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Cutting Edge: Lipopolysaccharide Induces Physical Proximity Between CD14 and Toll-Like Receptor 4 (TLR4) Prior to Nuclear Translocation of NF-B¹

Qingqi Jiang^*, Sachiko Akashi, Kensuke Miyake and Howard R. Petty^2,*

^* Department of Biological Sciences, Wayne State University, Detroit, MI 48202; and Department of Immunology, Saga Medical School, Saga, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Recent studies have identified the Toll-like receptor 4 (TLR4) as a membrane cofactor in LPS-mediated transmembrane signaling in cytokine induction, although the mechanism responsible for this cooperation is unknown. Using fluorescence resonance energy transfer (RET) techniques, we demonstrate that LPS triggers a physical association between CD14 and TLR4. Because LPS stimulation up-regulates CD14 and TLR4 expression, it was necessary to control for the possibility that these newly expressed molecules were associated with one another independent of LPS stimulation. Although the calcium ionophore A23187 increased the expression of CD14 and TLR4, they did not exhibit energy transfer. However, following A23187 treatment, LPS promoted physical proximity between CD14 and TLR4. Therefore, we suggest that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-B.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Lipopolysaccharide, the primary component of the outer membrane of Gram-negative bacteria, is responsible for the overwhelming innate immune response of the sepsis syndrome. Septicemia leads to multiple organ failure and death in about one-third of the patients. CD14, a GPI-linked cell surface glycoprotein, is the main LPS receptor of leukocytes and contributes to host sensitivity (1). LPS rapidly promotes a large array of leukocyte functions including adherence and reactive oxygen metabolite (ROM) production as well as priming functions such as phagocytosis (2, 3, 4, 5, 6). LPS also affects gene expression within hours of receptor engagement by triggering monocytes and macrophages to produce the inflammatory cytokines TNF-, IL-1, and IL-6 (7, 8). Inasmuch as CD14 lacks transmembrane and intracellular domains, the mechanism(s) of CD14-mediated transmembrane signaling has remained elusive. CD14 physically associates with the leukocyte integrin CR3 (9) and may thus promote cell adherence, binding, and phagocytosis (2, 3, 4, 5, 6, 10, 11, 12). Recently, Toll-like receptor 4 (TLR4)³ has been identified as another LPS signaling partner. Hyporesponsiveness and hyperresponsiveness to LPS have been traced to TLR4 (13, 14, 15). Because LPS and CD14 complexes transiently come into the proximity of CR3 before cell adherence (9), we hypothesized that LPS/CD14 complexes physically associate with TLR4 before the nuclear translocation of NF-B.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Materials

LPS (Escherichia coli serotype 026:B6), lipid A (F583), PMA, polymyxin B, and A23187 were purchased from Sigma Chemical Company (St. Louis, MO). FITC and tetramethylrhodamineisothiocyanate (TRITC) were obtained from Molecular Probes (Eugene, OR). Anti-NF-B mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). mAbs directed against Mo-5 and CD87 (urokinase receptor) were the generous gifts of Dr. Robert Todd (University of Michigan, Ann Arbor, MI).

Preparation of monocytes

Monocytes were isolated from peripheral blood using Ficoll-Hypaque (Sigma) density gradient centrifugation at 300 x g for 45 min at room temperature. Cells were suspended in pyrogen-free and sterile HBSS containing Ca²⁺/Mg²⁺ (Life Technologies, Grand Island, NY) and 50 ng/ml polymyxin B. Cells were allowed to adhere to glass coverslips at 37°C for 15 min then washed with HBSS.

Preparation of FITC- and TRITC-conjugated Abs

F(ab')₂ of anti-CD14 (clone 26ic) and mAb for hTLR4 (clone HTA125) were used (16, 17). Abs were dialyzed against 0.15 M carbonate-bicarbonate buffer at pH 9.3 overnight at 4°C. Samples were incubated with dyes at a fluorophore-protein ratio of 40 µg rhodamine or 30 µg FITC per milligram protein at room temperature for 4 h. The fluorescent conjugates were separated from unreacted fluorochromes by Sephadex G-25 (Sigma) column chromatography. Purified conjugates were dialyzed against PBS at pH 7.4 overnight at 4°C.

