Eur. J. Biochem. 267, 37±45 (2000) q FEBS 2000

Important role of membrane-associated CD14 in the induction of IFN-b andsubsequent nitric oxide production by murine macrophages in response tobacterial lipopolysaccharide

Shinji Saito, Motohiro Matsuura, Kaoru Tominaga, Teruo Kirikae and Masayasu Nakano

Department of Microbiology, Jichi Medical School, Tochigi, Japan

The surface antigen CD14 is known to play a central role in the recognition of lipopolysaccharide by

macrophages. We characterized a mutant cell line, J7.DEF.3, derived from a murine macrophage-like cell line,

J774.1, to be defective in the ability to express the membrane-associated form of CD14 (mCD14) but not in the

ability to release the soluble form of CD14 (sCD14), and used these parent and mutant cells to investigate the role

of CD14 in lipopolysaccharide signaling. In response to lipopolysaccharide stimulation, mutant cells produced

slightly less tumor necrosis factor than parent cells, and produced much less (negligible level) nitric oxide than

parent cells. Production of both tumor necrosis factor and nitric oxide by parent cells upon lipopolysaccharide

stimulation was suppressed by anti-CD14 serum. Expression of interferon-b mRNA by stimulation with

lipopolysaccharide, detected in parent cells, was barely detectable in mutant cells and in enzymatically mCD14-

eliminated parent cells. Lipopolysaccharide-induced nitric oxide production in parent cells was suppressed by

anti-(murine interferon-b), and its production in the mutant cells appeared and increased dose dependently on

exogenously supplied murine interferon-b in the presence of lipopolysaccharide. These results provide new

insight into the lipopolysaccharide signaling pathway, indicating that the lipopolysaccharide signal for

interferon-b production is transduced through a mCD14-dependent pathway and that the endogenously generated

interferon-b is an essential cofactor leading to nitric oxide production. Nuclear translocation of a transcription

factor, nuclear factor kB, was observed in both parent and mutant cells following stimulation with a low dose of

lipopolysaccharide, and mitogen-activated protein kinases were also activated in both types of cell, although a

higher dose of lipopolysaccharide was required by the mutant cells than by the parent cells. These results indicate

that these signaling factors may participate in the mCD14-independent lipopolysaccharide signaling pathway

rather than in the mCD14-dependent interferon-b-producing pathway.

Keywords: cell-surface molecule; MAPK; monocyte/macrophage; TNF.

Bacterial lipopolysaccharide (LPS)/endotoxin, a cell-wallcomponent of gram-negative bacteria, is a potent activator ofmammalian cells such as macrophages [1,2]. In response toLPS, macrophages produce various cytokines such as tumornecrosis factor (TNF), interleukin (IL)-1, IL-6, IL-8, IL-12 andinterferon (IFN)-b, in addition to chemical mediators such asprostaglandins and nitric oxide (NO) [3]. These cytokines andmediators have pleiotropic biological activities and play crucialroles in the immune response and inflammation. A 53±55 kDacell-surface glycoprotein, CD14, has been reported to be themain LPS receptor on leukocytes [4]. LPS first binds to a

specific soluble serum protein, LPS-binding protein (LBP),which accelerates the binding of LPS to CD14 [5,6].Membrane-associated CD14 (mCD14) is anchored to the cellmembrane by glycosylphosphatidylinositol (GPtdIns) linkageand lacks transmembrane and cytoplasmic domains [7]. It istherefore considered difficult for mCD14 to transduce a signalto the cell interior by itself, and additional signal transductionmolecules are assumed to be required for transduction of theLPS signal into cells [8,9]. It has been reported that cells arealso activated by an mCD14-independent mechanism at highLPS doses [4,10]. In addition to mCD14, CD14 is found in thesera and culture supernatant of monocytes and monocytic celllines as two soluble proteins lacking the GPtdIns-anchor,sCD14a and sCD14b [7,11±14]. It has been found that severalcell types that do not express mCD14, such as epithelial cells,endothelial cells and astrocytes, respond to LPS only inthe presence of sCD14 [15,16], indicating that sCD14 canalso mediate cell activation by LPS. sCD14±LPS com-plexes stimulate not only mCD14-negative cells, but alsomCD14-positive cells, such as neutrophils and monocytes[8,17,18]. Several reports have characterized the cell-surface structures that bind to sCD14±LPS complexes[19±21], although the molecular mechanisms are unclear.Macrophages from CD14-deficient knockout mice respond tohigh concentrations of LPS [22], also indicating that LPSreceptors can respond to LPS in the absence of mCD14 and

Correspondence to S. Saito, Department of Microbiology,

Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi,

Tochigi, 329-0498, Japan. Fax: + 81 285 44 1175, Tel.: + 81 285 58 7332,

E-mail: sinsaito@jichi.ac.jp

Abbreviations: EMSA, electrophoretic mobility shift assay; ERK,

extracellular signal-regulated kinase; GPtdIns,

glycosylphosphatidylinositol; IFN, interferon; IL, interleukin; iNOS,

inducible nitric oxide synthase; LBP, LPS-binding protein; LPS,

lipopolysaccharide; MAPK, mitogen-activated protein kinase; mCD14,

membrane associated CD14; NF-kB, nuclear factor kB; NO, nitric oxide;

PtdIns-PLC, phosphatidylinositol-specific phospholipase C; PVDF,

poly(vinylidene fluoride); sCD14, soluble form CD14; TNF, tumor necrosis

factor; uPAR, urokinase-type plasminogen activator receptor.

Received 22 September 1999, accepted 20 October 1999

38 S. Saito et al. (Eur. J. Biochem. 267) q FEBS 2000

LBP. As for LPS signal transduction via mCD14-dependent andmCD14-independent pathways, it has not been clarifiedwhether there are differences between these pathways.

In the present study, we characterized a mutant J7.DEF.3 cellline [23] derived from a murine macrophage-like J774.1 cellline and found that this mutant lost the ability to expressmCD14 but not to produce and release sCD14, and wedemonstrated by using the mutant and parent cell lines that thesignal for LPS-induced NO production is transduced throughthe mCD14-dependent pathway via IFN-b production, whereasthat for LPS-induced TNF production is transduced throughthe mCD14-independent pathway. Participation of nuclearfactor-kB (NF-kB) and mitogen-activated protein kinases(MAPK) in the signal transduction of these pathways is alsodiscussed.

E X P E R I M E N T A L P R O C E D U R E S

Reagents

The LPS used was a Re chemotype from Salmonella minnesotaR595, kindly provided by K. Hisatsune (Johsai University,Sakado, Japan). The LPS dissolved in pyrogen-free water at1 mg´mL21 as a stock solution was diluted appropriately intoculture medium for experimental use. Mouse IFN-b waspurchased from Calbiochem-Novabiochem Co. (La Jolla, CA,USA). Polyclonal rabbit anti-(murine CD14) serum was kindlyprovided by S. Yamamoto (Ohoita University, Ohoita, Japan).Monoclonal antibodies against mouse IFN-b (7F-D3) andmouse CD14 (rmC5-3) were purchased from Japan Immuno-Monitoring Center Inc. (Tokyo, Japan) and Pharmingen (SanDiego, CA, USA), respectively. Polyinosinic-polycytidylicacid (polyI´polyC) and polydeoxyinosinic-deoxycytidylic acid[poly(dI±dC)] were purchased from Sigma Chemical Co. (StLouis, MO, USA) and Pharmacia LKB Biotechnology(Uppsala, Sweden), respectively. Phosphatidylinositol-specificphospholipase C (PtdIns-PLC) was purchased from BoehringerMannheim Biochemica (Mannheim, Germany). The anti-MAPK mAb was obtained from Seikagaku Co. (Tokyo,Japan) and rabbit antibodies to p38 MAPK, phospho-specificMAPK and phospho-specific p38 MAPK were all from NewEngland Biolabs, Inc. (Beverly, MA).

