inhibition by a cd14 monoclonal antibody of lipopolysaccharide binding to murine macrophages

8
INTRODUCTION CD14 is a myeloid cell differentiation antigen expressed primarily on monocytes, 1 macrophages, 2 and neutrophils. 3 CD14 has been shown to be a receptor for a LPS and LPS binding protein (LBP) complex, 4 an acute phase serum protein found in normal serum in trace amounts. In the presence of LBP, LPS strongly activates monocytes via CD14 as measured by TNFα secretion. 1,5 This pathway of monocyte activation is thought to be a major contributor to the symptoms of endotoxin shock. 2 Recently, it has been demonstrated that CD14 also binds other bacterial products, 6–9 interleukin-2, 10 and endogenous phospho- lipids. 11 This evidence supports the contention that CD14 can have other functions in addition to that as a receptor for endotoxin. A number of investigators have studied the biochemical characteristics of human CD14 using anti- human CD14 mAb, but few studies have been done on mice using anti-murine CD14 mAb, although murine studies are useful to evaluate physiological roles of CD14 by in vivo experiments. 12 In this report, we analyzed a newly established mAb, 4C1. We demonstrate that 4C1 recognizes murine CD14, and blocks LPS-induced cytokine synthesis, similar to some human CD14 mAbs. 5 MATERIALS AND METHODS Animals ICR mice (male, 6 weeks old) and DA rats (male, 6 weeks old) were purchased from Japan SLC (Hamamatsu, Japan). DA rats were kept under specific pathogen-free conditions during immunization. Immunization and cell fusion RAW 264.7 (5 x 10 7 cells) was cultured in RPMI 1640 containing 10% fetal calf serum and 250 μg/ml (13)- © W. S. Maney & Son Ltd Journal of Endotoxin Research, Vol. 5, No. 3, 1999 Research article Inhibition by a CD14 monoclonal antibody of lipopolysaccharide binding to murine macrophages Y. Adachi 1 , C. Satokawa 1 , M. Saeki 1 , N. Ohno 1 , H. Tamura 2 , S. Tanaka 2 , T. Yadomae 1 1 Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan 2 Seikagaku Corporation, Tokyo Institute, Tokyo, Japan We have established an anti-CD14 mAb named 4C1 against murine macrophages. 4C1 can bind to thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages and casein- induced peritoneal neutrophils. Immunostaining with 4C1 was inhibited by treatment of the cells with phosphatidylinositol specific phospholipase C, suggesting that the antigen is GPI-anchored. Immunoprecipitates from biotin-labeled RAW264.7 cell lysate with 4C1 were around 55 kDa and were visualized with rmC5-3, the only commercially available anti-murine CD14 mAb. 4C1 positively stained COS7 cells transfected with an expression vector containing cDNA of murine CD14. Pretreatment of macrophages with 4C1 reduced LPS-mediated production of TNFα, IL-6, and nitrite. The binding of FITC-LPS to RAW264.7 cells was blocked by pretreatment with 4C1 but not with rmC5. Pretreatment of cells with unlabeled 4C1 mAb but not unlabeled rmC5-3 reduced binding of FITC-4C1. These results suggest that the 4C1 epitope on murine CD14 plays an important role in LPS binding and is distinct from the rmC5-3 epitope. Received 25 March 1999 Revised 24 June 1999 Accepted 25 June 1999 Correspondence to: Toshiro Yadomae, Laboratory of Immunopharma- cology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Tel: +81 426 76 5561; Fax: +81 426 76 5570 E-mail: [email protected] at UCSF LIBRARY & CKM on December 17, 2014 ini.sagepub.com Downloaded from

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Page 1: Inhibition by a CD14 monoclonal antibody of lipopolysaccharide binding to murine macrophages

INTRODUCTION

CD14 is a myeloid cell differentiation antigen expressedprimarily on monocytes,1 macrophages,2 and neutrophils.3

CD14 has been shown to be a receptor for a LPS and LPSbinding protein (LBP) complex,4 an acute phase serumprotein found in normal serum in trace amounts. In thepresence of LBP, LPS strongly activates monocytes viaCD14 as measured by TNFα secretion.1,5 This pathway ofmonocyte activation is thought to be a major contributorto the symptoms of endotoxin shock.2 Recently, it hasbeen demonstrated that CD14 also binds other bacterialproducts,6–9 interleukin-2,10 and endogenous phospho-lipids.11 This evidence supports the contention that CD14can have other functions in addition to that as a receptor

for endotoxin. A number of investigators have studied thebiochemical characteristics of human CD14 using anti-human CD14 mAb, but few studies have been done onmice using anti-murine CD14 mAb, although murinestudies are useful to evaluate physiological roles of CD14by in vivo experiments.12 In this report, we analyzed anewly established mAb, 4C1. We demonstrate that 4C1recognizes murine CD14, and blocks LPS-inducedcytokine synthesis, similar to some human CD14 mAbs.5

MATERIALS AND METHODS

Animals

ICR mice (male, 6 weeks old) and DA rats (male, 6 weeksold) were purchased from Japan SLC (Hamamatsu,Japan). DA rats were kept under specific pathogen-freeconditions during immunization.