NF-B nuclear staining

Monocytes were allowed to adhere to glass coverslips for 15 min at 37°C, then treated with LPS as described below. After fixation with methanol at -20°C and being made permeable with 2% Nonidet P-40, cells were blocked with 20% normal goat serum at room temperature for 20 min. Cells were incubated with 1 µg/ml rabbit anti-human NF-B Ab at room temperature for 60 min, rinsed with PBS, labeled with 1 µg/ml fluorescein-conjugated secondary Ab in the dark at 4°C for 45–60 min, and then washed with buffer.

Fluorescence microscopy

An axiovert-inverted fluorescence microscope with HBO-100 mercury illumination (Carl Zeiss, New York, NY) interfaced to a Dell 410 workstation via Scion SG-7 video card (Vay Tek, Fairfield, IA) was used. The fluorescence images were collected by an intensified charge-coupled device camera, model XC-77 (Hamamatsu, Hamamatsu City, Japan) and processed with ScionImage Software. A narrow bandpass-discriminating filter set was used with excitation at 485DF20 nm and emission of 530DF30 nm for FITC. For rhodamine, an excitation of 540DF20 nm and an emission of 590DF30 nm were used (Omega Optical, Brattleboro, VT). Long-pass dichroic mirrors at 510 and 560 nm were used for FITC and rhodamine, respectively. For energy transfer imaging, the 485DF22, 510LP, and 590DF30 filter combination was used (18).

Single-cell imaging spectrophotometry

Single-cell spectra were obtained using a microscope/imaging spectrophotometer system (18, 19). Labeled cells were illuminated with an excitation filter at 485DF22 nm and a 510LP dichroic mirror for FITC and resonance energy transfer (RET) experiments. For rhodamine emission spectra, excitation was provided with a 540DF20-nm filter and a 560LP dichroic mirror. The emission spectra were obtained with an Acton-150 (Acton, MA) imaging spectrophotometer. The input side of the spectrophotometer was fiberoptically coupled to the microscope. The exit port of the spectrophotometer was attached to a Gen-II intensifier that was coupled with an I-MAX-512 camera (Princeton Instruments, Trenton, NJ). Spectra collection was controlled by a high-speed Princeton ST-133 interface and a Stanford Research Systems (Sunnyvale, CA) DG-535 delay-gate generator. The systems were interfaced to a Dell 410 workstation running Winspec software (Princeton Instruments) to manage and analyze the data.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Previous studies have shown that CD14 is up-regulated by LPS (20, 21). To determine whether TLR4 expression is affected by LPS, adherent monocytes were stimulated with 200 ng/ml LPS or unstimulated for 0 to 30 min, fixed, then labeled with rhodamine-conjugated TLR4. The results showed that TLR4 was expressed on the membrane of unstimulated cells, and that TLR4 expression was increased by LPS, reaching a peak value at 15–30 min. Moreover, the inclusion of serum was found to enhance TLR4 expression. Cells were incubated with 200 ng/ml LPS in the presence or absence of 10% heat-inactivated serum. In the absence of serum, controls and LPS-treated cells demonstrated fluorescence intensities of 10,430 ± 7,810 and 28,670 ± 9,160 counts/min (n = 3; p < 0.05), respectively. In the presence of serum, control and LPS-treated samples exhibited intensities of 11,000 ± 8,520 and 96,860 ± 7,270 counts/min (n = 3; p < 0.001). Therefore, TLR4 expression is up-regulated by LPS.

To study the physical proximity of CD14 and TLR4, RET microscopy was used. RET microscopy, in both imaging and spectroscopic formats, can be used to detect lateral proximity of membrane proteins. This is accomplished by detecting the migration of excitation energy from donor (FITC) to acceptor (TRITC) chromophores attached to different proteins (22). Monocytes were allowed to adhere to endotoxin-free glass coverslips. Cells were incubated with either 200 ng/ml LPS or medium alone for 30 min at 37°C. Cells were fixed and stained with FITC-conjugated anti-CD14, rhodamine-conjugated anti-TLR4, or both at 4°C in the dark. The fluorescence images showed that anti-CD14 and anti-TLR4 were expressed on monocytes, and that the fluorescence of both receptors was enhanced after LPS stimulation (Fig. 1). Moreover, RET emission images were only observed on cells treated with LPS, suggesting a role for LPS in promoting the physical proximity between these two molecules.

