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Bacterial Lipopolysaccharide Activates Nuclear Factor-κB through Interleukin-1 Signaling Mediators in Cultured Human Dermal Endothelial Cells and Mononuclear Phagocytes*

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-κB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-α) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-κB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-κB-luciferase activity. LPS-induced NF-κB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-κB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.

Lipopolysaccharide (LPS), 1 or endotoxin, is the major component of the outer surface of Gram-negative bacteria. LPS is a potent activator of cells of the immune and inflammatory systems, including macrophages, monocytes, and endothelial cells, and contributes to systemic changes known as septic shock (1,2). LPS-induced activation of monocytes/macrophages is mediated through a cell surface receptor glycoprotein, known as membrane CD14 (mCD14). The binding of LPS to mCD14 is enhanced by LPS-binding protein, a plasma protein (3). On the other hand, vascular endothelial cells do not express mCD14 and respond to LPS only in the presence of soluble CD14 (4 -6). We (7) and others (8 -12) have shown that protein tyrosine phosphorylation and activation of ERK1, ERK2, p38 mitogenactivated protein kinase, and c-Jun N-terminal kinase appear to be important for LPS-induced cellular activation. LPS rapidly induces nuclear factor-B (NF-B) in both monocytic (13,14) and endothelial cells (15). Activation of NF-B is required for release of proinflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) (16,17). However, the molecular mechanisms of the signaling cascade induced by LPS to activate NF-B are unknown. Furthermore, the signaling LPS receptor is still unidentified.
The toll gene controls dorsoventral pattern formation during the early embryonic development of Drosophila melanogaster (18). Toll initiates a signaling pathway homologous to the mammalian NF-B activation cascade (18). The toll family of receptors is defined by homology to the Drosophila toll protein (19,20). The mammalian IL-1 receptor is a member of the toll family (18). Five other mammalian family members (toll-like receptors 1 through 5, TLR1-5) have been identified, but their function is uncertain. Several TLRs, similar to IL-1R, have been observed to signal through the NF-B pathway (19 -22). LPS signaling also leads to activation of NF-B, and recent studies suggested that a toll-like receptor (TLR) might be a signaling receptor that is activated by LPS (22,23). In these reports, expression of TLR2 in LPS-unresponsive human embryonic kidney cells (293 cells) enabled these cells to respond to LPS stimulation (22,23). These investigators observed that LPS binds to a TLR2 extracellular domain and suggested that TLR2 is a candidate for a long sought LPS receptor, although the data were generated from a transfected and normally LPSunresponsive cell line (22,23). A recent study in the LPSresistant C3H-HeJ mice has implicated another toll homologue (TLR4) as a signal-transducing component in the LPS receptor complex (24).
It is known that the IL-1 signaling pathway in mammals is strikingly similar to the toll signaling pathway in Drosophila (19 -22). The molecular events linking the IL-1 receptor (IL-1R) signaling complex to the induction of NF-B have been recently characterized. Upon binding of IL-1 to its receptors (IL-1R), IL-1R associates with the IL-1 receptor accessory protein (IL-1RAcP) (26,27). The complex then recruits and activates an * This work was supported by National Institutes of Health Grant AI40275 (to M. A.), by funds from MURST and AIRC, and European Community Grants BIO4CT972107 and BMH4CT983277 (to M. M). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In this study, we tested the hypothesis that LPS activates NF-B through IL-1 signaling molecules, namely MyD88, IRAK, IRAK2, and TRAF6 in two LPS-responsive cell types. Dominant negative mutants of these signaling elements were transfected into human monocytes (THP-1 cells) and human dermal endothelial cells together with a NF-B-responsive reporter gene, and LPS-induced NF-B luciferase activity was measured. Our results indicate that LPS transduces signals of NF-B activation utilizing the IL-1 signaling molecules in both monocytic and endothelial cells.

EXPERIMENTAL PROCEDURES
Cell Culture-Human THP-1 cells (from ATCC) were cultured in RPMI medium with 10% fetal calf serum. The immortalized human dermal endothelial cells (generous gift of Dr. Candal of the Center for Disease Control, Atlanta) were cultured in MCDB-131 medium with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 100 g/ml penicillin and streptomycin in 6-well plates.