Immunization and cell fusion

RAW 264.7 (5 x 107 cells) was cultured in RPMI 1640containing 10% fetal calf serum and 250 µg/ml (1→3)-

© W. S. Maney & Son LtdJournal of Endotoxin Research, Vol. 5, No. 3, 1999

Research article

Inhibition by a CD14 monoclonal antibody of lipopolysaccharidebinding to murine macrophages

Y. Adachi1, C. Satokawa1, M. Saeki1, N. Ohno1, H. Tamura2, S. Tanaka2, T. Yadomae1

1Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan

2Seikagaku Corporation, Tokyo Institute, Tokyo, Japan

We have established an anti-CD14 mAb named 4C1 against murine macrophages. 4C1 can bind tothioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages and casein-induced peritoneal neutrophils. Immunostaining with 4C1 was inhibited by treatment of the cellswith phosphatidylinositol specific phospholipase C, suggesting that the antigen is GPI-anchored.Immunoprecipitates from biotin-labeled RAW264.7 cell lysate with 4C1 were around 55 kDa andwere visualized with rmC5-3, the only commercially available anti-murine CD14 mAb. 4C1positively stained COS7 cells transfected with an expression vector containing cDNA of murineCD14. Pretreatment of macrophages with 4C1 reduced LPS-mediated production of TNFα, IL-6,and nitrite. The binding of FITC-LPS to RAW264.7 cells was blocked by pretreatment with 4C1 butnot with rmC5. Pretreatment of cells with unlabeled 4C1 mAb but not unlabeled rmC5-3 reducedbinding of FITC-4C1. These results suggest that the 4C1 epitope on murine CD14 plays animportant role in LPS binding and is distinct from the rmC5-3 epitope.

Received 25 March 1999Revised 24 June 1999Accepted 25 June 1999

Correspondence to: Toshiro Yadomae, Laboratory of Immunopharma-cology of Microbial Products, School of Pharmacy, Tokyo Universityof Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo192-0392, JapanTel: +81 426 76 5561; Fax: +81 426 76 5570E-mail: [email protected]

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β-D-glucan (grifolan13) for 18 h at 37°C. The cells werecentrifuged and then injected intraperitoneally into DArats every 2 weeks. Three days after the 4th immunization,the spleen was teased and prepared as antibody producingcells. Mouse myeloma NS-1 cells (5 x 108 cells) were pre-pared simultaneously and mixed with the antibody pro-ducing cells (3 x 107 cells) and centrifuged. The packedcells were fused by mixing with Dulbecco’s MEM(DMEM; Nissui, Japan) containing 50% polyethyleneglycol (Sigma). The mixed cells were washed and cul-tured in DMEM containing 20% FCS overnight and thencultured in DMEM containing 20% FCS and HAT(Sigma). Half the volume of culture medium was replacedwith fresh medium every other day until screening.

Screening of hybridoma

To test if the hybridoma supernatant (20% v/v) inhibits pro-duction of TNFα in RAW264.7 culture, 5 x 105 RAW264.7cells were cultured with 1 µg/ml LPS (Escherichia coliO111; Sigma) for 18 h in a flat-bottomed 48-well cultureplate (Sumilon). The macrophage culture supernatants werethen subjected to TNFα ELISA assay.

Hybridoma cloning

Hybridomas were cloned by limiting dilutions. Wells posi-tive for hybridoma growth were rescreened for staining ofRAW264.7 cells, and for inhibition of TNFα production.

Flow cytometry

Staining was performed in round-bottomed multiwellplates (NUNC) on 5 x 105 cells per sample. Washing wascarried out by centrifuging the plates 2–3 times betweenstaining steps with phosphate buffered saline (PBS) con-taining 2% FCS and 0.1% NaN3. Binding of rat mAbswas visualized with a two-step technique involvinghybridoma supernatants and FITC conjugate of goat IgGF(ab)′2 against anti-rat IgG (Cappel; 2.5 µg/ml).

