈmL)1 of aprotinin, lgỈmL)1 of leupeptin and lgỈmL)1 of pepstatin) After incubation for 35 on ice, Nonidet P-40 was added to the samples to a concentration of 0.1%, then the samples were left on ice for After centrifugation (5 min, 2000 g, °C), the cytoplasmic extracts were immediately adjusted to the condition for immunoprecipitation by adding the immunoprecipitation (2·) buffer described below The precipitates containing nuclear proteins were further washed once with buffer A containing 0.1% Nonidet P-40 and then extracted with buffer C (20 mm Hepes ⁄ KOH, pH 7.9, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, mm dithiothreitol, 0.2 mm phenylmethanesulfonyl fluoride and 25% glycerol) EMSA EMSA was performed as described previously [49] Binding reaction mixtures containing lg of nuclear extract protein, lg of poly(dI-dC) and 32P-labeled probe, were incubated for 20 at room temperature For the antibody-mediated supershift assay, nuclear extracts were pre-incubated with lg of anti-RelA for 20 at °C before adding the 32 P-labeled probe The reaction mixtures were analyzed electrophoresis through native 4% polyacrylamide gels The sequence of the NF-jB probe was 5¢-AATTCTCAGAG GGGACTTTCCGAGAGG-3¢ The sequence of the Oct-1 probe was 5¢-CTAGATATGCAAATCATTG-3¢ Immunoblotting Cells were washed with NaCl ⁄ Pi and cell extracts were prepared using the SDS ⁄ PAGE sample buffer described below After normalization of protein content according to the protein assay, samples were resolved by SDS ⁄ PAGE and FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS 2063 Biological action of hLF is mediated through TLR4 K Ando et al subjected to immunoblotting analyses The immunocomplexes on the poly(vinylidene difluoride) membranes were visualized using enhanced chemiluminescence detection Quantification of the bands was performed using densitometric 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, endogenous 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 delipidated 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 Acknowledgements We thank T Hiraga, K Ihashi, K Sato, N Komagata, H Majima, Y Namekawa, A Suzuki, A Kashimura, M Yamamuro, M Watanabe, K Ito, Y Murase and T Setoguchi for technical assistance We also thank Dr S Miyamoto for providing us with the x jB-Luc luciferase reporter vector This work was supported in part by a grant from the Japan Private School Promotion Foundation References Legrand D, Elass E, Carpentier M & Mazurier J (2005) Lactoferrin: a modulator of immune and inflammatory responses Cell Mol Life Sci 62, 2549–2559 Ward PP, Uribe-Luna S & Conneely OM (2002) Lactoferrin and host defense Biochem Cell Biol 80, 95–102 Vorland LH (1999) 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J 10, 2247–2258 50 DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E & Karin M (1997) A cytokine-responsive IjB kinase 2066 that activates the transcription factor NF-jB Nature 388, 548–554 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 induce NF-jB activation This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 2051–2066 ª 2010 The Authors Journal compilation ª 2010 FEBS ... reached the conclusion that TLR4 is indispensable TLR4 is an essential receptor for LPS recognition [ 34, 35] In addition, TLR4 is implicated in the recognition of taxol, a diterpene purified from the. .. instead of the CD 14? ??LPS complex, may be presented to TLR4, thereby triggering TLR4 activation through the carbohydrate chains of hLF However, hLF may interfere with the formation of the CD 14? ??LPS complex,... LPS monomer is then transferred to CD 14 CD 14 is required for the efficient transfer of the LPS monomer to MD-2, which is either soluble form or associated with the ectodomain of TLR4 The formation