Critically Regulates Lipopolysaccharide-Binding Protein

Lipopolysaccharide-Binding Protein Critically Regulates Lipopolysaccharide-Induced IFN-β Signaling Pathway in Human Monocytes This information is current as of October 6, 2021. Atsushi Kato, Takahisa Ogasawara, Toshiki Homma, Hirohisa Saito and Kenji Matsumoto J Immunol 2004; 172:6185-6194; ; doi: 10.4049/jimmunol.172.10.6185

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Lipopolysaccharide-Binding Protein Critically Regulates Lipopolysaccharide-Induced IFN-␤ Signaling Pathway in Human Monocytes1

Atsushi Kato,*† Takahisa Ogasawara,* Toshiki Homma,* Hirohisa Saito,*‡ and Kenji Matsumoto2*‡

LPS binding to Toll-like receptor 4 induces a large number of genes through activation of NF-␬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 Downloaded from 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-␤ itself by LPS in human monocytes. The mRNAs for IFN-␤ 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-␬B and http://www.jimmunol.org/ 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-␤ by LPS. The Journal of Immunology, 2004, 172: 6185–6194.

nnate immune responses have recently been implicated not Administration of LPS elicits several clinical phenomena, in- only in the host defense mechanisms against pathogenic bac- cluding endotoxic shock, inﬂammation, endotoxin tolerance, and I terial and viral microbes, but also in subsequent induction of Th1-type immune responses through massive production of proin- acquired immune responses, especially Th1 type (1). The most ﬂammatory cytokines and chemokines (5). LPS also induces large important receptors for the recognition of pathogen-associated mo- amounts of antiviral proteins (6, 7) and CXCR3 chemokines (8), by guest on October 6, 2021 lecular patterns (PAMPs)3 are a family of Toll-like receptors all of which are known to be regulated by type I or II IFNs (9). (TLRs), molecules that are conserved across species (2, 3). Among Transcriptional regulation of these cytokines and chemokines is all TLRs, TLR4, a receptor for LPS, is the most well-studied mol- mediated by NF-␬B and IFN-regulatory factor (IRF)-3 (7). ecule in both murine and human subjects (4). A number of studies have attempted to clarify the molecular mechanisms by which TLR4-bound LPS transduces its signals to NF-␬B and IRF-3. Four adaptor molecules with Toll-IL-1 receptor (TIR) domains are known to mediate intracellular signals in TLR4 *Department of Allergy and Immunology, National Research Institute for Child activation: myeloid differentiation factor-88 and TIR domain- Health and Development, Tokyo, Japan; †Nikken Chemicals, Saitama, Japan; and ‡Research Team for Allergy Transcriptome, RIKEN Research Center for Allergy and containing adapter protein/myeloid differentiation factor-88 adapt- Immunology, Yokohama, Japan er-like protein mediate the NF-␬B signal, but not IRF-3 (10), Received for publication November 6, 2003. Accepted for publication February whereas the most recent study revealed that TIR domain-containing 27, 2004. adapter-inducing IFN-␤-related adapter molecule/TIR- The costs of publication of this article were defrayed in part by the payment of page containing adapter molecule-2 and TIR domain-containing adapter- charges. This article must therefore be hereby marked advertisement in accordance inducing IFN-␤/TIR-containing adapter molecule-1 mediate the with 18 U.S.C. Section 1734 solely to indicate this fact. IRF-3 signal (11Ð14). The transmembrane signal of LPS is mediated 1 This work was supported in part by a grant from the Organization for Pharmaceu- tical Safety and Research and the Ministry of Health, Labour, and Welfare (the Mil- through a complex of molecules including CD14 (15), TLR4, and lennium Genome Project, MPJ-5), and by a grant from RIKEN Research Center for myeloid differentiation protein-2 (16). Myeloid differentiation pro- Allergy and Immunology. tein-2 is itself required for cell surface TLR4 expression. However, 2 Address correspondence and reprint requests to Dr. Kenji Matsumoto, Department no previous reports have tested the role of serum proteins in the of Allergy and Immunology, National Research Institute for Child Health and De- velopment, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan. E-mail address: NF-␬B-dependent or IRF-3-dependent signaling by LPS. [email protected] Several serum proteins and lipids have been reported to be ca- 3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; pable of binding to LPS (17, 18). Among them, soluble CD14 COX, cyclooxygenase; ERK, extracellular signal-regulated kinase; GRO3, growth- (sCD14) (19) and LPS-binding protein (LBP) (20) play key roles related oncogene 3; IFIT1, IFN-induced protein with tetratricopeptide repeats 1; IP- 10, IFN-␥-inducible protein-10; IRF, IFN-regulatory factor; ISG15, IFN-stimulated in the recognition of LPS. sCD14 can substitute membrane-bound gene 15; JNK, c-Jun N-terminal kinase; LBP, LPS-binding protein; mCD14, mem- CD14 in part to support binding of LPS to TLR4. sCD14 is re- brane-bound CD14; MAPK, mitogen-activated protein kinase; ODN, oligodeoxynucleotide; PGN, peptidoglycan; sCD14, soluble CD14; TIR, Toll-IL-1 receptor; ported to facilitate the LPS signal at low concentrations, but sup- TLR, Toll-like receptor; Tyk2, tyrosine kinase 2. presses it at high concentrations (21).

