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The Journal of Immunology, 2004, 172: 6185-6194.
Copyright © 2004 by The American Association of Immunologists

Lipopolysaccharide-Binding Protein Critically Regulates Lipopolysaccharide-Induced IFN-{beta} Signaling Pathway in Human Monocytes1

Atsushi Kato*,{dagger}, Takahisa Ogasawara*, Toshiki Homma*, Hirohisa Saito*,{ddagger} and Kenji Matsumoto2,*,{ddagger}

* Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, Japan; {dagger} Nikken Chemicals, Saitama, Japan; and {ddagger} Research Team for Allergy Transcriptome, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LPS binding to Toll-like receptor 4 induces a large number of genes through activation of NF-{kappa}B and IFN-regulatory factor-3 (IRF-3). However, no previous reports have tested the role of serum proteins in LPS-induced gene expression profiles. To investigate how serum proteins affect LPS-induced signaling, we investigated LPS-inducible genes in PBMC using an oligonucleotide probe-array system. Approximately 120 genes up-regulated by LPS were hierarchically divided into two clusters. Induction of one cluster, containing only IFN-inducible genes, was serum dependent. Real-time PCR analysis confirmed that IFN-inducible genes were induced only in the presence of serum, whereas inflammatory genes were induced both in the presence and absence of serum. Further analysis demonstrated that addition of LPS-binding protein (LBP), but not of soluble CD14 to the serum-free medium enabled the induction of IFN-inducible genes and IFN-{beta} itself by LPS in human monocytes. The mRNAs for IFN-{beta} and IFN-inducible genes were induced by LPS only in the presence of serum from LBP+/+ mice, and not in the presence of serum from LBP–/– mice. Blocking experiments also confirmed the involvement of LBP in this phenomenon. Immunoblotting analysis showed that phosphorylation of c-Jun N-terminal kinase, p38, IRF-3, tyrosine kinase 2, and STAT1 by LPS, but not of NF-{kappa}B and extracellular signal-regulated kinase was abrogated in the absence of LBP. This critical role for LBP implies the presence of possible mechanisms linking LBP to the intracellular signaling between Toll-like receptor 4 and IRF-3, leading to the induction of IFN-{beta} by LPS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Innate immune responses have recently been implicated not only in the host defense mechanisms against pathogenic bacterial and viral microbes, but also in subsequent induction of acquired immune responses, especially Th1 type (1). The most important receptors for the recognition of pathogen-associated molecular patterns (PAMPs)3 are a family of Toll-like receptors (TLRs), molecules that are conserved across species (2, 3). Among all TLRs, TLR4, a receptor for LPS, is the most well-studied molecule in both murine and human subjects (4).

Administration of LPS elicits several clinical phenomena, including endotoxic shock, inflammation, endotoxin tolerance, and Th1-type immune responses through massive production of proinflammatory cytokines and chemokines (5). LPS also induces large amounts of antiviral proteins (6, 7) and CXCR3 chemokines (8), all of which are known to be regulated by type I or II IFNs (9). Transcriptional regulation of these cytokines and chemokines is mediated by NF-{kappa}B and IFN-regulatory factor (IRF)-3 (7).

A number of studies have attempted to clarify the molecular mechanisms by which TLR4-bound LPS transduces its signals to NF-{kappa}B and IRF-3. Four adaptor molecules with Toll-IL-1 receptor (TIR) domains are known to mediate intracellular signals in TLR4 activation: myeloid differentiation factor-88 and TIR domain-containing adapter protein/myeloid differentiation factor-88 adapter-like protein mediate the NF-{kappa}B signal, but not IRF-3 (10), whereas the most recent study revealed that TIR domain-containing adapter-inducing IFN-{beta}-related adapter molecule/TIR-containing adapter molecule-2 and TIR domain-containing adapter-inducing IFN-{beta}/TIR-containing adapter molecule-1 mediate the IRF-3 signal (11, 12, 13, 14). The transmembrane signal of LPS is mediated through a complex of molecules including CD14 (15), TLR4, and myeloid differentiation protein-2 (16). Myeloid differentiation protein-2 is itself required for cell surface TLR4 expression. However, no previous reports have tested the role of serum proteins in the NF-{kappa}B-dependent or IRF-3-dependent signaling by LPS.

Several serum proteins and lipids have been reported to be capable of binding to LPS (17, 18). Among them, soluble CD14 (sCD14) (19) and LPS-binding protein (LBP) (20) play key roles in the recognition of LPS. sCD14 can substitute membrane-bound CD14 in part to support binding of LPS to TLR4. sCD14 is reported to facilitate the LPS signal at low concentrations, but suppresses it at high concentrations (21).

LBP, a 452-aa protein with a molecular mass of 60 kDa, is an acute-phase reactant produced during Gram-negative bacterial infections (22). It is synthesized mainly in the liver and also in pulmonary and gastrointestinal epithelial cells, and is thought to function as a carrier for LPS and to help control LPS-dependent monocyte responses. Recently, it has been shown that LBP also binds to glycolipids of mycobacteria (23) and lipoteichoic acid of Gram-positive bacteria (24).