Expression Vectors and Transfection of THP-1 Cells-Dominant negative expression vectors of MyD88, IRAK, IRAK2, TRAF2, TRAF6, and NIK have been characterized and described before (30,32,33). Cells were used for transfection with FuGene 6 Transfection Reagent (Boehringer Mannheim) following the manufacturer's instructions in RPMI with 10% serum. Reporter genes pCMV-␤-galactosidase (0.5 g) and ELAM-NF-B-luciferase (2 g) and pcDNA3 empty vector or dominant negative mutants of MyD88, IRAK, TRAF6, TRAF2, and NIK (3 g each) were used. After a 24-h transfection, cells were stimulated for 6 h with 100 ng/ml LPS. Cells were then lysed, and luciferase activity was measured with a Promega kit (Promega, Madison, WI) and a luminometer. ␤-Galactosidase activity was determined by the calorimetric method to normalize transfection efficiency as described earlier (30). Data shown are means of two independent experiments.
Transfection of Human Dermal Endothelial Cells-Transfection was carried out using the same method described above. The amount of NF-B luciferase construct DNA was 1.5 g, and empty vector and various dominant negative constructs were 2 g each. Cells were transfected for 24 h and stimulated for 6 h with 100 ng/ml LPS, human TNF-␣ (200 units/ml, Genzyme, Cambridge, MA), recombinant human IL-1␤ (400 units/ml, Genzyme), and recombinant IL-1 receptor antagonist (100 ng/ml, R&D Systems, Minneapolis, MN) in 2 ml of serumcontaining MCDB-131 medium.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis-Total RNA was isolated from HDMEC and THP-1 cells using a Qiagen kit (Valencia, CA) and treated with RNase-free DNase I. For RT reaction, the SuperScript TM Preamplification system (Life Technologies, Inc.) was applied. PCR amplification was performed with Taq polymerase (Qiagen, Valencia, CA) for 28 cycles at 95°C for 40 s, 54°C for 40 s, and 72°C for 1 min. PCR primers for TLR2 were 5Ј-GC-CAAAGTCTTGATTGATTGG and 5Ј-TTGAAGTTCTCCAGCTCCTG. PCR primers for TLR4 were 5Ј-TGGATACGTTTCCTTATAAG and 5Ј-GAAATGGAGGCACCCCTTC. GAPDH primers were obtained from CLONTECH.
Immunostaining and Immunoblotting-Smeared THP-1 cell and cultured HDMEC on slides were fixed with acetone for 5 min and then stained with rabbit anti-TLR2 and TLR-4 antibody (1:100) and rabbit IgG following the instructions on a DACO immunostaining kit. The anti-TLR2 and anti-TLR4 antisera were raised against synthetic peptides (extracellular domains of TLR2 and TLR4) by BABCO (Richmond, CA). The sequence of the synthetic peptide for TLR2 was a 27-amino acid peptide, starting at amino acid residue 277 of the mature hTLR2 (FRASDNDRVIDPGKVETLTIRRLHIPR), whereas the peptide for TLR4 was a 23-amino acid peptide, starting at amino acid 201 of mature hTLR4 (FKEIRHKLTLRNNFDLSLNVMKT). Following immunoperoxidase staining, the representative fields were photographed.
THP-1 and HDMEC cells were lysed in Laemmli buffer and separated with a 10% SDS-PAGE gel. The protein was then transferred onto a polyvinylidene difluoride membrane, and then the membrane was probed with anti-TLR2, anti-TLR4 antibodies, and prebleeds corresponding to each antibody (1:2,000). After incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody, the membrane was developed with an enhanced chemiluminescence ECL detection kit.

RESULTS AND DISCUSSION
A series of defense mechanisms are triggered in vertebrates and invertebrates in response to Gram-negative bacterial infections by sensing the presence of LPS (1-3). LPS-induced signal relay is thought to be initiated following its binding to specific cellular receptors which then triggers intracellular signaling pathways leading to the activation of NF-B (4 -12) in various LPS-responsive cell types. To date, the identification of a functional, signal-transducing component of the putative LPS receptor complex and the signaling pathways involved in LPSinduced activation of NF-B have remained elusive. Recent findings suggested that LPS might use signaling molecules of the TLR and IL-1R superfamilies to transduce signals (23,24).
To investigate the potential involvement of IL-1 and TNF signal transducers in LPS signaling in two LPS-responsive cell types, HDMEC and THP-1 cells, we cotransfected dominant negative constructs of various components of the NF-B signaling cascades for IL-1 and TNF together with NF-B-luciferase reporter gene. LPS induced the activation of NF-B in a time- (Fig. 1A) and dose-dependent (Fig. 1B) manner in THP-1 cells. Activation of NF-B reached a maximum at a LPS concentration of 100 ng/ml and when cells were stimulated with LPS for 6 h (Fig. 1, A and B). Similar results were also obtained from endothelial cells.