Cells and cell culture preparation

A mouse macrophage-like J774.1 cell line was kindly providedby T. Suzuki (University of Kansas Medical Center) and amutant J7.DEF.3 cell line of the J774.1 cell line defective inLPS binding [23] was used. As culture medium, RPMI-1640medium (ICN Biomedicals, Costa Mesa, CA, USA) supple-mented with 0.2% NaHCO3, 2 mm l-glutamine, 100 U´mL21

penicillin and 100 mg´mL21 streptomycin was used. The cellswere grown in 175-cm2 flasks (Nunc) in culture mediumsupplemented with 8% heat-inactivated fetal bovine serum at37 8C in a humidified atmosphere of 5% CO2 and 95% air.When these cells reached confluence, the culture medium andnonadherent cells were removed and replaced by fresh culturemedium with 8% fetal bovine serum. After an additional 24 hof culture, the cells were collected by vigorous pipetting,centrifuged, suspended in the culture medium with 2% fetalbovine serum and plated onto culture plates or dishes. The cellswere cultured for 3 h for complete adhesion then used forexaminations.

Immunoblot analysis of CD14

To obtain crude membrane fractions, adherent cells werewashed with NaCl/Pi containing 1 mm sodium orthovanadate,suspended in lysis buffer (1% Triton X-100, 20 mm Tris/HCl,pH 8.0, 137 mm NaCl, 10% glycerol, 1 mm Na3VO4, 2 mmEDTA, 1 mm PhCH2SO2F, 20 mm leupeptin, 0.15 U´mL21

aprotinin), and placed on ice for 20 min. Insoluble materialswere removed by centrifugation (1000 g for 10 min at 4 8C),and the supernatant fraction further centrifuged at 100 000 gfor 60 min at 4 8C. The pellets were suspended in lysis bufferand used as crude membrane fractions. To obtain concentratedculture supernatants, adherent cells were cultured in freshculture medium without fetal bovine serum. After an additional16 h of culture, the culture supernatants were collected,concentrated to 1/20 vol. using a Centricon 10 concentrator(Amicon, Inc., Beverly, MA, USA), and used as concentratedculture supernatant. Ten micrograms protein of the crudemembrane and 1 mg protein of the concentrated culturesupernatant were separated on 10% SDS/PAGE [24], trans-ferred to poly(vinylidene fluoride) (PVDF) membrane(Immobilon, Millipore, Bedford, MA, USA), and detectedCD14 with anti-(murine CD14) mAb and ECL Western blottingdetection reagent (Amersham, Buckinghamshire, UK).

Native PAGE assay

Binding of LPS to sCD14 released from J774.1 or J7.DEF.3cells was assessed basically according to a previously describednative PAGE assay [6,25]. The concentrated supernatants(described above) containing 1 mg protein were mixed with1 mg LPS, incubated at room temperature for 90 min andseparated on 10% native PAGE. Proteins on the gel weretransferred to a PVDF membrane and immunoblot analysis ofCD14 performed as described above.

Northern blot analysis

Total RNA (15 mg) isolated from cells using the acidguanidinium thiocyanate±phenol±chloroform method [26]was electrophoresed on 1% agarose gel containing 2.2 mformaldehyde, transferred to a nylon membrane (Hybond-N+;Amersham), fixed using the alkali fixation method andhybridized with probes for detection of specific mRNAs. Theprobes used were cDNAs of murine CD14, murine b-actin,murine TNF-a (a gift from S. Natori, University of Tokyo),murine inducible nitric oxide synthase (iNOS; a gift from Y.Ogiso, National Institute of Radiological Science, Chiba,Japan) and murine d-glyceraldehyde 3-phosphate dehydro-genase. The probes were labeled using the random primermethod with [a-32P]dCTP. Hybridization was performed by therapid hybridization system (Amersham) according to theinstruction manual. Results were read with a Bio-imaginganalyzer BAS 2000 (Fuji Photo Film, Co. Ltd, Tokyo, Japan).

TNF assay

The activity of TNF in the test samples was measured bycytolytic activity to actinomycin D-treated L929 cells [27]. Theviability of the cells was determined by quantitative colori-metric staining with crystal violet. TNF activity (U´mL21) wascalculated from the dilution factor of test samples necessary tocause 50% cell lysis, with collection by an internal standard ofa recombinant human TNF-a in each assay.

q FEBS 2000 Specific role of mCD14 in LPS signaling (Eur. J. Biochem. 267) 39

NO assay

NO production in the test sample supernatants was assessed bymeasuring nitrite, a stable end product of NO, using colori-metric assay with Griess reagent [28]. Briefly, 50 mL testsamples in 96-well plates were mixed with equal volumes ofGriess reagent (1% sulfanilamide and 0.1% N-1-naphthylethyl-endiamine dihydrochloride in 2.5% H3PO4 solution) and left atroom temperature for 10 min. The A540 value was measuredusing an ELISA reader, and the nitrite concentration wasquantified (in mm) from the standard curve of sodium nitritesolution in each assay.

Rt-pcr

Total RNA was isolated from the cells as described above.cDNAs were synthesized from 1 mg of the total RNAs bypriming with 200 ng of random hexamer, 1.25 mm dNTP and60 U of SuperScript II reverse transcriptase (Gibco BRL LifeTechnologies, Inc., Gaithersburg, MD, USA) at 37 8C for 1 h.The sequences of the primers used in RT-PCR for mouse IFN-bwere 5 0-CTCCAGCTCCAAGAAAGGACG-3 0 and 5 0-GAAG-TTTCTGGTAAGTCTTCG-3 0, and those for mouse b-actinwere 5 0-GTGGGCCGCTCTAGGCACCA-3 0 and 5 0-CGG-TTGGCCTTAGGGTTCAGGGGGG-3 0. PCR was carried outat 94 8C for 1 min, 55 8C for 1 min and 72 8C for 2 min as onecycle. Thirty-five cycles were performed. The PCR productswere analyzed after electrophoresis on 1% agarose gel byethidium bromide staining.