In the present study, we have comprehensively investigated LPS-inducible genes in PBMC in the presence or absence of serum. We found that several genes, all of which are known to be IFN inducible (9), were induced by LPS only in the presence of serum factor. Further examination revealed that LBP, a soluble factor, plays a critical role in the regulation of the IFN-{beta} signaling pathway by LPS. Our findings imply the presence of possible mechanisms linking LBP to the intracellular signaling between TLR4 and IRF-3, leading to the induction of IFN-{beta} by LPS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Abs

LPS (Salmonella typhimurium), DMSO, cycloheximide, and PMA were purchased from Sigma-Aldrich (St. Louis, MO). Phosphorothioate oligodeoxynucleotides (ODNs) were synthesized at Sawady Technology (Tokyo, Japan). The sequence of the CpG ODN 2006 was 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'. Recombinant human sCD14 (Biometec, Greifswald, Germany) and LBP (R&D Systems, Minneapolis, MN) were negative in the limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). Polyclonal Abs specific for phosphorylated and nonphosphorylated forms of three mitogen-activated protein kinases (MAPKs) (c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK)), NF-{kappa}B p65, STAT1, and tyrosine kinase 2 (Tyk2) were purchased from Cell Signaling Technology (Beverly, MA). Anti-IRF-3 Ab was purchased from IBL (Gunma, Japan). The JNK inhibitor SP600125, the p38 inhibitor SB202190, the mitogen-activated protein/ERK kinase inhibitor U0126, and the proteasome inhibitor MG132 were purchased from Calbiochem (San Diego, CA).

Preparation and cell culture

All human subjects in this study provided written informed consent, which was approved by the Ethical Review Board at the National Center for Child Health and Development (Tokyo, Japan). Human PBMC were isolated by centrifugation on a Ficoll-sodium metrizoate density gradient (density = 1.077 g/ml; lymphocyte separation medium; ICN Biomedicals, Aurora, OH). After washing with PBS (Life Technologies, Grand Island, NY), the cells were suspended in RPMI 1640 medium (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at a cell density of 2 x 106 cells/ml. PBMC were stimulated with 10 –0-10–4 g/ml LPS, 2 µg/ml peptidoglycan (PGN; Invivogen, San Diego, CA), or 2 µg/ml CpG ODN 2006 in the presence or absence of 10% heat-inactivated FBS (JRH Biosciences, Lenexa, KS) for up to 6 h.

Human monocytes were isolated from PBMC by an indirect magnetic labeling system using mAbs against anti-CD3, anti-CD7, anti-CD16, anti-CD19, anti-CD56, anti-CD123, and anti-glycophorin A, according to the manufacturer’s protocol (Monocyte Isolation Kit II; Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD14-positive monocytes was always >90%.

The THP-1 human monocytic cell line (ATCC, TIB-202) was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 µM 2-ME. Differentiated THP-1 cells were obtained by treatment with 5 nM PMA for 2 days and then starved overnight in 0.5% FBS containing RPMI 1640 medium in the presence of 5 nM PMA before stimulation. They were then stimulated with 1–1000 ng/ml LPS in the presence or absence of 1% heat-inactivated human AB serum (ICN Biomedicals) for up to 24 h. In some experiments, recombinant sCD14 and LBP were added to the serum-free medium 1 h before stimulation with LPS. In blocking experiments, 10 µg/ml mouse anti-human CD14 mAb (clone biG 13;Biometec) or 10 µg/ml mouse anti-human LBP mAb (clone biG 412; Biometec) were added to differentiated THP-1 cells in the presence of 1% human AB serum and incubated for 1 h. Then 10 ng/ml LPS was added and incubated continuously for additional 3 h. In some experiments, differentiated THP-1 cells were stimulated for 3 h with 100 ng/ml LPS in the presence or absence of 2% serum from LBP–/– mice (BALB/c background; Biometec) or LBP+/+ control mice (BALB/c; Biometec) (20). In some experiments, differentiated THP-1 cells were pretreated with 0.1% DMSO, 10 µM SP600125, 10 µM SB202190, 10 µM U0126, 1 µM MG132, or 10 µg/ml cycloheximide for 1 h, and then stimulated with 10 ng/ml LPS for 3 h.

GeneChip expression analysis

GeneChip analysis was performed, as described previously (25). Gene expression was analyzed with the GeneChip Human Genome U133A probe array (Affymetrix, Santa Clara, CA). Data analysis was performed with GeneSpring software version 4.2.1 (Silicon Genetics, Redwood City, CA). To normalize the staining intensity variations among chips, the average difference values for all genes on a given chip were divided by the median value for expression of all genes on the chip. To eliminate genes containing only a background signal, genes were selected only if the raw data were >300, and the gene expression was judged to be "present" by the Microarray Suite software version 5.0 (Affymetrix). A hierarchical clustering analysis was performed using a minimum distance value of 0.001, a separation ratio of 0.5, and the standard definition of the correlation distance.