LBP, a 452-aa protein with a molecular mass of 60 kDa, is an ground; Biometec) or LBPϩ/ϩ control mice (BALB/c; Biometec) (20). In acute-phase reactant produced during Gram-negative bacterial in- some experiments, differentiated THP-1 cells were pretreated with 0.1% ␮ ␮ ␮ ␮ fections (22). It is synthesized mainly in the liver and also in pul- DMSO, 10 M SP600125, 10 M SB202190, 10 M U0126, 1 M MG132, or 10 ␮g/ml cycloheximide for 1 h, and then stimulated with 10 monary and gastrointestinal epithelial cells, and is thought to func- ng/ml LPS for 3 h. tion as a carrier for LPS and to help control LPS-dependent monocyte responses. Recently, it has been shown that LBP also GeneChip expression analysis binds to glycolipids of mycobacteria (23) and lipoteichoic acid of GeneChip analysis was performed, as described previously (25). Gene ex- Gram-positive bacteria (24). pression was analyzed with the GeneChip Human Genome U133A probe In the present study, we have comprehensively investigated array (Affymetrix, Santa Clara, CA). Data analysis was performed with LPS-inducible genes in PBMC in the presence or absence of se- GeneSpring software version 4.2.1 (Silicon Genetics, Redwood City, CA). To normalize the staining intensity variations among chips, the average rum. We found that several genes, all of which are known to be difference values for all genes on a given chip were divided by the median IFN inducible (9), were induced by LPS only in the presence of value for expression of all genes on the chip. To eliminate genes containing serum factor. Further examination revealed that LBP, a soluble only a background signal, genes were selected only if the raw data were factor, plays a critical role in the regulation of the IFN-␤ signaling Ͼ300, and the gene expression was judged to be “present” by the Microar- ray Suite software version 5.0 (Affymetrix). A hierarchical clustering anal- pathway by LPS. Our findings imply the presence of possible ysis was performed using a minimum distance value of 0.001, a separation mechanisms linking LBP to the intracellular signaling between ratio of 0.5, and the standard definition of the correlation distance. TLR4 and IRF-3, leading to the induction of IFN-␤ by LPS. Semiquantitative real-time RT-PCR analysis Materials and Methods Primer sets for 10 genes, TNF-␣ (sense, 5Ј-ATCTACTCCCAGGTC Reagents and Abs CTCTTCAA-3Ј; antisense, 5Ј-GCAATGATCCCAAAGTAGACCT-3Ј), Downloaded from growth-relatedoncogene3(GRO3;sense,5Ј-GCAGGGAATTCACCTCAA 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 5Ј-TCGTCGTTTT GTCGTTTTGTCGTT-3Ј. Recombinant human sCD14 (Biometec, Greifs- wald, Germany) and LBP (R&D Systems, Minneapolis, MN) were negative in the limulus amebocyte lysate assay (BioWhittaker, Walkersville, http://www.jimmunol.org/ 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-␬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 by guest on October 6, 2021 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 ϭ 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 ␮g/ml streptomycin at a cell density of 2 ϫ 106 cells/ml. PBMC were stimulated with 10 Ϫ0-10Ϫ4 g/ml LPS, 2 ␮g/ml peptidoglycan (PGN; Invivogen, San Di- ego, CA), or 2 ␮g/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, Ber- gisch Gladbach, Germany). The purity of CD14-positive monocytes was always Ͼ90%. The THP-1 human monocytic cell line (ATCC, TIB-202) was main- tained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 20 ␮M 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 FIGURE 1. LPS-induced serum-dependent and -independent gene clus- RPMI 1640 medium in the presence of 5 nM PMA before stimulation. ters in PBMC. Two gene clusters containing 63 and 57 genes (a total of 138 They were then stimulated with 1Ð1000 ng/ml LPS in the presence or probe sets) were up-regulated more than 3-fold in PBMC after stimulation absence of 1% heat-inactivated human AB serum (ICN Biomedicals) for with 100 ng/ml LPS in both 3 and6hofculture in the presence of 10% up to 24 h. In some experiments, recombinant sCD14 and LBP were added FBS. Data were analyzed by applying a hierarchical-tree algorithm to the to the serum-free medium 1 h before stimulation with LPS. In blocking normalized intensity. The color code for the signal strength in the classi- ␮ experiments, 10 g/ml mouse anti-human CD14 mAb (clone biG 13; fication scheme is shown in the box on the left. Induced genes are indicated Biometec) or 10 ␮g/ml mouse anti-human LBP mAb (clone biG 412; Biometec) were added to differentiated THP-1 cells in the presence of 1% by shades of red; repressed genes are indicated by shades of blue. Exactly human AB serum and incubated for 1 h. Then 10 ng/ml LPS was added and the same experiments were performed with PBMCs from three individual incubated continuously for additional 3 h. In some experiments, differen- donors, and equal amounts of mRNA were mixed and hybridized with a tiated THP-1 cells were stimulated for 3 h with 100 ng/ml LPS in the single microarray (GeneChip). Gene symbols are shown in the right presence or absence of 2% serum from LBPϪ/Ϫ mice (BALB/c back- column. The Journal of Immunology 6187 Downloaded from http://www.jimmunol.org/ by guest on October 6, 2021