Preparation of nuclear extract and electrophoretic mobilityshift assay (EMSA) to detect NF-kB translocation

Cells were incubated with various amounts of LPS for 1 h,washed, harvested and resuspended by vortexing for 10 s inhypotonic buffer A (10 mm Hepes at pH 7.8, 10 mm KCl,0.1 mm EDTA, 0.5% NP-40, 1 mm dithiothreitol, 0.5 mmPhCH2SO2F, 5 mg´mL21 aprotinin, 5 mg´mL21 pepstatin and5 mg´mL21 leupeptin). The nuclei in the suspension wereseparated from the cytosol by microcentrifugation at 1600 g for1 min, resuspended in buffer C (50 mm Hepes at pH 7.8,0.42 m KCl, 0.1 mm EDTA, 5 mm MgCl2, 20% glycerol, 1 mmdithiothreitol, 0.5 mm PhCH2SO2F, 5 mg´mL21 aprotinin,5 mg´mL21 pepstatin and 5 mg´mL21 leupeptin) and kept onice for 30 min while being vortexed occassionally. Nuclearextracts were obtained from the suspension by microcentrifu-gation at 15 000 g for 15 min [29]. Translocated NF-kB innuclear extracts was detected using an oligonucleotide contain-ing mouse IL-6 NF-kB-binding sequence as a probe [30]. Thesequence was as follows: 5 0-AGCTTAAATGTGGGATTTTCC-CATGAGA-3 0. The oligonucleotide was annealed then labeledwith [a-32P]dCTP. For the binding reaction of NF-kB to theprobe, 20 mL reaction mixtures containing nuclear extracts(5 mg protein), 10 mm Hepes (pH 7.8), 50 mm KCl, 1 mmEDTA, 5 mm MgCl2, 10% glycerol, 5 mm dithiothreitol,0.7 mm PhCH2SO2F and 2 mg´mL21 of poly(dI±dC) with orwithout a competitor cold oligonucleotide (�100) were lefton ice for 10 min. A radiolabeled oligonucleotide probe(20 000 c.p.m.) was then added and the mixture maintainedat room temperature for 30 min. The samples were subjected toelectrophoresis on a 5% polyacrylamide gel with 0.25 � TBEbuffer (22.5 mm Tris, 22.2 mm borate and 0.5 mm EDTA) andanalyzed by autoradiography.

Immunoblot analysis of ERK1, ERK2 and p38 MAPK

Cells stimulated with or without LPS were lysed in lysis buffer(1% Triton X-100, 20 mm Tris/HCl at pH 7.5, 120 mm NaCl,10% glycerol, 1 mm Na3VO4, 2 mm EDTA, 1 mm PhCH2SO2F,20 mm leupeptin, 0.15 U´mL21 aprotinin and 5 mg´mL21

pepstatin) by keeping on ice for 20 min. The Triton X-100-soluble protein was collected by microcentrifugation at15 000 g for 10 min at 4 8C [31]. The solubilized protein(10 mg) was separated on 10% SDS/PAGE and transferred to aPVDF membrane. MAPKs (ERK1, ERK2 and p38 MAPK)were analyzed for whole proteins and phosphorylated proteinsusing antibodies against MAPKs and their phosphorylatedforms, respectively, using ECL Western blotting detectionreagent (Amersham).

R E S U L T S

Lack of mCD14 in J7.DEF.3 cells that preserve the ability torelease sCD14

The mutant cell line J7.DEF.3 was isolated from J774.1 cellsbased on a lack of specific LPS binding in the presence ofserum [23]. The available data indicate that, in these mutantcells, the defect is a lack of CD14 expression on the cell surface[32]. To confirm and further characterize the lack of CD14expression in the mutant cells, mRNA and protein expressionsof CD14 were analyzed. As shown in Fig. 1A, CD14 mRNA

Fig. 1. Biochemical characterization of CD14 expression in mutant

J7.DEF.3 and parental J774.1 cells. (A) Total RNAs extracted from J774.1

(J7) and J7.DEF.3 (DEF.3) cells were examined for CD14 mRNA using

Northern blot analysis. Total RNA from L929 cells (CD14 negative) was

used as a negative control. Murine b-actin mRNA was determined as the

control of constitutively expressed mRNA. (B) CD14 on cell membranes

and in culture supernatants was detected by immunoblot analysis. Crude

membrane fraction (membrane fr.) and concentrated culture supernatant

(culture sup.) were prepared as described in Experimental procedures.

Membrane fraction containing 10 mg protein and culture supernatant

containing 1 mg protein prepared from J774.1 and J7.DEF.3 cells were

subjected to 10% SDS/PAGE and transferred to a PVDF membrane. CD14

was detected with anti-(murine CD14) mAb and an enhanced chemilumi-

nescence system. (C) Binding of LPS to sCD14 released into culture

supernatant was detected by a native-PAGE assay. The culture supernatant

(J7 sup. or DEF.3 sup.) containing 1 mg protein was incubated with (+) or

without (±) 1 mg of LPS for 90 min at room temperature. After incubation,

the mixture was electrophoresed on 10% native-PAGE and transferred to a

PVDF membrane. The electromobility shift of sCD14 was detected by anti-

CD14 mAb. The position of CD14 alone or the LPS±CD14 complex is

indicated by an arrow. Similar results were obtained in two separate

experiments. M, medium control.

40 S. Saito et al. (Eur. J. Biochem. 267) q FEBS 2000

was expressed in both mutant and parent cells. As for CD14proteins, they were detected in both the crude membranefraction and the culture supernatant of parent J774.1 cells at< 55 kDa, whereas they were detected only in the culturesupernatant of mutant J7.DEF.3 cells and not in the crudemembrane fraction (Fig. 1B). The CD14 molecules in theculture supernatants are considered to be produced by the cellsbecause they were cultured in the absence of fetal bovineserum, which might contain sCD14. We then analyzed theLPS-binding capacity of the CD14 molecules using a nativePAGE assay [6,25]. As shown in Fig. 1C, CD14 in thesupernatants of both J774.1 and J7.DEF.3 cells shifted theirelectrophoretic mobility by the addition of LPS. This resultindicates that the CD14 binds to LPS as described previously[6,25]. Taken together, J7.DEF.3 cells are characterized as cellsthat lose the ability to produce mCD14 but not the ability toproduce and release sCD14 possessing LPS binding capacity.

Production of TNF and NO by J774.1 and J7.DEF.3 cells inresponse to LPS

J774.1 and J7.DEF.3 cells were cultured for 3 h in culturemedium with 2% fetal bovine serum for completeadhesion, and LPS was then added at final concentrationsof 0.1±100 ng´mL21 for cell activation. The TNF activity andnitrite concentration in the culture supernatants were deter-mined. J774.1 cells responded dose dependently to LPS andproduced TNF (Fig. 2A) and NO (Fig. 2B) quite well. J7.DEF.3cells also responded to LPS for TNF production, althoughsomewhat more weakly than the parent cells (Fig. 2A),however, J7.DEF.3 cells hardly responded to LPS for NOproduction (Fig. 2B). As reported previously [23], J7.DEF.3cells produced NO upon LPS stimulation when the cells werestimulated immediately with LPS just after cell preparation(immediate LPS stimulation) without waiting for 3 h tocomplete cell adhesion. Production of NO by the parentJ774.1 cells was also enhanced upon immediate LPS stimu-lation compared with later LPS stimulation (after the cells werestably adhered), and the enhanced amount of NO wascomparable with or higher than that produced by the mutantcells upon immediate LPS stimulation (data not shown). Theseresults suggest the existence of a NO-producing mechanismwhich functions transiently upon LPS stimulation in both the

parent and mutant cells before cell adhesion is completed. ThismCD14-independent mechanism is currently under investi-gation. Here, we carried out the experiments using stablyadhered cells, which are generally used for macrophageexperiments, and investigated the role of CD14 in LPSsignaling pathways.

Northern blot analysis of TNF-a and iNOS mRNA expres-sion in LPS-stimulated J774.1 and J7.DEF.3 cells wasperformed (Fig. 3). TNF-a mRNA expression upon LPSstimulation in both cell types was similar in both quantityand time kinetics. Expression of iNOS mRNA upon LPSstimulation could not be detected at any time in the mutantcells, while significant expression was observed at 8±16 h inthe parent cells. The reduced LPS responsiveness of the mutantcells for NO production was shown to occur in the mRNAexpression step. These results indicate that the mutant cells stillhave active pathways for the transduction of some LPS signalswhich do not require the expression of mCD14.