Semiquantitative real-time RT-PCR analysis

Primer sets for 10 genes, TNF-{alpha} (sense, 5'-ATCTACTCCCAGGTCCTCTTCAA-3'; antisense, 5'-GCAATGATCCCAAAGTAGACCT-3'), growth-related oncogene 3 (GRO3; sense, 5'-GCAGGGAATTCACCTCAAGA-3'; antisense, 5'-GGTGCTCCCCTTGTTCAGTA-3'), IL-6 (sense, 5'-CCACACAGACAGCCACTCAC-3'; antisense, 5'-AGGTTGTTTTCTGCCAGTGC-3'), cyclooxygenase-2 (COX2; sense, 5'-CCACTTCAAGGGATTTTGGA-3'; antisense, 5'-GAGAAGGCTTCCCAGCTTTT-3'), IFN-induced protein with tetratricopeptide repeats 1 (IFIT1; sense, 5'-GCCATTTTCTTTGCTTCCCCTA-3'; antisense, 5'-TGCCCTTTTGTAGCCTCCTTG-3'), IFN-induced protein of 10 kDa (IP-10; sense, 5'-CGCTGTACCTGCATCAGCATT-3'; antisense, 5'-GCTCCCCTCTGGTTTTAGGAG-3'), IFN-stimulated gene 15 (ISG15; sense, 5'-GGACAAATGCGACGAACCTCT-3'; antisense, 5'-CCCTCGAAGGTCAGCCAGA-3'), IRF-7 (sense, 5'-TGACACCCCCATCTTCGACTT-3'; antisense, 5'-CACCAGGACCAGGCTCTTCTC-3'), IFN-{beta} (sense, 5'-TGCTCTCCTGTTGTGCTTCTCC-3'; antisense, 5'-CATCTCATAGATGGTCAATGCGG-3'), and GAPDH (sense, 5'-GAAGGTGAAGGTCGGAGTC-3'; antisense, 5'-GAAGATGGTGATGGGATTTC-3') were synthesized at Sawady Technology. Total RNA was extracted by RNeasy (Qiagen, Hilden, Germany) and was digested by DNase I (Qiagen), according to the manufacturer’s instructions. Single-strand cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Semiquantitative real-time RT-PCR was performed with a dsDNA-binding dye, SYBR Green I, using an Applied Biosystems 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) (25). The expression levels of mRNA were normalized by the median expression of a housekeeping gene (GAPDH).

Measurement of chemokines in the culture supernatant

The cytokine and chemokine concentrations in the supernatant were measured with ELISA kits specifically recognizing TNF-{alpha} or IP-10 (R&D Systems). The minimal detection limits for the two kits were 15.6 and 7.8 pg/ml for TNF-{alpha} and IP-10, respectively.

Western blot analysis

Cells were harvested at the indicated time points, and then whole cell lysates were dissolved in Laemmli’s sample buffer. To detect NF-{kappa}B p65, p38, ERK, JNK, STAT1, or Tyk2, 15 µg of whole cell lysates was resolved on a 4–20% gradient SDS-PAGE. Proteins were transferred onto Hybond-P (Amersham Biosciences, Arlington Heights, IL), which was blocked in Block Ace (Dainippon Seiyaku, Osaka, Japan). Western blotting was performed according to the procedure supplied with the ECL Western blotting reagents kit (LumiGLO; Cell Signaling Technology). Native PAGE was performed, as described previously (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of serum-dependent LPS-inducible genes

To examine the effect of serum proteins, we incubated PBMC with LPS in serum-free or 10% FBS-containing culture medium and measured mRNA expression with the GeneChip system. Using hierarchical clustering analysis of the gene expression profiles of ~20,000 genes, we identified two gene clusters (Fig. 1 and supplementary data).4 In the first gene cluster, inflammatory genes such as TNF-{alpha}, IL-1{beta}, IL-6, and COX2, and neutrophil chemoattractant genes such as GRO3 and IL-8 were up-regulated by LPS both in the presence and absence of serum. In contrast, the second gene cluster contained genes that were up-regulated by LPS only in the presence of serum. These included antiviral genes such as IFIT1, 2',5'-oligoadenylate synthetase 1, myxovirus (influenza virus) resistance 1, ISG15, and protein kinase, IFN-inducible dsRNA dependent; antitumor genes such as TRAIL; and Th1 chemoattractant genes such as IP-10. Interestingly, the majority of genes in the second gene cluster are known to be regulated by type I IFNs (7, 9).



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  		FIGURE 1. LPS-induced serum-dependent and -independent gene clusters in PBMC. Two gene clusters containing 63 and 57 genes (a total of 138 probe sets) were up-regulated more than 3-fold in PBMC after stimulation with 100 ng/ml LPS in both 3 and 6 h of culture in the presence of 10% FBS. Data were analyzed by applying a hierarchical-tree algorithm to the normalized intensity. The color code for the signal strength in the classification scheme is shown in the box on the left. Induced genes are indicated by shades of red; repressed genes are indicated by shades of blue. Exactly the same experiments were performed with PBMCs from three individual donors, and equal amounts of mRNA were mixed and hybridized with a single microarray (GeneChip). Gene symbols are shown in the right column.