View larger version (67K): [in this window] [in a new window]   FIGURE 1. Representative fluorescence micrographs of labeled human monocytes. Cells were incubated with 200 ng/ml LPS in 10% heat-inactivated FCS (LPS+) or buffer alone (LPS-) at 37°C for 30 min. A–F, Cells were fixed and labeled with both anti-CD14 and anti-TLR4 at 4°C for 30 min. Using appropriate optical filters, CD14 (FITC) fluorescence (A and B), TLR4 (rhodamine) fluorescence (C and D), and RET (E and F) are shown. Enhanced labeling and RET are observed in the presence of LPS. G and H, After fixation, other samples were blocked with normal goat serum and stained with FITC-conjugated anti-NF-B mAbs. Substantial translocation into the nucleus is seen after 60 min at 37°C in LPS-treated cells (H), but not in control cells, which display cytoplasmic staining (G) (n = 3–8, where n = the number of independent experiments) (x1000).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Representative emission spectra of labeled monocytes are shown. A–C, Cells were incubated with either 200 ng/ml LPS or culture medium only at 37°C for 30 min. Cells were stained with FITC-conjugated anti-CD14 (donor) and/or rhodamine-conjugated anti-TLR4 (acceptor). LPS enhances TRL4 and CD14 expression as illustrated in A and B, respectively. Although RET cannot be discerned in the absence of LPS, substantial amounts of RET can be observed in the presence of LPS (C). D–F, Cells were treated with 200 ng/ml LPS at 37°C for 30 min then labeled with both reagents. After fixation, cells were stained with anti-CD14/anti-TLR4 at ratios of 1:5, 1:1, or 5:1. Spectral features are clearly dependent upon the donor/acceptor-labeling ratio of the cells (n = 4).

Although these data support an LPS-dependent physical association of CD14 and TLR4, they do not discriminate between the possibilities that LPS binding triggers 1) a lateral association of CD14 and TLR4 in the plasma membrane, and 2) the delivery of preassociated CD14 and TLR4 from an intracellular pool to the plasma membrane. To address this issue, we sought to up-regulate CD14 and TLR4 expression in the absence of LPS. Therefore, we treated cells with the calcium ionophore A23187 (2 µM, 20 min, 37°C) in HBSS containing 5 mM Ca²⁺. This procedure up-regulated CD14 and TLR4 expression to the same extent as LPS (Fig. 3, A and B). However, CD14-to-TLR4 RET was not observed in these cells (Fig. 3C, A23187 control). Thus, CD14 and TLR4 are not delivered to the plasma membrane in a preassociated form. When LPS (200 ng/ml) is incubated with A23187-treated cells, RET is again observed (Fig. 3C). Hence, the physical proximity of CD14 and TLR4 is induced at the plasma membrane.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Effect of A23187 on expression of CD14 and TLR4, and their association. Monocytes were incubated at 37°C for 15 min in the presence or absence of 2 µM A23187 then labeled with anti-CD14 and/or anti-TLR4 reagents as described above. Cells were fixed with 3.7% glutaraldyhyde. A23187 dramatically enhances the expression of both CD14 (A) and TLR4 (B) on cells. When cells were labeled with both reagents, RET was not observed (C). However, RET was detected when A23187-treated cells were incubated with LPS (C) (n = 4).

To correlate the physical association of CD14 and TLR4 with downstream elements of signal transduction, we examined NF-B translocation to the nucleus. In these experiments, monocytes were incubated with 200 ng/ml LPS or culture medium alone for 60 min at 37°C. Cells were fixed as described in Materials and Methods. After blocking with 10% normal goat serum, cells were stained with FITC-conjugated anti-NF-B mAb. As expected, LPS induced NF-B staining inside the nucleus (Fig. 1H). In contrast, there was no nuclear staining for cells exposed to HBSS alone (Fig. 1G). RET was observed at the earliest time points tested, whereas roughly 1 h was required to obtain nuclear staining for NF-B. Thus, physical proximity of CD14 and TLR4 precedes NF-B nuclear translocation.