Suppression of LPS-induced production of TNF and NO byanti-CD14 serum

Production of TNF and NO by the parent J774.1 cells wasobserved by stimulating the cells with low LPS concentrations,

Fig. 2. LPS-induced production of TNF and NO by J774.1 and

J7.DEF.3 cells. J774.1 (X) and J7.DEF.3 (W) cells were stimulated with

various concentrations of LPS in the presence of 2% fetal bovine serum. (A)

Culture supernatants were collected 4 h after stimulation and TNF activity

was assessed by L929 cytotoxic assay. (B) To assess NO production, culture

supernatants were collected 48 h after stimulation. NO production was

measured as the stable end product nitrite in the culture supernatants by

Griess reagent. Data points are the mean of triplicate samples ^ SD.

Results of one representative experiment from four are shown.

Fig. 3. Northern blot analysis of LPS-induced expression of iNOS-

specific or TNF-a-specific mRNA in J774.1 (J7) and J7.DEF.3 (DEF.3)

cells. J774.1 and J7.DEF.3 cells (1 � 107 cells) were exposed to LPS

(10 ng´mL21) for 0±16 h. Total RNAs (15 mg per lane) extracted from

cells at the indicated times were electrophoresed on a 1% agarose gel,

transferred to a Hybond-N+ membrane and hybridized sequentially with32P-labeled cDNAs specific to mRNAs for iNOS (top), TNF-a (middle) and

glyceraldehydephosphate dehydrogenase: d-glyceraldehyde 3-phosphate

dehydrogenase (bottom). Three additional experiments gave similar results.

Fig. 4. Suppressive effect of anti-CD14 serum on LPS-induced produc-

tion of TNF and NO by J774.1 cells. J774.1 cells were pre-incubated with

various concentrations of rabbit anti-(murine CD14) polyclonal serum

(IgG) (W) or rabbit normal IgG (X) for 1 h, and then stimulated with

10 ng´mL21 LPS in the presence of 2% fetal bovine serum. Production of

TNF (A) and NO (B) in the culture supernatant were measured as described

in Fig. 2. Data points are the mean of triplicate samples ^ SD. Results of

one representative experiment from four are shown.

q FEBS 2000 Specific role of mCD14 in LPS signaling (Eur. J. Biochem. 267) 41

e.g. 0.1±100 ng´mL21 in the presence of fetal bovine serum (asshown in Fig. 2). It has been suggested that such a response ofmonocytes and macrophages to low concentrations of LPS islargely CD14 dependent [4,33,34]. To clarify the participationof CD14 in the LPS-induced TNF and NO production by J774.1cells, the inhibitory effect of anti-CD14 serum on productionwas examined. J774.1 cells were stimulated with 10 ng´mL21

LPS in the presence of various concentrations of rabbitanti-(murine CD14) polyclonal serum or normal rabbit IgG.Anti-CD14 serum, but not the control IgG, suppressed theLPS-induced production of both TNF and NO by J774.1 cells ina dose-dependent manner (Fig. 4). These results, together withthose presented above (Figs 1±3), indicate that CD14 plays acritical role in the signaling pathways for the LPS-inducedproduction of TNF and NO, and that mCD14 is indispensablefor NO production but not for TNF production, in which sCD14probably plays a role complementary to mCD14.

Participation of IFN-b as an essential cofactor forLPS-induced NO production

It has been suggested that the production of NO fromLPS-stimulated macrophages is modulated by IFN-b, and thatIFN-b is a necessary cofactor for the transduction of the LPSsignal through a pathway leading to NO production [35±37].Therefore, we examined the IFN-b-producing activities of theparent and mutant cells upon LPS stimulation. IFN-b mRNAexpression in LPS-stimulated cells was analyzed by RT-PCR.As shown in Fig. 5, IFN-b mRNA was expressed strongly inJ774.1 cells in response to LPS at doses of 10±1000 ng´mL21

and to (polyI´polyC) at 100 mg´mL21. In the mutant J7.DEF.3cells, however, only weak expression of IFN-b mRNA wasdetected even at the highest LPS dose (1000 ng´mL21), whilesignificant expression was detected in response to (polyI´polyC). The inhibitory effect of anti-IFN-b mAb on NOproduction by LPS-stimulated J774.1 cells was then examined.Dose-dependent inhibition of anti-IFN-b mAb against NOproduction was observed (Fig. 6A). In the following experi-ment, the effect of exogenously supplied IFN-b on NOproduction by LPS-stimulated J7.DEF.3 cells was examined.As shown in Fig. 6B, NO was produced dose dependently onIFN-b supplied in the presence of LPS but was not produced inthe absence of LPS. These results strongly indicate that IFN-bis an essential cofactor for LPS-induced NO production in thesecells and that the inability of J7.DEF.3 cells to produce NO

upon LPS stimulation is due to the lack of signaling for theproduction of IFN-b through mCD14.

Reduction in LPS-induced IFN-b production by J774.1 cellsafter the enzymatic elimination of mCD14

To eliminate mCD14 from cell surfaces, J774.1 cells werepretreated with PtdIns-PLC and then stimulated with LPS.Expression of IFN-b mRNA was assessed by RT-PCR. Asshown in Fig. 7, the LPS-induced mRNA expression of IFN-bin J774.1 cells disappeared after PtdIns-PLC treatment, and inJ7.DEF.3 cells it was negligible independent of LPS stimulationand PtdIns-PLC treatment. Although PtdIns-PLC treatmentof the cells also eliminates GPtdIns-anchored proteins, withthe exception of mCD14, participation of such proteins inLPS-induced cell activation has not been indicated. Strongexpression of TNF-a mRNA upon LPS stimulation in bothtypes of cell (Fig. 3) was not affected by PtdIns-PLC treatment(data not shown), indicating that this treatment barelyinfluences LPS signals independent of mCD14. These resultsindicate that enzymatic elimination of mCD14 (and the otherGPtdIns-anchored proteins) from J774.1 cells makes thembehave like J7.DEF.3 cells in response to LPS and confirms thepossibility that expression of mCD14 is important formacrophages to produce IFN-b upon LPS stimulation.

Fig. 5. RT-PCR analysis of LPS-induced expression of IFN-b

mRNA. J774.1 (J7) and J7.DEF.3 (DEF.3) cells were exposed to

LPS (0±1000 ng´mL21) or (polyI´polyC) (100 mg´mL21) for 2 h. Total

RNAs extracted from stimulated cells were reverse-transcribed. The

reverse-transcribed products were subjected to 35-cycle PCR using sense

upstream and antisense downstream primers specific for murine IFN-b or

b-actin. PCR products were subjected to agarose gel electrophoresis and

visualized by ethidium bromide staining. Similar results were obtained in

two separate experiments.

Fig. 6. Participation of IFN-b in NO production by LPS-stimulated

J774.1 or J7.DEF.3 cells. (A) J774.1 cells were pre-incubated with various

concentrations of anti-(murine IFN-b) mAb (X) or control normal rat IgG

(W) for 1 h, and stimulated with LPS (10 ng´mL21) for a further 48 h.

Neutralizing activity of anti-(murine IFN-b) mAb is 4 � 105 NU´mg21.

(B) J7.DEF.3 cells were stimulated with (B) or without (A) 10 ng´mL21

LPS in the presence of the indicated concentrations of mouse natural IFN-b

for 48 h. NO was measured as nitrite in the culture supernatants. Data

points are the mean of triplicate samples ^ SD. Representative data of three

separate experiments are shown.