 
Quantitative analysis of LPS-inducible genes

To confirm the GeneChip data, we applied a real-time PCR method. Inflammatory genes such as TNF-{alpha}, GRO3, IL-6, and COX2 were strongly up-regulated by LPS in a dose-dependent manner both in the presence and absence of 10% FBS (Fig. 2A). In contrast, IFN-inducible genes such as IFIT1, IP-10, ISG15, and IRF-7 were strongly up-regulated by LPS only in the presence of 10% FBS (Fig. 2B). Even at the highest concentration of LPS (100 µg/ml), up-regulation of these genes was not observed when cells were cultured without serum. However, induction of IFN-inducible genes by CpG ODN 2006, a TLR9 ligand, did not require serum proteins (Fig. 2B). In addition, induction of IFN-inducible genes by a TLR3 ligand, poly(I:C), or a TLR7 and 8 ligand, R-848, also did not require serum proteins (data not shown). Moreover, PGN up-regulated TNF-{alpha}, GRO3, IL-6, and COX2 both in the presence and absence of serum (Fig. 2A), but did not induce IFIT1, IP-10, ISG15, or IRF-7 either in the presence or in the absence of serum (Fig. 2B).



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  		FIGURE 2. LPS-mediated up-regulation of mRNA expression for TNF-{alpha}, GRO3, IL-6, COX2, IFIT1, IP-10, ISG15, and IRF-7 in PBMC and monocytes. PBMC were incubated for 6 h with different concentrations of LPS, 2 µg/ml PGN, or 2 µg/ml CpG ODN 2006 in the presence ({square}) or absence ({blacksquare}) of 10% FBS (A and B). Human primary monocytes were incubated for 3 h with 100 ng/ml LPS in the presence ({square}) or absence ({blacksquare}) of 10% FBS (C). The mRNA levels of TNF-{alpha}, GRO3, IL-6, COX2, IFIT1, IP-10, ISG15, and IRF-7 were determined by SYBR green-based real-time quantitative RT-PCR. The results are shown as the mean ± SEM of four independent experiments. The copy number is expressed as the number of transcripts/ng total RNA.

 
TLR4 is known to be mainly expressed on the monocytes in PBMC (27), and to determine whether this phenomenon in PBMC is mainly attributable to activation of monocytes, we incubated human primary monocytes with LPS in serum-free or 10% FBS-containing culture medium. The results showed that IFN-inducible genes such as IFIT1 and ISG15 were also up-regulated by LPS only in the presence of FBS, whereas inflammatory genes such as TNF-{alpha} and IL-6 were up-regulated by LPS under both conditions (Fig. 2C). This suggests that monocytes play a major role in the critical regulation of LPS-inducible genes under both conditions.

Serum-dependent induction in THP-1 cells

To characterize the effect of serum proteins on the induction of IFN-inducible genes, we established PMA-differentiated THP-1 cells to use as a substitute for human monocytes (28). In the presence of 1% human AB serum, mRNAs for inflammatory genes such as TNF-{alpha} and GRO3, and IFN-inducible genes such as IFIT1, IP-10, ISG15, and IRF-7 were up-regulated by LPS in a dose-dependent manner (Fig. 3A, and data not shown). In contrast, in the absence of serum, IFIT1 and IP-10 mRNAs were not induced by LPS, whereas TNF-{alpha} and GRO3 mRNA were (Fig. 3A). This finding indicates that PMA-differentiated THP-1 cells closely mimic human monocytes in terms of responses to LPS. This phenomenon was further confirmed by measuring the concentration of proteins in the supernatant. TNF-{alpha} protein was induced both in the presence and absence of serum, but IP-10 protein was induced by LPS only in the presence of human AB serum (Fig. 3B). In the time-kinetic analysis (Fig. 3A), mRNA for TNF-{alpha} and GRO3 peaked at 3–6 h of culture both in the presence and absence of serum. In contrast, mRNA for IFIT1 or IP-10 was not induced by LPS in the absence of serum even after 24 h of culture. These results indicate that lack of induction of mRNA for IFIT1 or IP-10 is not simply due to the magnitude of stimulation nor to differences in the time kinetics.



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  		FIGURE 3. Serum-dependent and -independent induction of TNF-{alpha}, GRO3, IFIT1, IP-10, and IFN-{beta} by LPS. Differentiated THP-1 cells were incubated at different concentrations of LPS for up to 24 h in the presence ({square}) or absence ({blacksquare}) of 1% AB serum (A–C). Human primary monocytes were incubated for 3 h with 100 ng/ml LPS in the presence ({square}) or absence ({blacksquare}) of 1% AB serum (D). The mRNA levels of TNF-{alpha}, GRO3, IFIT1, and IP-10 (A), or IFN-{beta} (C and D) were determined by SYBR green-based real-time quantitative RT-PCR. Concentrations of TNF-{alpha} and IP-10 protein (B) in the culture supernatant were measured by ELISA. The results are shown as the mean ± SEM of three independent experiments (A–D).

 
Looking at IFN-inducible genes in more detail, we found that in the presence of serum, the induction of mRNA for IFIT1 and IP-10 reached a peak at 6 h after stimulation with LPS, whereas induction of mRNA for IFN-{beta} and inflammatory genes had peaked within 3 h (Fig. 3, A and C). This suggests that IFN-{beta} and inflammatory genes were directly induced by LPS via TLR4. Previous reports indicate that induction of IFIT1 and IP-10 by LPS is regulated by IFN-{beta}, implying that up-regulation of IFN-inducible genes is regulated by LPS-induced IFN-{beta} (29). Notably, induction of mRNA for IFN-{beta} by LPS was completely abolished in the absence of serum in differentiated THP-1 cells and human monocytes (Fig. 3, C and D). Taken together, these results indicate that serum proteins are essential components of the LPS-induced IFN-{beta} signaling pathway.