In this study we have shown that LPS triggers close physical proximity between CD14 and TLR4. TLR4 is one member of the Toll-like receptor family. TLR4 requires CD14 to participate in the process of LPS-induced signaling, including NF-B activation (21, 24). The positive RET signal between TLR4 and CD14 indicates that the donor and acceptor chromophores are 7 nm apart; on a molecular level, this would roughly correspond to CD14 and TLR4 being nearest protein neighbors. A CD14/LPS/TLR4 complex is consistent with other reports suggesting that LPS is in close physical proximity with TLR4 during signaling, although the binding affinity of LPS for TLR4 might be low (25). We suggest a model wherein LPS induces physical proximity between CD14 and TLR4. LPS is delivered from the serum protein LPS-binding protein to membrane-bound CD14. LPS-charged CD14 has been previously shown to interact with the leukocyte integrin CD11b/CD18 before cell adherence (9); this may contribute to certain immediate responses of leukocytes to LPS. The close physical proximity of CD14 and CD11b/CD18 is lost after adherence. It now appears that LPS-charged CD14 may then promote changes in gene expression by physically interacting with TLR4. We suggest that the LPS/CD14 complex can associate with various membrane partners to elicit different aspects of the inflammatory response. The ability of CD14 to shuttle between CR3 and TLR4 is analogous to urokinase receptor (another GPI-linked protein) shuttling between CR3 and CR4 during neutrophil polarization (26, 27).

We have also shown that the up-regulation of CD14 and TLR4 are very early events in LPS stimulation of human monocytes. Although the up-regulation of CD14 has been previously reported (20, 21), the up-regulation of human TLR4 has not. However, TLR4 expression declines after LPS stimulation in murine peritoneal macrophages (28, 29). It is possible that this difference could be accounted for by species or cell type; differences in human and murine TLR4 responses have been reported (e.g., 25). However, this difference could also be accounted for by the fact that we are studying changes that take place over several minutes, whereas the down-regulation of TLR4 expression occurs over a period of hours and is temporally associated with LPS internalization and changes in TLR4 gene expression.

Although these studies have established a molecular proximity between CD14 and TLR4, they have not established the supramolecular composition of these signaling complexes. That is, there may be other components of the signaling complex. One possibility is CD11b/CD18. However, attempts to measure RET between TLR4 and CD11b/CD18 have not been successful (our unpublished data). It may be that these proteins are part of the same membrane domain or present in an array on a membrane, but too far apart to be detected with RET. However, other microscopic methods, such as fluctuation correlation spectroscopy, might contribute to further dissecting these signaling complexes within the membranes of intact living cells.

	Footnotes

 
¹ This work has been supported by National Institutes of Health Grant AI27409 (to H.R.P). Q.J. has been supported by National Institutes of Health Training Grant T-32 AI07527.

² Address correspondence and reprint requests to Dr. Howard R. Petty, Department of Biological Sciences, Wayne State University, Detroit, MI 48202.

³ Abbreviations used in this paper: TLR4, Toll-like receptor 4; RET, resonance energy transfer; TRITC, tetramethylrhodamineisothiocyanate.

Received for publication June 27, 2000. Accepted for publication July 27, 2000.

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				A. Lentschat, H. Karahashi, K. S. Michelsen, L. S. Thomas, W. Zhang, S. N. Vogel, and M. Arditi Mastoparan, a G Protein Agonist Peptide, Differentially Modulates TLR4- and TLR2-Mediated Signaling in Human Endothelial Cells and Murine Macrophages J. Immunol., April 1, 2005; 174(7): 4252 - 4261. [Abstract] [Full Text] [PDF]

				J. A. Whitsett, C. J. Bachurski, K. C. Barnes, P. A. Bunn Jr., L. M. Case, D. N. Cook, D. Crooks, M. W. Duncan, L. Dwyer-Nield, R. C. Elston, et al. Functional Genomics of Lung Disease Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2/S1): S1 - S81. [Full Text] [PDF]