Fig. 7. Effect of PtdIns-PLC treatment on LPS-stimulated expression

of IFN-b mRNA. J774.1 (J7) and J7.DEF.3 (DEF.3) cells were pre-

incubated with (+) or without (±) 1 U´mL21 PtdIns-PLC for 90 min and

stimulated with (+) or without (±) 10 ng´mL21 LPS for a further 2 h. Total

RNAs extracted from the cells were analyzed for mRNA expression of IFN-

b and control b-actin by RT-PCR as described in the legend to Fig. 5.

Similar results were obtained in two separate experiments.

42 S. Saito et al. (Eur. J. Biochem. 267) q FEBS 2000

LPS-induced NF-kB activation in the parent and mutant cells

Translocation of NF-kB, a transcription factor, from the cytosolto the nucleus is known to be the result of activation of thisfactor, and occurs during the process of LPS-induced macro-phage activation. Translocation of NF-kB upon LPS stimu-lation in J774.1 cells was compared with that in J7.DEF.3 cells.Nuclear extracts were prepared from cells stimulated with LPSfor 1 h and subjected to EMSA. As shown in Fig. 8, a LPSconcentration as low as 0.1 ng´mL21 was sufficient to activateNF-kB in both the parent and mutant cells. As shown in manyother reports, two bands of NF-kB members were also detectedin our experiments. These two bands corresponded to a p50/p50homodimer and a p50/p65 (or Rel A) heterodimer of NF-kBmembers, judging from data reported previously [29] and ourpreliminary data obtained with specific antibodies (data notshown). The intensities of NF-kB bands, estimated by the ratioof the densities of stimulated samples to that of a controlunstimulated sample, in both the parent and mutant cells uponstimulation with LPS at concentrations over 0.1 ng´mL21, werein the range 3.0±3.8. No clear differences in the intensitieswere observed by changing the LPS doses or cell lines from the

parent to the mutant. These results indicate that expression ofmCD14 is not necessary for LPS-induced NF-kB activation.

LPS-induced protein phosphorylation of MAPKs in parent andmutant cells

Tyrosine phosphorylation of several members of the MAPKfamily, including ERK1, ERK2 and p38 MAPK, is known toplay an important role in LPS signaling in macrophages.The behavior of tyrosine phosphorylations of ERK1, ERK2and p38 upon LPS stimulation in J774.1 and J7.DEF.3cells was investigated by immunoblot analysis. As shownin Fig. 9A, tyrosine phosphorylated ERK1, ERK2 and p38MAPKs were detected in J774.1 cells upon stimulationwith 1±1000 ng´mL21 LPS, and in J7.DEF.3 cells uponstimulation with higher doses ($ 100 ng´mL21) of LPS. Thetime kinetics for the phosphorylation of three MAPKs, ERK1,ERK2 and p38, upon stimulation with LPS (100 ng´mL21) areshown in Fig. 9B. Similar time kinetics for the phosphorylationwere seen in both types of cell. The phosphorylation of all threeMAPKs was maximal at 15 min, and decreased thereafter.These results suggest that activation of MAPKs upon LPSstimulation in macrophages is dependent only partly, if at all,on the expression of mCD14.

D I S C U S S I O N

J7.DEF.3 cells were isolated from J774.1 cells based on a lackof specific LPS binding in the presence of serum [23] andinsufficient expression of CD14 on their cell surface wassuggested [32]. More detailed biochemical characterization ofCD14 expression on J7.DEF.3 and parent J774.1 cellsperformed in this study revealed that J7.DEF.3 cells lackedmCD14 production while CD14 mRNA expression and sCD14production were preserved (Fig. 1A,B). sCD14 molecules fromboth parent and mutant cells were detected as a single bandwith a relative molecular mass (Mr) of < 55 000, which was

Fig. 8. LPS-induced NF-kB translocation in J774.1 and J7.DEF.3 cells.

J774.1 (J7) and J7.DEF.3 (DEF.3) cells were stimulated with various

concentrations (0±10 ng´mL21) of LPS for 1 h. The DNA-binding

activities of NF-kB in nuclear extracts of the cells were detected by

EMSA using a radiolabeled oligonucleotide containing the sequence of kB

site on murine IL-6 gene as a probe. Similar results were obtained in three

separate experiments. C, competitor cold oligonucleotide to the probe

(100-fold excess to hot probe).

Fig. 9. Analysis of LPS-induced MAPK activation in J774.1 and J7.DEF.3 cells by immunoblot using specific antibodies against phosphorylated

MAPKs. J774.1 (J7) and J7.DEF.3 (DEF.3) cells were stimulated with indicated concentrations of LPS (0±1000 ng´mL21) for 15 min (A) or stimulated with

100 ng´mL21 LPS for the indicated times (0±60 min) (B). After the stimulation, cell lysates (containing 20 mg protein) were prepared and

subjected to SDS/PAGE. Separated proteins on the gel were transferred to a PVDF membrane and analyzed by immunoblotting using antibodies against

MAPKs as indicated in the left-hand side of the panels. Phosphorylated forms of ERKs were detected by anti-(phospho-MAPK) serum (top row; Anti-

pMAPK) and whole ERKs by anti-MAPK serum (second row; Anti-MAPK). Phosphorylated and whole p38 were detected by anti-(phosphorylated-p38)

serum (third row; Anti-pp38) and anti-p38 serum (bottom row; Anti-p38), respectively. The positions of ERK1, ERK2 and p38 are indicated by arrows.

Similar results were obtained in three separate experiments.

q FEBS 2000 Specific role of mCD14 in LPS signaling (Eur. J. Biochem. 267) 43

similar to that for mCD14. In human monocytic cell cultures, ithas been reported that two different forms of sCD14 with Mr

values of 48 000 and 56 000 are released into the culturesupernatant [12±14]. It has been suggested that the low-Mr

form is derived from mCD14 by limited proteolysis, whereasthe high-Mr form is secreted directly by escaping theattachment of the GPtdIns-anchor, which gives membraneassociation ability to the CD14 protein. In the murinemacrophage cell cultures used in this study, the high-Mr formof sCD14 was detected but the low Mr form was not. Murinemonocytic cells may not have the protease-dependent mechan-isms necessary to produce the low Mr form of sCD14, unlikehuman monocytic cells, although further analysis of sCD14 inculture supernatant from diverse types of murine monocyticcells is required to confirm this. The amount of sCD14 in theculture supernatant of the mutant J7.DEF.3 cells was observedto increase somewhat from that of the parent J774.1 cells(Fig. 1B). It is possible that the mutant cells are defectiveat some point in the process before attachment of theGPtdIns-anchor to the CD14 protein, leading to theproduction of a high-Mr sCD14 identical or similar tothat of the constitutive sCD14 produced by both the parent andmutant cells.