LBP-dependent IFN-{beta} induction

Two serum proteins, sCD14 and LBP, have been shown to play critical roles in LPS signaling (17). To elucidate whether sCD14 or LBP was required for activation of IFN-{beta} signaling by LPS, we added recombinant sCD14 and LBP to the serum-free medium before LPS stimulation. Induction of TNF-{alpha} by LPS was enhanced by the addition of either sCD14 or LBP to the serum-free medium (Fig. 4A). In contrast, IFIT1 and IP-10 were induced by LPS on the addition of LBP, but not on the addition of sCD14 (Fig. 4, B and C). However, concomitant addition of sCD14 synergistically enhanced LBP-dependent induction of IFN-inducible genes (Fig. 4, B and C). Exactly the same pattern was observed with induction of mRNA for IFN-{beta} by LPS in differentiated THP-1 cells and human monocytes (Fig. 4, D and E). These results indicate that LBP has an essential role in the activation of IFN-{beta} signaling and that sCD14 facilitates this function.



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  		FIGURE 4. Effects of recombinant sCD14 and LBP on up-regulation of mRNA by LPS. Differentiated THP-1 cells were incubated for 3 h with 10 ng/ml LPS ({blacksquare}) or vehicle control ({square}) in the presence or absence of indicated concentrations of recombinant sCD14 and LBP in serum-free medium or 1% AB serum-containing medium (A–C). Differentiated THP-1 cells (D) and human monocytes (E) were incubated with 100 ng/ml LPS or vehicle control for 2 (D) or 3 (E) h in the presence or absence of 100 ng/ml recombinant sCD14 and LBP in serum-free medium. The mRNA levels of TNF-{alpha} (A), IFIT1 (B), IP-10 (C), and IFN-{beta} (D and E) were determined by SYBR green-based real-time quantitative RT-PCR. The copy number is expressed as the number of transcripts/ng total RNA. The results are shown as the mean ± SEM of three independent experiments.

 
To clarify the effects of LBP and CD14 on the activation of IFN-{beta} signaling by LPS, we performed blocking experiments using neutralizing mAbs against CD14 or LBP. In the presence of human AB serum, induction of TNF-{alpha} by LPS was strongly inhibited by anti-CD14 mAb and partially inhibited by anti-LBP mAb (Fig. 5A). In contrast, induction of IFN-{beta} and IFIT1 by LPS was completely abolished by either anti-LBP mAb or anti-CD14 mAb (Fig. 5A).



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  		FIGURE 5. Effects of neutralizing mAbs against CD14 and LBP on up-regulation of mRNA expression in differentiated THP-1 cells by LPS. Differentiated THP-1 cells were preincubated with 10 µg/ml mAbs against CD14 and LBP or control IgG1 for 1 h in 1% AB serum-containing medium before stimulation with 10 ng/ml LPS for 3 h (A). To determine the effect of cell surface CD14 or LBP, differentiated THP-1 cells were incubated with 10 µg/ml mAbs against CD14 and LBP or control IgG1 for 1 h in serum-free medium, washed three times, and then stimulated for 3 h with 10 ng/ml LPS in 1% AB serum-containing medium (B). Differentiated THP-1 cells were incubated for 3 h with 100 ng/ml LPS ({blacksquare}) or vehicle control ({square}) in the presence or absence of 2% serum from LBP–/– mice or LBP+/+ control mice (C). The mRNA levels of TNF-{alpha}, IFN-{beta}, and IFIT1 were determined by SYBR green-based real-time quantitative RT-PCR. The copy number is expressed as the number of transcripts/ng total RNA. The results are shown as the mean ± SEM of three independent experiments.

 
Previous reports have indicated that the interaction of LPS with membrane-bound CD14 (mCD14) and membrane-associated LBP is an important step in LPS-mediated activation (30). To test whether mCD14 and membrane-associated LBP are involved in our system or not, we washed differentiated THP-1 cells three times after preincubation with anti-CD14 mAb or anti-LBP mAb for 1 h in the absence of serum, and then added 1% AB serum-containing medium. In these THP-1 cells, induction of IFN-{beta} and IFIT1 by LPS was inhibited in anti-CD14 mAb-treated cells, but not in anti-LBP mAb-treated cells (Fig. 5B). The same inhibitory pattern was observed with another clone for anti-CD14 mAb (18D11; Diatech.com AS, Oslo, Norway), and anti-LBP mAb (biG 33; Biometec) (data not shown). Thus, a combination of serum LBP, but not membrane-associated LBP, and CD14, either mCD14 or sCD14, must be an essential component of the LPS-induced IFN-{beta} signaling pathway.

We also used serum from LBP–/– mice to investigate the possible involvement of other serum proteins in the induction of IFN-{beta} and IFN-inducible genes by LPS. mRNAs for IFN-{beta} and IFIT1 were induced by LPS only in the presence of serum from LBP+/+ control mice, and not in the presence of serum from LBP–/– mice (Fig. 5C). This suggests that LBP is capable of enhancing LPS-induced IFN-{beta} expression, but other serum proteins are not.