FIGURE 2. LPS-mediated up-regulation of mRNA expression for TNF-␣, 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 ␮g/ml PGN, or 2 ␮g/ml CpG ODN 2006 in the presence (Ⅺ)or absence (f) of 10% FBS (A and B). Human primary monocytes were incubated for 3 h with 100 ng/ml LPS in the presence (Ⅺ) or absence (f)of 10% FBS (C). The mRNA levels of TNF-␣, 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 Ϯ SEM of four independent experiments. The copy number is expressed as the number of transcripts/ng total RNA.

GA-3Ј; antisense, 5Ј-GGTGCTCCCCTTGTTCAGTA-3Ј), IL-6 (sense, 5Ј- Hilden, Germany) and was digested by DNase I (Qiagen), according to the CCACACAGACAGCCACTCAC-3Ј; antisense, 5Ј-AGGTTGTTTTCTG manufacturer’s instructions. Single-strand cDNA was synthesized with Su- CCAGTGC-3Ј),cyclooxygenase-2(COX2;sense,5Ј-CCACTTCAAGGGA perScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Semiquanti- TTTTGGA-3Ј; antisense, 5Ј-GAGAAGGCTTCCCAGCTTTT-3Ј), IFN- tative real-time RT-PCR was performed with a dsDNA-binding dye, SYBR induced protein with tetratricopeptide repeats 1 (IFIT1; sense, 5Ј-GCCA Green I, using an Applied Biosystems 7700 Sequence Detection System TTTTCTTTGCTTCCCCTA-3Ј;antisense,5Ј-TGCCCTTTTGTAGCCTCC (Applied Biosystems, Foster City, CA) (25). The expression levels of TTG-3Ј), IFN-induced protein of 10 kDa (IP-10; sense, 5Ј-CGCT mRNA were normalized by the median expression of a housekeeping gene GTACCTGCATCAGCATT-3Ј; antisense, 5Ј-GCTCCCCTCTGGTTTTA (GAPDH). GGAG-3Ј), IFN-stimulated gene 15 (ISG15; sense, 5Ј-GGACAAATGCG ACGAACCTCT-3Ј; antisense, 5Ј-CCCTCGAAGGTCAGCCAGA-3Ј), IRF-7 (sense, 5Ј-TGACACCCCCATCTTCGACTT-3Ј; antisense, 5Ј- Measurement of chemokines in the culture supernatant CACCAGGACCAGGCTCTTCTC-3Ј), IFN-␤ (sense, 5Ј-TGCTCTCCT GTTGTGCTTCTCC-3Ј; antisense, 5Ј-CATCTCATAGATGGTCAAT The cytokine and chemokine concentrations in the supernatant were GCGG-3Ј), and GAPDH (sense, 5Ј-GAAGGTGAAGGTCGGAGTC-3Ј; measured with ELISA kits speciﬁcally recognizing TNF-␣ or IP-10 antisense, 5Ј-GAAGATGGTGATGGGATTTC-3Ј) were synthesized at (R&D Systems). The minimal detection limits for the two kits were 15.6 Sawady Technology. Total RNA was extracted by RNeasy (Qiagen, and 7.8 pg/ml for TNF-␣ and IP-10, respectively. 6188 CRITICAL ROLE OF LBP IN LPS SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 3. Serum-dependent and -independent induction of TNF-␣, GRO3, IFIT1, IP-10, and IFN-␤ by LPS. Differentiated THP-1 cells were incubated at different concentrations of LPS for up to 24 h in the presence (Ⅺ) or absence (f) of 1% AB serum (AÐC). Human primary monocytes were incubated for 3 h with 100 ng/ml LPS in the presence (Ⅺ) or absence (f) of 1% AB serum (D). The mRNA levels of TNF-␣, GRO3, IFIT1, and IP-10 (A), or IFN-␤ (C and D) were determined by SYBR green-based real-time quantitative RT-PCR. Concentrations of TNF-␣ and IP-10 protein (B) in the culture supernatant were measured by ELISA. The results are shown as the mean Ϯ SEM of three independent experiments (AÐD). by guest on October 6, 2021 Western blot analysis in the second gene cluster are known to be regulated by type I IFNs (7, 9). Cells were harvested at the indicated time points, and then whole cell