Recently, CD14-deficient knockout (CD14KO) mice havebeen established [38] and the LPS-responsivenesses of macro-phages from the CD14KO mice has been reported [22]. Incontrast to mutant J7.DEF.3 cells, which preserve the ability toproduce sCD14, the CD14KO macrophages did not produceeither mCD14 or sCD14. They induced barely detectable levelsof mRNA, as well as TNF-a protein in response to lowconcentrations of LPS (1 and 10 ng´mL21) in the absence ofsCD14. It was also noted that the CD14KO macrophages couldrespond to sCD14±LPS complexes in the absence of mCD14.In the human monocytic cell line Mono-Mac-6 cells, it has beendemonstrated that an sCD14 purified from the urine ofnephrotic patients enhanced the CD14-mediated release ofTNF-a upon LPS stimulation [12]. In the present study,J7.DEF.3 cells released sCD14 possessing LPS-binding ability(Fig. 1C) and responded to LPS at low concentrations of0.1 ng´mL21 for the production of TNF (Fig. 2A), suggestingthat the response of these mutant cells is mediated by ansCD14-dependent LPS signal. The somewhat stronger responsefor LPS-induced TNF production observed in the parentJ774.1 cells may be due to the additional effects of themCD14-dependent LPS signal. Using the anti-(murine CD14)polyclonal serum, which suppressed LPS-induced TNF pro-duction by the parent cells (Fig. 4A), we tried to suppressLPS-induced TNF production by the mutant cells, however,suppression of TNF production was not clearly observed. Thisresult does not rule out the possibility of the above-mentionedsCD14-dependent LPS signal. The amount of sCD14 releasedfrom the mutant J7.DEF.3 cells (Fig. 1B) may be too much tobe neutralized by the anti-CD14 serum, or this antibody mayhave weak or no activity to neutralize the action of sCD14while having strong activity to mCD14. In fact, an anti-(humanCD14) serum, MY4, is frequently used to neutralize the actionof mCD14, while this antibody has been reported to be inactivefor the inhibition of LPS binding to sCD14 [39]. At present, wehave no direct evidence to indicate that LPS-induced TNFproduction by the mutant cells is dependent on sCD14,although we think that this mechanism is highly possible.Strong inhibitors against murine sCD14, including antibodies,are required to solve this problem.

Using the adherent cells from J774.1 and J7.DEF.3 cell lines,different LPS-induced NO production between the parent and

mutant cells was demonstrated (Figs 2B and 3). Unlike theabove-mentioned LPS-induced TNF production, NO pro-duction was shown to require mCD14, but not sCD14, as anessential factor. It has been indicated that endogenous IFN-bproduced by LPS-stimulated murine macrophages acts as anessential cofactor for LPS-induced NO production [35±37].The role of IFN-b in LPS-induced NO production wasconfirmed in the present experiment, which showed sup-pression of NO production by anti-IFN-b serum (Fig. 6A).Furthermore, the necessity of mCD14 expression for LPS-induced IFN-b production (Figs 5 and 7) was demonstrated forthe first time in the present study. NO production could beinduced even by mCD14-negative mutant cells when IFN-bwas added exogenously with LPS to stimulate the cells(Fig. 6B). This means that once IFN-b is produced, anmCD14-dependent signal is not necessary for NO productionand the dependence of LPS-induced NO production on mCD14is due to mCD14 dependence upon LPS stimulation in theprocess of IFN-b production. For the production of NO bymutant cells, the addition of IFN-b alone was not sufficient andthe additional presence of LPS was required (Fig. 6B). Thisindicates that signals from both LPS and IFN-b are necessaryfor the production of NO and that the signal from LPS maytransduce without mCD14 if IFN-b is present. Furthermore,iNOS was detected in the parent cells in response to LPS in thepresence of fetal bovine serum, but not in its absence (data notshown), indicating that some serum factor(s) such as LBP isalso required for LPS-induced NO production.

The mutation in J7.DEF.3 cells is probably in the machinerythat attaches GPtdIns tails to CD14 and may not be CD14specific. PtdIns-PLC treatment of J774.1 cells nonspecificallyremoves GPtdIns-anchored proteins, such as CD16b, CD55,CD59, urokinase-type plasminogen activator receptor (uPAR)and mCD14. Therefore, we cannot exclude the possibility thatGPtdIns-anchored proteins other than mCD14 act as somecomponents of the IFN-b production machinery. It has beenindicated that most of these GPtdIns-anchored proteinsassociate with b2 integrins and regulate the integrin functionof leukocytes by transducing similar signals [40]. Participationof these GPtdIns-anchored proteins, with the exception ofmCD14, in macrophage activation by LPS has, however, notbeen demonstrated, indicating the possibility that these proteinsplay some role in the LPS-induced IFN-b-production pathway.Recently, several reports have indicated that Toll-like receptor(TLR)2 and TLR4 act as signal transducing receptors for LPS[41±43]. Participation of these receptors in the IFN-b-production pathway is an interesting subject that remains tobe elucidated.

NF-kB is an important transcription factor for immuneand inflammatory responses, and it plays a central role inthe production of cytokines, including TNF-a and IL-6, byLPS-stimulated macrophages [44±47]. In the present study, thenuclear translocation of NF-kB was seen in both parent andmutant cells by stimulation with LPS at a dose as low as0.1 ng´mL21 (Fig. 8). This indicates that NF-kB is activatedsensitively to LPS stimulation without mCD14 as in the case ofTNF production (Fig. 2A). Participation of NF-kB in the LPSsignaling pathway for TNF production is considered highlypossible.

As for LPS-induced NO production in macrophages,expression of the iNOS gene is required. Several binding sitesfor transcription factors in the promoter region of the iNOSgene have been implicated in iNOS regulation [48,49], andseveral transcription factors, such as NF-kB, IRF-1, Oct-1 andStat1a, have been shown to interact with the promoter region. It

44 S. Saito et al. (Eur. J. Biochem. 267) q FEBS 2000

has been reported that nuclear translocation of NF-kB forbinding to the kB site located downstream of the promoterregion is essential but not sufficient for expression of the iNOSgene [50]. Another important factor, IRF-1, is known to benecessary for iNOS transcription [51,52]. Recently, Gao et al.[37] demonstrated that the activation of Stat1a by LPS-inducedIFN-ab is critical for the induction of murine iNOS geneexpression. Here, nuclear translocation of NF-kB in mutantcells was detected upon stimulation with LPS, whereas NO wasnot produced. Transcription factors required for expression ofthe iNOS gene in addition to NF-kB, such as IRF-1 andStat1a, may not be activated upon LPS stimulation in themutant cells.

Signaling pathways through MAPK family cascades are alsoindicated to play important roles in macrophage activation byLPS [2,3]. MAPKs in the mutant cells were still activated inresponse to LPS with time kinetics similar to those in the parentcell, although they required higher LPS doses than did theparent cells (Fig. 9). These findings suggest that MAPKs do notparticipate directly in the induction pathways for both IFN-band NO via mCD14. Considering the somewhat lower ability ofthe mutant cells in LPS-induced TNF production comparedwith the parent cells (Fig. 2), activation of MAPKs mayparticipate in the signal transduction for TNF production.

Here we demonstrated for the first time that the LPSsignal for the production of IFN-b is transduced through amCD14-dependent pathway in murine macrophages andthat this endogenously induced IFN-b is an essential cofactorfor LPS-induced NO production, indicating the dependence ofNO production on mCD14. These findings provide new insightinto the role of mCD14 in the LPS signaling pathway.

A C K N O W L E D G E M E N T S

We thank Dr Yoichi Ogiso, Dr Syunji Natori and Dr Syunsuke Yamamoto

for supplying the murine iNOS cDNA probe, the murine TNF-a cDNA

probe and rabbit anti-(murine CD14) polyclonal serum, respectively. This

work was supported in part by grants 08770197 (S. S.) and 09670295

(M. M.) from the Ministry of Education, Science and Culture of Japan.