Serum-dependent activation of JNK and p38

It is known that LPS is capable of activating three MAPKs, p38, ERK, and JNK, and NF-{kappa}B (31). To determine whether these MAPKs and NF-{kappa}B are critical for the activation of IFN-{beta} signaling, we investigated immunodetection of their phosphorylated forms. NF-{kappa}B and ERK were phosphorylated by LPS both in the presence and absence of serum. However, JNK and p38 were phosphorylated by LPS only in the presence of serum (Fig. 6A). Moreover, phosphorylation of STAT1 reached a peak at 3 h after stimulation with LPS only in the presence of serum (Fig. 6A).



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  		FIGURE 6. Serum-dependent and -independent phosphorylation of NF-{kappa}B, MAPKs, and STAT1 in differentiated THP-1 cells by LPS. Differentiated THP-1 cells were incubated for up to 6 h with 100 ng/ml LPS in the presence or absence of 1% AB serum (A). Differentiated THP-1 cells were incubated for 0.5 h with 100 ng/ml LPS in the presence or absence of 100 ng/ml recombinant sCD14 and LBP in serum-free medium (B). At the indicated time points, cells were harvested and the cell lysates were subjected to SDS-PAGE. After blotting to polyvinylidene difluoride membrane, total and phosphorylated NF-{kappa}B, MAPKs, and STAT1 were detected with specific mAbs. The results are representative of at least two separate experiments.

 
To determine the effect of LBP and sCD14 on the phosphorylation of JNK and p38 by LPS, we added LBP and sCD14 to serum-free medium before LPS stimulation. The results showed that JNK and p38 were phosphorylated by LPS only in the presence of LBP, and not in the presence of sCD14 alone or in the absence of serum (Fig. 6B).

JNK and p38 in LPS signaling

To investigate the upstream signal transduction pathways for IFN-{beta} and IFN-inducible genes by LPS, we performed experiments in which differentiated THP-1 cells were treated with SP600125, SB202190, U0126, MG132, or DMSO, a vehicle control, and then stimulated with LPS for 3 h in the presence of serum. Up-regulation of mRNA for TNF-{alpha} was strongly blocked by MG132 and U0126 (Fig. 7). However, up-regulation of the mRNA for IFN-{beta} was strongly blocked by SP600125 and SB202190, but not by MG132, U0126, or cycloheximide (Fig. 7). In contrast, up-regulation of mRNAs for IFIT1 and IP-10 was strongly blocked by SP600125, SB202190, and cycloheximide (Fig. 7).



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  		FIGURE 7. Effects of signal transduction inhibitors on up-regulation of mRNA expression in differentiated THP-1 cells by LPS. Differentiated THP-1 cells were pretreated with 0.1% DMSO, 10 µM SP600125 (JNK inhibitor), 10 µM SB202190 (p38 inhibitor), 10 µM U0126 (mitogen-activated protein/ERK kinase inhibitor), 1 µM MG132 (proteasome inhibitor), or 10 µg/ml cycloheximide (CHX; protein synthesis inhibitor) for 1 h, and then stimulated with 10 ng/ml LPS for 3 h. The mRNA levels of TNF-{alpha}, IFN-{beta}, IFIT1, or IP-10 were determined by SYBR green-based real-time quantitative RT-PCR. The copy number is expressed as the number of transcripts/ng total RNA. The results are shown as the mean ± SEM of three independent experiments.

 
LBP-dependent IRF-3 activation

To clarify the effects of LBP and sCD14 on the activation of IFN-{beta} signaling by LPS, we added LBP and sCD14 to the serum-free medium before LPS stimulation. STAT1 and Tyk2 were phosphorylated by LPS only in the presence of LBP, but not in the presence of sCD14 alone (Fig. 8A). It is known that induction of IFN-{beta} is regulated by IRF-3 activation and dimerization (32). We examined the formation of phosphorylated IRF-3 dimers in LPS-stimulated THP-1 cells by native PAGE. IRF-3 was also dimerized by LPS only in the presence of LBP (Fig. 8B).



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  		FIGURE 8. Effects of sCD14 and LBP on the phosphorylation of STAT1, Tyk2, or IRF-3 in differentiated THP-1 cells by LPS. Differentiated THP-1 cells were incubated with 100 ng/ml LPS in the presence or absence of 100 ng/ml recombinant sCD14 and LBP in serum-free medium. After 3 h of incubation, cells were harvested and the cell lysates were subjected to SDS-PAGE. After blotting to polyvinylidene difluoride membrane, total and phosphorylated STAT1 and Tyk2 were detected with specific mAbs (A). After 2 h of incubation, cells were harvested and cell lysates were subjected to native PAGE, and monomeric and dimeric IRF-3 were detected with anti-IRF-3 mAb (B). The results are representative of at least two separate experiments.