				S. Epelman, D. Stack, C. Bell, E. Wong, G. G. Neely, S. Krutzik, K. Miyake, P. Kubes, L. D. Zbytnuik, L. L. Ma, et al. Different Domains of Pseudomonas aeruginosa Exoenzyme S Activate Distinct TLRs J. Immunol., August 1, 2004; 173(3): 2031 - 2040. [Abstract] [Full Text] [PDF]

				M. Komai-Koma, L. Jones, G. S. Ogg, D. Xu, and F. Y. Liew TLR2 is expressed on activated T cells as a costimulatory receptor PNAS, March 2, 2004; 101(9): 3029 - 3034. [Abstract] [Full Text] [PDF]

				J. Branger, S. Knapp, S. Weijer, J. C. Leemans, J. M. Pater, P. Speelman, S. Florquin, and T. van der Poll Role of Toll-Like Receptor 4 in Gram-Positive and Gram-Negative Pneumonia in Mice Infect. Immun., February 1, 2004; 72(2): 788 - 794. [Abstract] [Full Text] [PDF]

				M. A. Campos, G. M. S. Rosinha, I. C. Almeida, X. S. Salgueiro, B. W. Jarvis, G. A. Splitter, N. Qureshi, O. Bruna-Romero, R. T. Gazzinelli, and S. C. Oliveira Role of Toll-Like Receptor 4 in Induction of Cell-Mediated Immunity and Resistance to Brucella abortus Infection in Mice Infect. Immun., January 1, 2004; 72(1): 176 - 186. [Abstract] [Full Text] [PDF]

				J. Y. Lee, J. Ye, Z. Gao, H. S. Youn, W. H. Lee, L. Zhao, N. Sizemore, and D. H. Hwang Reciprocal Modulation of Toll-like Receptor-4 Signaling Pathways Involving MyD88 and Phosphatidylinositol 3-Kinase/AKT by Saturated and Polyunsaturated Fatty Acids J. Biol. Chem., September 26, 2003; 278(39): 37041 - 37051. [Abstract] [Full Text] [PDF]

				E. S. Van Amersfoort, T. J. C. Van Berkel, and J. Kuiper Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clin. Microbiol. Rev., July 1, 2003; 16(3): 379 - 414. [Abstract] [Full Text] [PDF]

				S. B. Mizel, A. P. West, and R. R. Hantgan Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin J. Biol. Chem., June 20, 2003; 278(26): 23624 - 23629. [Abstract] [Full Text] [PDF]

				B. Su, P. J. M. Ceponis, S. Lebel, H. Huynh, and P. M. Sherman Helicobacter pylori Activates Toll-Like Receptor 4 Expression in Gastrointestinal Epithelial Cells Infect. Immun., June 1, 2003; 71(6): 3496 - 3502. [Abstract] [Full Text] [PDF]

				A J Kvalsvig and D J Unsworth The immunopathogenesis of meningococcal disease J. Clin. Pathol., June 1, 2003; 56(6): 417 - 422. [Abstract] [Full Text] [PDF]

				V. L. Vega and A. De Maio Geldanamycin Treatment Ameliorates the Response to LPS in Murine Macrophages by Decreasing CD14 Surface Expression Mol. Biol. Cell, February 1, 2003; 14(2): 764 - 773. [Abstract] [Full Text] [PDF]

				Y. I. Miller, S. Viriyakosol, C. J. Binder, J. R. Feramisco, T. N. Kirkland, and J. L. Witztum Minimally Modified LDL Binds to CD14, Induces Macrophage Spreading via TLR4/MD-2, and Inhibits Phagocytosis of Apoptotic Cells J. Biol. Chem., January 10, 2003; 278(3): 1561 - 1568. [Abstract] [Full Text] [PDF]

				K. R. Cooke, K. Olkiewicz, N. Erickson, and J. L.M. Ferrara The role of endotoxin and the innate immune response in the pathophysiology of acute graft versus host disease Innate Immunity, December 1, 2002; 8(6): 441 - 448. [Abstract] [PDF]

H. Bosshart and M. Heinzelmann Arginine-Rich Cationic Polypeptides Amplify Lipopolysaccharide-Induced Monocyte Activation Infe