lysates were dissolved in Laemmli’s sample buffer. To detect NF-␬B p65, p38, ERK, JNK, STAT1, or Tyk2, 15 ␮g of whole cell lysates was Quantitative analysis of LPS-inducible genes resolved on a 4Ð20% gradient SDS-PAGE. Proteins were transferred onto Hybond-P (Amersham Biosciences, Arlington Heights, IL), which To confirm the GeneChip data, we applied a real-time PCR was blocked in Block Ace (Dainippon Seiyaku, Osaka, Japan). Western method. Inflammatory genes such as TNF-␣, GRO3, IL-6, and blotting was performed according to the procedure supplied with the COX2 were strongly up-regulated by LPS in a dose-dependent ECL Western blotting reagents kit (LumiGLO; Cell Signaling Technol- manner both in the presence and absence of 10% FBS (Fig. 2A). In ogy). Native PAGE was performed, as described previously (26). contrast, IFN-inducible genes such as IFIT1, IP-10, ISG15, and Results IRF-7 were strongly up-regulated by LPS only in the presence of Identification of serum-dependent LPS-inducible genes 10% FBS (Fig. 2B). Even at the highest concentration of LPS (100 ␮g/ml), up-regulation of these genes was not observed when cells To examine the effect of serum proteins, we incubated PBMC with were cultured without serum. However, induction of IFN-induc- LPS in serum-free or 10% FBS-containing culture medium and ible genes by CpG ODN 2006, a TLR9 ligand, did not require measured mRNA expression with the GeneChip system. Using serum proteins (Fig. 2B). In addition, induction of IFN-inducible hierarchical clustering analysis of the gene expression profiles of genes by a TLR3 ligand, poly(I:C), or a TLR7 and 8 ligand, R-848, ϳ 20,000 genes, we identified two gene clusters (Fig. 1 and sup- also did not require serum proteins (data not shown). Moreover, 4 plementary data). In the first gene cluster, inflammatory genes PGN up-regulated TNF-␣, GRO3, IL-6, and COX2 both in the ␣ ␤ such as TNF- , IL-1 , IL-6, and COX2, and neutrophil chemoat- presence and absence of serum (Fig. 2A), but did not induce IFIT1, tractant genes such as GRO3 and IL-8 were up-regulated by LPS IP-10, ISG15, or IRF-7 either in the presence or in the absence of both in the presence and absence of serum. In contrast, the second serum (Fig. 2B). gene cluster contained genes that were up-regulated by LPS only TLR4 is known to be mainly expressed on the monocytes in in the presence of serum. These included antiviral genes such as PBMC (27), and to determine whether this phenomenon in PBMC Ј Ј IFIT1, 2 ,5 -oligoadenylate synthetase 1, myxovirus (influenza vi- is mainly attributable to activation of monocytes, we incubated rus) resistance 1, ISG15, and protein kinase, IFN-inducible dsRNA human primary monocytes with LPS in serum-free or 10% FBS- dependent; antitumor genes such as TRAIL; and Th1 chemoat- containing culture medium. The results showed that IFN-inducible tractant genes such as IP-10. Interestingly, the majority of genes genes such as IFIT1 and ISG15 were also up-regulated by LPS only in the presence of FBS, whereas inflammatory genes such as 4 The on-line version of this article contains supplementary material. TNF-␣ and IL-6 were up-regulated by LPS under both conditions The Journal of Immunology 6189