NF-B activation induced by various cytokine receptors is mediated by members of the TRAF adapter family. While TRAF2 plays a crucial role in NF-B activation by TNFR-1 and TNFR-2 (37, 38), TRAF6 has been implicated in IL-1 signaling (31,32). Therefore, we next determined whether dominant negative versions of TRAF6 (⌬TRAF6) or TRAF2 (⌬TRAF2) could act to inhibit LPS-induced NF-B activity. ⌬TRAF-6 but not ⌬TRAF2 significantly impaired LPS-induced NF-B activation, suggesting that TRAF6 may act as an additional downstream mediator of LPS-induced NF-B activation cascade (Figs. 1D and 2A). ⌬TRAF2 blocked TNF-induced NF-B activation in endothelial cells (Fig. 2C), but not LPS-induced NF-B activation (Figs. 1D and 2A). Because the pathways for IL-1 and TNF-␣ signaling converge at the level of NIK for NF-B activation, we next investigated whether dominant negative NIK mutant (⌬NIK) would block LPS-induced, as well as IL-1-and TNF-induced, NF-B activation. As expected, ⌬NIK blocked NF-B activation induced by LPS, IL-1, and TNF-␣ (Fig. 2).
IL-1 receptor antagonist had no effect on LPS-induced NF-B activation ( Fig. 2A) but inhibited IL-1-induced NF-B activation in endothelial cells (Fig. 2B). This observation suggests that NF-B activation that we measured following 6 h of  1 and 3) and THP-1 cells (lanes 2 and 4) was analyzed by PCR following reverse transcription (RT; lanes 1 and 2) or without RT (lanes 3 and 4). RT-PCR analysis of GAPDH expression was used as control (lower panel, 983 base pairs). Labels of base pairs at the right are DNA standard markers. LPS stimulation of cells is not due to an autocrine effect such as LPS-induced IL-1 release from endothelial cells.
These data suggest that LPS stimulation of endothelial cells and THP-1 cells triggers an IL-1R-like signal relay leading to activation of NF-B (Fig. 5). Further support for this concept is provided by the observation of a 15-year-old girl with recurrent infections who was found to be resistant to both LPS and IL-1 stimulation in vivo and in vitro (39). The authors suggested that resistance to LPS and IL-1 was due to a defect very early in the common signaling pathway for LPS and IL-1 (39).
The experiments with IL-1R antagonist also suggest that LPS does not use IL-1 receptor to transduce signals in endothelial cells. Although recent findings imply that TLR2 or other members of the TLR family, which use the IL-1R signaling pathway, might be an important mediator for LPS signaling (23,24), further studies are needed to identify naturally existing LPS receptors in LPS-responsive cells. Beutler and coworkers (25) recently reported that TLR4 is the protein encoded by the LPS gene, which is mutated in the LPS-non-responsive C3H-HeJ mice. To investigate the expression of the TLR2 and TLR4 message in HDMEC and THP-1 cells, we used RT-PCR. Human THP-1 and HDMEC were found to express significant levels of both TLR2 and TLR4 mRNA (Fig. 3). Expression levels of TLR2 appeared to be stronger in THP-1 whereas the expression level of TLR4 appeared to be stronger in HDMEC. Expression of TLR2 and TLR4 was confirmed with Northern analysis in THP-1 cells. Immunohistochemistry and immunoblotting data demonstrate that TLR2 and TLR4 proteins are expressed on THP-1 cells and HDMEC (Fig. 4). Staining was absent in THP-1 and HDMEC incubated without the first antibody or incubated with rabbit IgG. These results suggest that TLR2 and TLR4 are expressed in endothelial and monocytic cells and may represent a signaling component of a cellular receptor for LPS which signals through an IL-1-like pathway (Fig. 5).
In summary, we have demonstrated in endothelial and THP-1 cells that LPS-induced NF-B activation is mediated by IL-1R signaling molecules, namely MyD88, IRAK, IRAK2, and TRAF6, but not the TNF signaling molecule, TRAF2. We have also shown that TLR2 and TLR4 are expressed on the cell surface of two LPS-responsive cell types, endothelial cells and THP-1 cells. These data strongly suggest that a crucial signaling component in the LPS receptor complex may belong to the IL-1 receptor/TLR superfamily, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1. MyD88 appears to represent the most upstream mediator of the LPS-mediated signaling cascade, which ultimately activates NF-B, thus driving transcriptional activation of several cytokines. Thus, MyD88 may represent a potentially useful therapeutic target to control the molecular switch from innate to the adaptive immune response.