R E F E R E N C E S

1. Raetz, C.R.H., Ulevitch, R.J., Wright, S.D., Sibley, C.H., Ding, A. &

Nathan, C.F. (1991) Gram-negative endotoxin: an extraordinary lipid

with profound effects on eukaryotic signal transduction. FASEB J. 5,

2652±2660.

2. Ulevitch, R.J. & Tobias, P.S. (1995) Receptor-dependent mechanisms

of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13,

437±457.

3. Sweet, M.J. & Hume, D.A. (1996) Endotoxin signal transduction in

macrophages. J. Leukoc. Biol. 60, 8±26.

4. Wright, S.D., Romos, R.A., Tobias, P.S., Ulevitch, R.J. & Mathison,

J.C. (1990) CD14, a receptor for complexes of lipopolysaccharide

(LPS) and LPS binding protein. Science 249, 1431±1433.

5. Schumann, R.R., Leong, S.R., Flaggs, G.W., Gray, P.W., Wright, S.D.,

Mathison, J.C., Tobias, P.S. & Ulevitch, R.J. (1990) Structure

and function of lipopolysaccharide binding protein. Science 249,

1429±1431.

6. Hailman, E., Lichfnstein, H.S., Wurfel, M.M., Miller, D.S., Johnson,

D.A., Kelley, M., Busse, L.A., Zukowski, M.M. & Wright, S.D.

(1994) Lipopolysaccharide (LPS)-binding protein accelerates the

binding of LPS to CD14. J. Exp. Med. 179, 269±277.

7. Haziot, A., Chen, S., Ferrero, E., Low, M.G., Silber, R. & Goyert, S.M.

(1988) The monocyte differentiation Ag CD14 is anchored to the

cell membrane by a phosphatidylinositol linkage. J. Immunol.

141, 547±552.

8. Lee, J.-D., Kravchenko, V., Kirkland, T.N., Han, J., Mackman, N.,

Moriarty, A., Leturcq, D., Tobias, P.S. & Ulevitch, R.J. (1993)

Glycosyl-phosphatidylinositol-anchored or integral membrane forms

of CD14 mediate identical cellular response to endotoxin. Proc. Natl

Acad. Sci. USA 90, 9930±9934.

9. Weinstein, S.L., June, C.H. & Defranco, A.L. (1993) Lipopolysacchar-

ide-induced protein tyrosine phosphorylation in human macrophages

is mediated by CD14. J. Immunol. 151, 3829±3838.

10. Beaty, C.D., Franklin, T.L., Uehara, Y. & Wilson, C.B. (1994)

Lipopolysaccharide-induced cytokine production in human mono-

cytes: role of tyrosine phosphorylation in transmembrane signal

transduction. Eur. J. Immunol. 24, 1278±1284.

11. Bazil, V., Horejsi, V., Baudys, M., Kristofova, H., Strominger, J.,

Kostka, W. & Hilgert, I. (1986) Biochemical characterization of a

soluble form of 53-kDa monocyte surface Ag. Eur. J. Immunol. 16,

1583±1589.

12. Labeta, M.O., Durieux, J.-J., Fernandez, N., Herrmann, R. & Ferrara, P.

(1993) Release from a human monocyte-like cell line of two different

soluble forms of the lipopolysaccharide receptor, CD14. Eur. J.

Immunol. 23, 2144±2151.

13. Durieux, J.-J., Vita, N., Popescu, O., Guette, F., Clazada-Wack, J.,

Munker, R., Schmidt, R.E., Lupker, J., Ferrara, P., Ziegler-Heitbrock,

H.W.L. & Labeta, M.O. (1994) The two soluble forms of the

lipopolysaccharide receptor, CD14: characterization and release by

normal human monocytes. Eur. J. Immunol. 24, 2006±2012.

14. Bufler, P., Stiegler, G., Schuchmann, M., Hess, S., KruÈger, C., Stelter,

F., Eckerskorn, C., SchuÈtt, C. & Engelmann, H. (1995) Soluble

lipopolysaccharide receptor (CD14) is released via two different

mechanisms from human monocytes and CD14 transfectants. Eur. J.

Immunol. 25, 604±610.

15. Frey, E.A., Miller, D.S., Jahr, T.G., Sundan, A., Bazil, V., Espevik, T.,

Finlay, B.B. & Wright, S.D. (1992) Soluble CD14 participates in

the response of cells to lipopolysaccharide. J. Exp. Med. 176,

1665±1671.

16. Pugin, J., Schurer-Maly, C.C., Leturcq, D., Moriarty, A., Ulevitch, R.J.

& Tobias, P.S. (1993) Lipopolysaccharide activation of human

endothelial and epithelial cells is mediated by lipopolysaccharide-

binding protein and soluble CD14. Proc. Natl Acad. Sci. USA 90,

2744±2748.

17. Detmers, P.A., Zhou, D. & Powell, D.E. (1994) Different signaling

pathways for CD18-mediated adhesion and Fc-mediated phagocy-

tosis: response of neutrophils to LPS. J. Immunol. 153, 2137±2145.

18. Hailman, E., Vasselon, T., Kelley, M., Busse, L.A., Hu, M.C.-T.,

Lichenstein, H.S., Detmers, P.A. & Wright, S.D. (1996) Stimulation

of macrophages and neutrophils by complexes of lipopolysaccharide

and soluble CD14. J. Immunol. 156, 4384±4390.

19. Schletter, J., Brade, H., Brade, L., KruÈger, C., Loppnow, H., Kusumoto,

S., Rietschel, E.T., Flad, H.-D. & Ulmer, A.J. (1995) Binding of

lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of

human cells is mediated by soluble CD14 and LPS-binding protein.

Infect. Immun. 63, 2576±2580.

20. Vita, N., Lefort, S., Sozzani, P., Reeb, R., Richards, S., Borysiewicz,

L.K., Ferrara, P. & LabeÂta, M.O. (1997) Detection and biochemical

characteristics of the receptor for complexes of soluble CD14 and

bacterial lipopolysaccharide. J. Immunol. 158, 3457±3462.

21. Haziot, A., Katz, I., Rong, G.W., Lin, X.Y., Silver, J. & Goyert, S.M.

(1997) Evidence that the receptor for soluble CD14: LPS complexes

may not be the putative signal-transducing molecule associated with

membrane-bound CD14. Scand. J. Immunol. 46, 242±245.

22. Perera, P.-Y., Vogel, S.N., Detore, G.R., Haziot, A. & Goyert, S.M.

(1997) CD14-dependent and CD14-independent signaling pathways

in murine macrophages from normal and CD14 knockout mice

stimulated with lipopolysaccharide or Taxol. J. Immunol. 158,

4422±4429.

23. Kirikae, T., Schade, F.U., Kirikae, F., Rietschel, E.T. & Morrison, D.C.

(1993) Isolation of a macrophage-like cell line defective in binding of

lipopolysaccharide: influence of serum and lipopolysaccharide chain

length on macrophage activation. J. Immunol. 151, 2742±2752.

24. Laemmli, U.K. (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227, 680±685.

q FEBS 2000 Specific role of mCD14 in LPS signaling (Eur. J. Biochem. 267) 45

25. Juan, T.S.-C., Hailman, E., Kelley, M.J., Busse, L.A., Davy, E., Empig,

C.J., Narhi, L.O., Wright, S.D. & Lichenstein, H.S. (1995)

Identification of a lipopolysaccharide binding domain in CD14

between amino acids 57 and 64. J. Biol. Chem. 270, 5219±5224.

26. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA

isolation by acid guanidinium thiocyanate-phenol-chloroform extrac-

tion. Anal. Biochem. 162, 156±159.

27. Ruff, M.R. & Gifford, G.E. (1980) Purification and physiochemical

characterization of rabbit tumor necrosis factor. J. Immunol. 125,

1671±1677.

28. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S.

& Tannenbaum, S.R. (1982) Analysis of nitrate, nitrite, and

[15N]nitrate in biological fluids. Anal. Biochem. 126, 131±138.

29. Delude, R.L., Fenton, M.J., Savedra, R. Jr, Perera, P.-Y., Vogel, S.N.,

Thieringer, R. & Golenbock, D.T. (1994) CD14-mediated trans-

location of nuclear factor-kB induced by lipopolysaccharide does not

require tyrosine kinase activity. J. Biol. Chem. 269, 22253±22260.

30. Libermann, T.A. & Baltimore, D. (1990) Activation of interleukin-6

gene expression through the NF-kB transcription factor. Mol. Cell.

Biol. 10, 2327±2334.

31. Han, J., Lee, J.D., Tobias, P.S. & Ulevitch, R.J. (1993) Endotoxin

induces rapid protein tyrosine phosphorylation in 70Z/3 cells

expressing CD14. J. Biol. Chem. 268, 25009±25014.

32. Goyert, S.M., Kirikae, T., Kirikae, F. & Morrison, D.C. (1992)

Identification of defective molecule in LPS-binding mutant J7.DEF3

cells isolated from macrophage-like J774.1 cells. In 2nd Conference

of the International Endotoxin Society, p. 100. Vienna, Austria.

33. Heumann, D., Gallay, P., Barras, C., Zaech, P., Ulevitch, R.J., Tobias,

P.S., Glauser, M.-P. & Baumgartner, J.D. (1992) Control of

lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis

factor secretion in human peripheral blood monocytes. J. Immunol.

148, 3505±3512.

34. Kitchens, R.L., Ulevitch, R.J. & Munford, R.S. (1992) Lipopolysac-

charide (LPS) partial structures inhibit response to LPS in human

macrophage cell line without inhibiting uptake by a CD14-mediated

pathway. J. Exp. Med. 176, 485±494.

35. Fujihara, M., Ito, N., Pace, J.L., Watanabe, Y., Russell, S.W. &

Suzuki, T. (1994) Role of endogenous interferon-b in lipopoly-

saccharide-triggered activation of the inducible nitric-oxide

synthase gene in a mouse macrophage cell line, J774. J. Biol.

Chem. 269, 12773±12778.

36. Zhang, X., Alley, E.W., Russell, S.W. & Morrison, D.C. (1994)

Necessity and sufficiency of b interferon for nitric oxide production

in mouse peritoneal macrophages. Infect. Immun. 62, 33±40.

37. Gao, J.J., Filla, M.B., Fultz, M.J., Vogel, S.N., Russell, S.W. & Murphy,

W.J. (1998) Autocrine/paracrine IFN-ab mediates the lipo-

polysaccharide-induced activation of transcription factor Stat1a in

mouse macrophages: pivotal role of Stat1a in induction of the

inducible nitric oxide synthase gene. J. Immunol. 161, 4803±4810.

38. Haziot, A., Ferrero, E., KoÈntgen, F., Hijiya, N., Yamamoto, S., Silver,

J., Stewart, C.L. & Goyert, S.M. (1996) Resistance of CD14-deficient

mice to endotoxin shock and reduced dissemination of Gram-

negative bacteria in CD14-deficient mice. Immunity 4, 407±414.

39. Blondin, C., Dur, A.L., Cholley, B., Caroff, M. & Haeffner-Cavaillon,

N. (1997) Lipopolysaccharide complexed with soluble CD14 binds to

normal human monocytes. Eur. J. Immunol. 27, 3303±3309.

40. Sendo, F., Suzuki, K., Watanabe, T., Takeda, Y. & Araki, Y. (1998)

Modulation of leukocyte transendotherial migration by integrin-

associated glycosyl phosphatidyl inositol (GPI)-anchored proteins.

Inflamm. Res. 47 (Suppl. 3), s133±s136.

41. Yang, R.B., Mark, M.R., Gray, A., Huang, A., Xie, M.H., Zhang, M.,

Goddard, A., Wood, W.I., Gurney, A.L. & Godowski, P.J. (1998)

Toll-like receptor-2 mediates lipopolysaccharide-induced cellular

signalling. Nature 395, 284±288.

42. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Huffel, C.V., Du, X.,

Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M.,

Ricciardi-Castagnoli, P., Layton, B. & Beutler, B. (1998) Defective

LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in

Tlr4 gene. Science 282, 2085±2088.

43. Hoshino, K., Takeuchi, O., Kawai, T., Ogawa, T., Takeda, Y., Takeda,

K. & Akira, S. (1999) Toll-like receptor 4 (TLR4)-deficient mice are

hyporesponsive to lipopolysaccharide: evidence for TLR4 as the lps

gene product. J. Immunol. 162, 3749±3752.

44. Shakhov, A.N., Collart, M.A., Vassalli, P., Nedospasov, S.A. &

Jongeneel, C.V. (1990) kB-type enhancers are involved in lipopoly-

saccharide-mediated transcriptional activation of tumor necrosis

factor a gene in primary macrophages. J. Exp. Med. 171, 35±47.

45. Drouet, C., Shakhov, A.N. & Jongeneel, C.V. (1991) Enhancers and

transcription factors controlling the inducibility of the tumor

necrosis factor-a promoter in primary macrophages. J. Immunol.

147, 1694±1700.

46. Collart, M.A., Baeuerle, P. & Vassalli, P. (1990) Regulation of tumor

necrosis factor a transcription in macrophages: involvement of four

kB-like motifs and of constitutive and inducible forms of NF-kB.

Mol. Cell. Biol. 10, 1498±1506.

47. Muller, J.M., Ziegler-Heitbrock, H.W. & Baeuerle, P.A. (1993) Nuclear

factor kB, a mediator of lipopolysaccharide effects. Immunobiology

187, 233±256.

48. Xie, Q.W., Whisnant, R. & Nathan, C. (1993) Promoter of the mouse

gene encoding calcium-independent nitric oxide synthase confers

inducibility by interferon g and bacterial lipopolysaccharide. J. Exp.

Med. 177, 1779±1784.

49. Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, A.M., Snyder, S.H.,

Russell, S.W. & Murphy, W.J. (1993) Macrophage nitric oxide

synthase gene: two upstream regions mediate induction by interferon

g and lipopolysaccharide. Proc. Natl Acad. Sci. USA 90, 9730±9734.

50. Xie, Q.W., Kashiwabara, Y. & Nathan, C. (1994) Role of transcription

factor NF-kB/Rel in induction of nitric oxide synthase. J. Biol. Chem.

269, 4705±4708.

51. Kamijo, R., Harada, H., Matsuyama, T., Bosland, M., Gerecitano, J.,

Shapiro, D., Le, J., Koh, S.I., Kimura, T., Green, S.J., Mak, T.W.,

Taniguchi, T. & Vilcek, J. (1994) Requirement for transcription

factor IRF-1 in NO synthase induction in macrophages. Science

263, 1612±1615.

52. Martin, E., Nathan, C. & Xie, Q.W. (1994) Role of interferon

regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med.

180, 977±984.