(Fig. 2C). This suggests that monocytes play a major role in the Looking at IFN-inducible genes in more detail, we found that in critical regulation of LPS-inducible genes under both conditions. the presence of serum, the induction of mRNA for IFIT1 and IP-10 reached a peak at 6 h after stimulation with LPS, whereas induc- Serum-dependent induction in THP-1 cells tion of mRNA for IFN-␤ and inflammatory genes had peaked To characterize the effect of serum proteins on the induction of within 3 h (Fig. 3, A and C). This suggests that IFN-␤ and inflam- IFN-inducible genes, we established PMA-differentiated THP-1 matory genes were directly induced by LPS via TLR4. Previous cells to use as a substitute for human monocytes (28). In the pres- reports indicate that induction of IFIT1 and IP-10 by LPS is reg- ence of 1% human AB serum, mRNAs for inflammatory genes ulated by IFN-␤, implying that up-regulation of IFN-inducible such as TNF-␣ and GRO3, and IFN-inducible genes such as IFIT1, genes is regulated by LPS-induced IFN-␤ (29). Notably, induction IP-10, ISG15, and IRF-7 were up-regulated by LPS in a dose- of mRNA for IFN-␤ by LPS was completely abolished in the ab- dependent manner (Fig. 3A, and data not shown). In contrast, in the sence of serum in differentiated THP-1 cells and human monocytes absence of serum, IFIT1 and IP-10 mRNAs were not induced by (Fig. 3, C and D). Taken together, these results indicate that serum LPS, whereas TNF-␣ and GRO3 mRNA were (Fig. 3A). This find- proteins are essential components of the LPS-induced IFN-␤ sig- ing indicates that PMA-differentiated THP-1 cells closely mimic naling pathway. human monocytes in terms of responses to LPS. This phenomenon ␤ was further confirmed by measuring the concentration of proteins LBP-dependent IFN- induction in the supernatant. TNF-␣ protein was induced both in the pres- Two serum proteins, sCD14 and LBP, have been shown to play ence and absence of serum, but IP-10 protein was induced by LPS critical roles in LPS signaling (17). To elucidate whether sCD14 or only in the presence of human AB serum (Fig. 3B). In the time- LBP was required for activation of IFN-␤ signaling by LPS, we Downloaded from kinetic analysis (Fig. 3A), mRNA for TNF-␣ and GRO3 peaked at added recombinant sCD14 and LBP to the serum-free medium 3Ð6 h of culture both in the presence and absence of serum. In before LPS stimulation. Induction of TNF-␣ by LPS was enhanced contrast, mRNA for IFIT1 or IP-10 was not induced by LPS in the by the addition of either sCD14 or LBP to the serum-free medium absence of serum even after 24 h of culture. These results indicate (Fig. 4A). In contrast, IFIT1 and IP-10 were induced by LPS on the that lack of induction of mRNA for IFIT1 or IP-10 is not simply addition of LBP, but not on the addition of sCD14 (Fig. 4, B and due to the magnitude of stimulation nor to differences in the time C). However, concomitant addition of sCD14 synergistically en- kinetics. hanced LBP-dependent induction of IFN-inducible genes (Fig. 4, http://www.jimmunol.org/ by guest on October 6, 2021

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 (f) or vehicle control (Ⅺ) in the presence or absence of indicated concentrations of recombinant sCD14 and LBP in serum-free medium or 1% AB serum-containing medium (AÐC). Differentiated THP-1 cells (D) and human monocytes (E) were incubated with 100 ng/ml LPS or vehicle control for 2 (D)or3(E) h in the presence or absence of 100 ng/ml recombinant sCD14 and LBP in serum-free medium. The mRNA levels of TNF-␣ (A), IFIT1 (B), IP-10 (C), and IFN-␤ (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 Ϯ SEM of three independent experiments. 6190 CRITICAL ROLE OF LBP IN LPS SIGNALING