 
These data suggest that LBP is an essential component of the LPS-induced IFN-{beta} signaling pathway, exerting its effect via TLR4, JNK, p38, and IRF-3.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LPS is known to induce a large number of genes in different cell types (33, 34, 35, 36), and consistent with this we identified ~120 genes up-regulated (>3-fold in normalized data) in PBMC after 3–6 h of culture with 100 ng/ml LPS. Almost all LPS-induced genes can be classified into two distinct clusters according to their regulation in the presence or absence of serum in the culture medium by using hierarchical clustering analysis. Interestingly, most of the genes in the serum-dependent cluster are known to be IFN inducible (9). Further analysis confirmed that this phenomenon is not simply due to differences in the magnitude of stimuli nor due to differences in time kinetics. Even at the highest concentrations of LPS (up to 100 µg/ml), LPS failed to induce IFN-inducible genes in the absence of serum (Fig. 2). At very high concentrations, LPS is reported to activate cells and to induce TNF-{alpha} production in a CD14-independent manner (37), and the induction is at least in part CD11b dependent (38). However, our results clearly demonstrate that regardless of the concentration of LPS, the presence of serum either switches on or off the expression of IFN-inducible genes in PBMC, primary monocytes, and differentiated THP-1 cells (Figs. 2 and 3). In addition, our time kinetics study, together with previous reports, shows that the induction of IFN-inducible genes by LPS is IFN-{beta} dependent (Fig. 3, A and C). Thus, LPS seems to induce IFN-{beta} signaling in a serum-dependent fashion. Unlike other IRF-3-activating PAMPs (poly(I:C), R-848, or CpG ODN) or the non-IRF-3-activating PAMP (PGN) tested, LPS exerts serum factor-dependent activation of IFN-inducible genes in PBMC, suggesting that such a phenomenon is TLR4 specific.

Several serum proteins and lipids have been reported to be capable of binding to LPS (17, 18). Among them, sCD14 (19) and LBP (20) play key roles in the recognition of LPS. In the present study, we revealed that LBP plays a critical role in the up-regulation of IFN-{beta} and IFN-inducible genes in three experimental settings: first, the addition of LBP to the serum-free medium enabled the induction of IFN-{beta} and IFN-inducible genes by LPS (Fig. 4); second, the inhibition of LBP by blocking mAb strongly reduced the induction of IFN-{beta} and IFN-inducible genes by LPS in the presence of serum (Fig. 5, A and B); and third, IFN-{beta} and IFN-inducible genes were not induced by LPS in the presence of serum from LBP–/– mice (Fig. 5C).

Because of the cross-species specificity (39) and heat resistance (50% conserved activity after heating at 56°C for 30 min) of LBP (40), addition of 10% FBS to the medium was consequently able to induce IFN-inducible genes. In our preliminary study, we compared FBS and human AB serum and titrated human AB serum in the context of their effects on the responses to LPS. The results showed a sufficient effect of 1% AB serum in our experiment system (data not shown), and using human serum allowed us to use neutralizing anti-human LBP mAb and anti-human CD14 mAb. In addition, 1% human AB serum, which we used in our experiments, has been reported to be capable of maximally inducing adhesion of neutrophils upon stimulation with LPS (41).

sCD14 and LBP played distinct roles in the induction of inflammatory genes and IFN-{beta} by LPS. The blocking experiments using anti-CD14 mAb clearly demonstrated that CD14 is essential for transducing the LPS signal to induce both TNF-{alpha} and IFN-{beta} (Fig. 4). However, data from a previous report (28), together with our blocking and washing experiments (Fig. 5B), strongly suggest that differentiated THP-1 cells express a substantial amount of mCD14. Thus, the induction of TNF-{alpha} by LPS did not require any additional serum proteins. Addition of LBP or sCD14 alone enhanced the expression of TNF-{alpha}. However, addition of sCD14 alone could not induce IFN-{beta} or IFIT1. Only in combination with LBP did sCD14 enhance the induction of IFN-{beta} and IFN-inducible genes by LPS (Fig. 4).

In contrast, addition of LBP alone up-regulated expression of IFN-{beta} and IFIT1 (Fig. 4). This is perhaps due to the expression of mCD14 on the cell surface of differentiated THP-1 cells. In other words, in cells with expression of CD14, the induction of IFN-{beta} by LPS is critically regulated by LBP.

A number of studies have attempted to clarify the molecular mechanisms by which TLR4-bound LPS transduces its signals to induce expression of inflammatory genes and IFN-inducible genes (16, 29). In the upstream part of the signal transduction pathways to the transcription factors NF-{kappa}B and IRF-3, activation of three MAPKs (31) has been documented. Among these molecules, JNK (42) and p38 (43) in particular are thought to be involved in the IRF-3 pathway. To investigate in what stages of the signal transduction pathways the absence of LBP switches off the LPS signal, we determined the phosphorylation of molecules in the upstream part in the signal transduction pathways of IFN-{beta} and IFN-inducible genes. As a result, phosphorylation of JNK, p38, IRF-3, Tyk2, and STAT1, but not NF-{kappa}B or ERK was diminished or abrogated in the absence of serum (Figs. 6A and 8). In addition, the phosphorylation of JNK, p38, IRF-3, Tyk2, and STAT1 was observed only when LBP was added to serum-free medium (Figs. 6B and 8). Thus, LBP seems to play critical roles in the activation of JNK, p38, IRF-3, Tyk2, and STAT1. Blocking experiments using specific inhibitors for the signal transduction pathways further demonstrated that, upon stimulation with LPS, expression of TNF-{alpha} was regulated by NF-{kappa}B, whereas induction of IFN-{beta}, IFIT1, and IP-10 was regulated by JNK and p38 (Fig. 7). It is noteworthy that the induction of IFIT1 and IP-10, but not of TNF-{alpha} or IFN-{beta}, seems to require de novo protein synthesis. This fact, along with the LBP-dependent Tyk2 and STAT1 phosphorylation, is consistent with previous reports that the induction of these molecules is regulated by IFN-{beta} (6, 7, 44).