B and C). Exactly the same pattern was observed with induction of inhibited in anti-CD14 mAb-treated cells, but not in anti-LBP mAb- mRNA for IFN-␤ by LPS in differentiated THP-1 cells and human treated cells (Fig. 5B). The same inhibitory pattern was observed with monocytes (Fig. 4, D and E). These results indicate that LBP has another clone for anti-CD14 mAb (18D11; Diatech.com AS, Oslo, an essential role in the activation of IFN-␤ signaling and that Norway), and anti-LBP mAb (biG 33; Biometec) (data not shown). sCD14 facilitates this function. Thus, a combination of serum LBP, but not membrane-associated To clarify the effects of LBP and CD14 on the activation of LBP, and CD14, either mCD14 or sCD14, must be an essential com- IFN-␤ signaling by LPS, we performed blocking experiments us- ponent of the LPS-induced IFN-␤ signaling pathway. ing neutralizing mAbs against CD14 or LBP. In the presence of We also used serum from LBPϪ/Ϫ mice to investigate the pos- human AB serum, induction of TNF-␣ by LPS was strongly in- sible involvement of other serum proteins in the induction of hibited by anti-CD14 mAb and partially inhibited by anti-LBP IFN-␤ and IFN-inducible genes by LPS. mRNAs for IFN-␤ and mAb (Fig. 5A). In contrast, induction of IFN-␤ and IFIT1 by LPS IFIT1 were induced by LPS only in the presence of serum from was completely abolished by either anti-LBP mAb or anti-CD14 LBPϩ/ϩ control mice, and not in the presence of serum from mAb (Fig. 5A). LBPϪ/Ϫ mice (Fig. 5C). This suggests that LBP is capable of en- Previous reports have indicated that the interaction of LPS with hancing LPS-induced IFN-␤ expression, but other serum proteins membrane-bound CD14 (mCD14) and membrane-associated LBP are not. is an important step in LPS-mediated activation (30). To test whether mCD14 and membrane-associated LBP are involved in our Serum-dependent activation of JNK and p38 system or not, we washed differentiated THP-1 cells three times after It is known that LPS is capable of activating three MAPKs, p38, preincubation with anti-CD14 mAb or anti-LBP mAb for1hinthe ERK, and JNK, and NF-␬B (31). To determine whether these absence of serum, and then added 1% AB serum-containing medium. MAPKs and NF-␬B are critical for the activation of IFN-␤ sig- Downloaded from In these THP-1 cells, induction of IFN-␤ and IFIT1 by LPS was naling, we investigated immunodetection of their phosphorylated http://www.jimmunol.org/

FIGURE 5. Effects of neutralizing mAbs against by guest on October 6, 2021 CD14 and LBP on up-regulation of mRNA expression in differentiated THP-1 cells by LPS. Differen- tiated THP-1 cells were preincubated with 10 ␮g/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 for3h(A). To determine the effect of cell surface CD14 or LBP, differentiated THP-1 cells were incubated with 10 ␮g/ml mAbs against CD14 and LBP or control IgG1 for1hin 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 (f) or vehicle control (Ⅺ) in the presence or absence of 2% serum from LBPϪ/Ϫ mice or LBPϩ/ϩ control mice (C). The mRNA levels of TNF-␣, IFN-␤, 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 Ϯ SEM of three independent experiments. The Journal of Immunology 6191 Downloaded from

FIGURE 6. Serum-dependent and -independent phosphorylation of NF- ␬B, MAPKs, and STAT1 in differentiated THP-1 cells by LPS. Differen- tiated THP-1 cells were incubated for up to 6 h with 100 ng/ml LPS in the http://www.jimmunol.org/ 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 FIGURE 7. Effects of signal transduction inhibitors on up-regulation of ng/ml recombinant sCD14 and LBP in serum-free medium (B). At the mRNA expression in differentiated THP-1 cells by LPS. Differentiated indicated time points, cells were harvested and the cell lysates were sub- THP-1 cells were pretreated with 0.1% DMSO, 10 ␮M SP600125 (JNK jected to SDS-PAGE. After blotting to polyvinylidene diﬂuoride mem- inhibitor), 10 ␮M SB202190 (p38 inhibitor), 10 ␮M U0126 (mitogen- brane, total and phosphorylated NF-␬B, MAPKs, and STAT1 were de- activated protein/ERK kinase inhibitor), 1 ␮M MG132 (proteasome inhib- tected with speciﬁc mAbs. The results are representative of at least two itor), or 10 ␮g/ml cycloheximide (CHX; protein synthesis inhibitor) for separate experiments. 1 h, and then stimulated with 10 ng/ml LPS for 3 h. The mRNA levels of ␣ ␤

TNF- , IFN- , IFIT1, or IP-10 were determined by SYBR green-based by guest on October 6, 2021 real-time quantitative RT-PCR. The copy number is expressed as the num- forms. NF-␬B and ERK were phosphorylated by LPS both in the ber of transcripts/ng total RNA. The results are shown as the mean Ϯ SEM presence and absence of serum. However, JNK and p38 were of three independent experiments. 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). To determine the effect of LBP and sCD14 on the phosphory- ylated by LPS only in the presence of LBP, but not in the presence lation of JNK and p38 by LPS, we added LBP and sCD14 to of sCD14 alone (Fig. 8A). It is known that induction of IFN-␤ is serum-free medium before LPS stimulation. The results showed regulated by IRF-3 activation and dimerization (32). We examined that JNK and p38 were phosphorylated by LPS only in the pres- the formation of phosphorylated IRF-3 dimers in LPS-stimulated ence of LBP, and not in the presence of sCD14 alone or in the THP-1 cells by native PAGE. IRF-3 was also dimerized by LPS absence of serum (Fig. 6B). only in the presence of LBP (Fig. 8B). These data suggest that LBP is an essential component of the JNK and p38 in LPS signaling LPS-induced IFN-␤ signaling pathway, exerting its effect via To investigate the upstream signal transduction pathways for TLR4, JNK, p38, and IRF-3. IFN-␤ and IFN-inducible genes by LPS, we performed experiments in which differentiated THP-1 cells were treated with Discussion SP600125, SB202190, U0126, MG132, or DMSO, a vehicle con- LPS is known to induce a large number of genes in different cell trol, and then stimulated with LPS for3hinthepresence of serum. types (33Ð36), and consistent with this we identified ϳ120 genes Up-regulation of mRNA for TNF-␣ was strongly blocked by up-regulated (Ͼ3-fold in normalized data) in PBMC after 3Ð6hof MG132 and U0126 (Fig. 7). However, up-regulation of the mRNA culture with 100 ng/ml LPS. Almost all LPS-induced genes can be for IFN-␤ was strongly blocked by SP600125 and SB202190, but classified into two distinct clusters according to their regulation in not by MG132, U0126, or cycloheximide (Fig. 7). In contrast, the presence or absence of serum in the culture medium by using up-regulation of mRNAs for IFIT1 and IP-10 was strongly blocked hierarchical clustering analysis. Interestingly, most of the genes in by SP600125, SB202190, and cycloheximide (Fig. 7). the serum-dependent cluster are known to be IFN inducible (9). Further analysis confirmed that this phenomenon is not simply due LBP-dependent IRF-3 activation to differences in the magnitude of stimuli nor due to differences in To clarify the effects of LBP and sCD14 on the activation of IFN-␤ time kinetics. Even at the highest concentrations of LPS (up to 100 signaling by LPS, we added LBP and sCD14 to the serum-free ␮g/ml), LPS failed to induce IFN-inducible genes in the absence of medium before LPS stimulation. STAT1 and Tyk2 were phosphor- serum (Fig. 2). At very high concentrations, LPS is reported to 6192 CRITICAL ROLE OF LBP IN LPS SIGNALING

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 com- pared 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-␤ by LPS. The blocking experiments using anti-CD14 mAb clearly demonstrated that CD14 is essential for transducing the LPS signal to induce both TNF-␣ and IFN-␤ (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. Downloaded from Thus, the induction of TNF-␣ by LPS did not require any additional serum proteins. Addition of LBP or sCD14 alone enhanced the expression of TNF-␣. However, addition of sCD14 alone could not induce IFN-␤ or IFIT1. Only in combination with LBP did sCD14 enhance the induction of IFN-␤ and IFN-inducible genes

by LPS (Fig. 4). http://www.jimmunol.org/ In contrast, addition of LBP alone up-regulated expression of FIGURE 8. Effects of sCD14 and LBP on the phosphorylation of IFN-␤ and IFIT1 (Fig. 4). This is perhaps due to the expression of STAT1, Tyk2, or IRF-3 in differentiated THP-1 cells by LPS. Differenti- mCD14 on the cell surface of differentiated THP-1 cells. In other ated THP-1 cells were incubated with 100 ng/ml LPS in the presence or words, in cells with expression of CD14, the induction of IFN-␤ by absence of 100 ng/ml recombinant sCD14 and LBP in serum-free medium. LPS is critically regulated by LBP. After3hofincubation, cells were harvested and the cell lysates were subjected to SDS-PAGE. After blotting to polyvinylidene diﬂuoride mem- A number of studies have attempted to clarify the molecular brane, total and phosphorylated STAT1 and Tyk2 were detected with spe- mechanisms by which TLR4-bound LPS transduces its signals to induce expression of inﬂammatory genes and IFN-inducible genes