The blocking and washing experiments (Fig. 5B) showed clearly that membrane-associated LBP is not involved in the induction of IFN-{beta} or IFN-inducible genes upon stimulation with LPS. Thus, a soluble form of LBP must play a critical role in this process. However, the mechanisms by which LBP, a soluble protein, transduces signals to IRF-3, an intracellular transcription factor, are still unclear. It is possible that other molecules may play roles in LBP-dependent induction of IFN-{beta} by LPS. This should be studied further in the future.

Among 10 human TLRs, stimulation through only TLR3, 4, 5, 7, 8, and 9 induces expression of IFN-inducible genes via induction of type I IFN (1, 4, 10, 45). To date, the products of several IFN-inducible genes have been identified to be up-regulated by LPS. These include antiviral proteins (7), antitumor proteins (46), Th1 cell-attracting chemokines (47), and type I IFN itself (7). Binding of type I IFN to its specific receptors can recruit STAT4 and induce subsequent Th1 responses (48). This fact, along with the production of Th1 cell-attracting chemokines (47), strongly suggests that the induction of IFN-inducible genes is responsible for Th1-skewed immune responses (49). Thus, the presence of LBP may play critical roles of LPS-related inflammatory reactions and immune responses, especially those of the Th1 type. Presumably such Th1-type immune responses support induction of immune memory to the pathogens at inflammatory sites, improving immune responses during subsequent Ag exposure and infections. In this respect, LBP seems to participate in the connection between innate immune responses and acquired immune responses through induction of IFN-inducible genes.

LBP is known as an acute-phase protein, but substantial levels of LBP are found in sera from normal individuals (5.7–18.1 µg/ml) (22, 50, 51). According to our in vitro experiments, the LBP concentration in normal serum is high enough to fully induce IFN-{beta} expression upon stimulation with LPS. However, in the case of cerebrospinal fluid (52), i.p. fluid (53), synovial fluid (54), or mucous membrane surfaces (55), it is still unknown whether or not increased concentrations of LBP may be involved in the inflammatory reactions or immune memory.

Very high concentrations of LBP have been reported to suppress NF-{kappa}B-dependent signals (56). However, the effect of high concentrations of LBP on the induction of IFN-inducible genes is still unknown. It has yet to be elucidated whether or not the presence of LBP may affect signaling with other TLR4 ligands, especially those of viral organisms such as the F protein of respiratory syncytial virus (57).

In the present study, we have demonstrated the critical role of LBP in the induction of IFN-{beta} by LPS. Its clinical relevance requires further investigation, but such a critical role implies the presence of possible mechanisms linking LBP to the intracellular signaling between TLR4 and IRF-3, leading to the induction of IFN-{beta} by LPS.


   Acknowledgments
 
We thank Dr. Jonathan Batchelor (National Center for Child Health and Development) for proofreading the manuscript. We thank Dr. Takashi Fujita (Tokyo Metropolitan Institute of Medical Science) for valuable advice regarding the IRF-3 immunoblotting. We also thank Noriko Hashimoto (National Research Institute for Child Health and Development) for skillful technical assistance.


   Footnotes
 
1 This work was supported in part by a grant from the Organization for Pharmaceutical Safety and Research and the Ministry of Health, Labour, and Welfare (the Millennium Genome Project, MPJ-5), and by a grant from RIKEN Research Center for Allergy and Immunology. Back

2 Address correspondence and reprint requests to Dr. Kenji Matsumoto, Department of Allergy and Immunology, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan. E-mail address: kmatsumoto{at}nch.go.jp Back

3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; COX, cyclooxygenase; ERK, extracellular signal-regulated kinase; GRO3, growth-related oncogene 3; IFIT1, IFN-induced protein with tetratricopeptide repeats 1; IP-10, IFN-{gamma}-inducible protein-10; IRF, IFN-regulatory factor; ISG15, IFN-stimulated gene 15; JNK, c-Jun N-terminal kinase; LBP, LPS-binding protein; mCD14, membrane-bound CD14; MAPK, mitogen-activated protein kinase; ODN, oligodeoxynucleotide; PGN, peptidoglycan; sCD14, soluble CD14; TIR, Toll-IL-1 receptor; TLR, Toll-like receptor; Tyk2, tyrosine kinase 2. Back

4 The on-line version of this article contains supplementary material. Back

Received for publication November 6, 2003. Accepted for publication February 27, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Airway Epithelial Cells Produce B Cell-Activating Factor of TNF Family by an IFN-beta-Dependent Mechanism
J. Immunol., November 15, 2006; 177(10): 7164 - 7172.
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 Innate ImmunityHome page
K. Miyake
Invited review: Roles for accessory molecules in microbial recognition by Toll-like receptors
Innate Immunity, August 1, 2006; 12(4): 195 - 204.
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S. Roes, F. Mumm, U. Seydel, and T. Gutsmann
Localization of the Lipopolysaccharide-binding Protein in Phospholipid Membranes by Atomic Force Microscopy
J. Biol. Chem., February 3, 2006; 281(5): 2757 - 2763.
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