cific mAbs (A). After2hofincubation, cells were harvested and cell by guest on October 6, 2021 lysates were subjected to native PAGE, and monomeric and dimeric IRF-3 (16, 29). In the upstream part of the signal transduction pathways were detected with anti-IRF-3 mAb (B). The results are representative of at to the transcription factors NF-␬B and IRF-3, activation of three least two separate experiments. MAPKs (31) has been documented. Among these molecules, JNK (42) and p38 (43) in particular are thought to be involved in the activate cells and to induce TNF-␣ production in a CD14-inde- IRF-3 pathway. To investigate in what stages of the signal trans- pendent manner (37), and the induction is at least in part CD11b duction pathways the absence of LBP switches off the LPS signal, dependent (38). However, our results clearly demonstrate that re- we determined the phosphorylation of molecules in the upstream gardless of the concentration of LPS, the presence of serum either part in the signal transduction pathways of IFN-␤ and IFN-induc- switches on or off the expression of IFN-inducible genes in PBMC, ible genes. As a result, phosphorylation of JNK, p38, IRF-3, Tyk2, primary monocytes, and differentiated THP-1 cells (Figs. 2 and 3). and STAT1, but not NF-␬B or ERK was diminished or abrogated In addition, our time kinetics study, together with previous reports, in the absence of serum (Figs. 6A and 8). In addition, the phos- ␤ shows that the induction of IFN-inducible genes by LPS is IFN- phorylation of JNK, p38, IRF-3, Tyk2, and STAT1 was observed dependent (Fig. 3, A and C). Thus, LPS seems to induce IFN-␤ only when LBP was added to serum-free medium (Figs. 6B and 8). signaling in a serum-dependent fashion. Unlike other IRF-3-acti- Thus, LBP seems to play critical roles in the activation of JNK, vating PAMPs (poly(I:C), R-848, or CpG ODN) or the non-IRF- p38, IRF-3, Tyk2, and STAT1. Blocking experiments using spe- 3-activating PAMP (PGN) tested, LPS exerts serum factor-depen- cific inhibitors for the signal transduction pathways further dem- dent activation of IFN-inducible genes in PBMC, suggesting that onstrated that, upon stimulation with LPS, expression of TNF-␣ such a phenomenon is TLR4 specific. ␬ ␤ Several serum proteins and lipids have been reported to be ca- was regulated by NF- B, whereas induction of IFN- , IFIT1, and pable of binding to LPS (17, 18). Among them, sCD14 (19) and IP-10 was regulated by JNK and p38 (Fig. 7). It is noteworthy that ␣ ␤ LBP (20) play key roles in the recognition of LPS. In the present the induction of IFIT1 and IP-10, but not of TNF- or IFN- , study, we revealed that LBP plays a critical role in the up-regula- seems to require de novo protein synthesis. This fact, along with tion of IFN-␤ and IFN-inducible genes in three experimental set- the LBP-dependent Tyk2 and STAT1 phosphorylation, is consis- tings: first, the addition of LBP to the serum-free medium enabled tent with previous reports that the induction of these molecules is the induction of IFN-␤ and IFN-inducible genes by LPS (Fig. 4); regulated by IFN-␤ (6, 7, 44). second, the inhibition of LBP by blocking mAb strongly reduced The blocking and washing experiments (Fig. 5B) showed clearly the induction of IFN-␤ and IFN-inducible genes by LPS in the that membrane-associated LBP is not involved in the induction of presence of serum (Fig. 5, A and B); and third, IFN-␤ and IFN- IFN-␤ or IFN-inducible genes upon stimulation with LPS. Thus, a inducible genes were not induced by LPS in the presence of serum soluble form of LBP must play a critical role in this process. How- from LBPϪ/Ϫ mice (Fig. 5C). ever, the mechanisms by which LBP, a soluble protein, transduces The Journal of Immunology 6193 signals to IRF-3, an intracellular transcription factor, are still un- 6. Malcolm, K. C., and G. S. Worthen. 2003. Lipopolysaccharide stimulates p38- clear. It is possible that other molecules may play roles in LBP- dependent induction of antiviral genes in neutrophils independently of paracrine factors. J. Biol. Chem. 278:15693. dependent induction of IFN-␤ by LPS. This should be studied 7. Doyle, S., S. Vaidya, R. O’Connell, H. Dadgostar, P. Dempsey, T. Wu, G. Rao, further in the future. R. Sun, M. Haberland, R. Modlin, and G. Cheng. 2002. IRF3 mediates a TLR3/ Among 10 human TLRs, stimulation through only TLR3, 4, 5, TLR4-specific antiviral gene program. Immunity 17:251. 8. Laich, A., M. Meyer, E. R. Werner, and G. 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