Isolation of mouse primary leukocytes

Spleen from ICR mouse was excised and teased on stain-less mesh to obtain a single cell suspension. The cells weresuspended in Eagle’s minimum essential medium(EMEM) and washed with EMEM by centrifugation at300 g for 5 min. The contaminating erythrocytes werelysed with lysing buffer containing 0.15 M ammoniumchloride, 1 mM KHCO3, 0.1 mM ethylenediamine-tetraacetic acid (pH 7.2). The spleen cells were washedtwice with EMEM and resuspended in RPMI 1640 con-

taining 10% fetal calf serum (RPMI-FCS). Bone marrowcells were obtained by flushing femoral shafts and thensuspended in RPMI-FCS. To obtain bone marrow-derivedmacrophages, the bone marrow cells were cultured inRPMI-FCS containing 10% L929 cell conditionedmedium as a source of M-CSF for 5 days. Thereafter, thecells were further cultured by changing the medium onalternate days until day 6. The macrophages were flushedwith cold PBS containing 0.5% lidocaine hydrochloride(Astra Japan Ltd, Japan) to detach the adherent cells, andresuspended in RPMI-FCS. Peritoneal neutrophils wereharvested from peritoneal lavage 6 h after intraperitonealinjection of 2 ml 8% casein solution into ICR mice. Theperitoneal cells were washed and resuspended in RPMI-FCS. The ratio of neutrophils in the cell suspension wasdetermined by microscopy as about 90% after Giemsastaining.

Preparation of COS7 transfectant

A vector-expressing murine CD14 was generated byamplification of full-length murine CD14 cDNA by PCR.The oligonucleotides 5′-CGCGGATCCACTTTCAGA-ATCTACCGACC-3′ and 5′-CGGAATTCGGACCCT-CAGAAACCAGGA-3′ were used as primers to theamino terminus and carboxyl terminus of CD14, respec-tively, in a PCR reaction that amplified CD14 from acDNA mixture prepared using M-MLV reverse transcrip-tase (Amersham) and oligo-dT primer (Gibco BRL). Theends of the amplified murine CD14 DNA were cleaved atthe newly introduced BamHI and EcoRI sites. The ampli-fied and cleaved murine CD14 was ligated together via theBamHI overhangs and inserted into the vector pcDNA3(RIKEN Gene Bank, Tsukuba, Japan) via the EcoRI sites.The resulting pcDNA3-CD14 vector (pmCD14neo) wasconfirmed by restriction enzyme mapping. COS7 cell lineexpressing murine CD14 and control transfectant weremade by transfecting COS7 cells with the vectorpmCD14neo and pcDNA3 empty vector, respectively.Transfections were performed by electroporating 4 x 106

cells in 0.8 ml of electroporation buffer (272 mM sucrose,7 mM sodium phosphate, 1 mM MgCl2 (pH 7.4)) at 260 Vand 25 µF for 7.5 ms in the presence of 40 µg of the appro-priate vector. The cells were incubated for 48 h inDulbecco’s MEM (Sigma) containing 10% FCS (Iwaki,Tokyo, Japan) and 50 µg/ml gentamicin. Upon recovery,CD14-transfected and control vector-transfected cells werestained for murine CD14 expression using the TRITC-labeled 4C1. The staining was confirmed by confocal laserfluorescence microscopy (µRadiance, BioRad). The fre-quency of cells expressing CD14 was also determined byFACS after staining the cells with 4C1 and FITC-labeledgoat IgG F(ab)′2 against anti-rat IgG (Cappel).

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Cytokine production assay

Quantification of the cytokines in the culture supernatantand lysate was performed by a specific sandwich ELISAsystem as described in other reports. Microtiter ELISAplates (Sumilon, Japan) were coated with the capture anti-body for TNFα (rat IgG1; Pharmingen) or IL-6 (rat-IgG1;Pharmingen). The antibody was diluted in bicarbonatebuffer (pH 9.6) to a final concentration of 1.5 µg/well, and50 µl was added to each well. Plates were incubated at4°C overnight, followed by washing 3 times with PBScontaining 0.05% Tween 20 (PBS-T). Blocking wasachieved by adding 100 µl/well of PBS-T containing0.5% BSA (BPBS-T) and then incubating the mixture at37°C for 40 min. After three more wash cycles, thermTNFα (Pharmingen) or rmIL-6 (R & D Systems) andsupernatant samples were added to the plate (50 µl/well,duplicate) and left to incubate for 40 min at 37°C. Afterthree washings with PBS-T and blocking with BPBS-Tfor 10 min, bound soluble cytokine was detected using ananti-mTNFα polyclonal antibody (Genzyme) or biotinlabeled anti-mIL-6 monoclonal antibody (Pharmingen).Antibodies were diluted in BPBS-T, and 50 µl/well wereadded for 40 min at 37°C. The plates were washed another3 times, and 50 µl of a 1:10000 diluted anti-rabbit IgG-horseradish peroxidase (Cappel™) (avidin-horseradishperoxidase for IL-6) was added to each well. After incu-bation for 40 min and three final washes with PBS-T, theplates were developed using 50 µl/well tetramethylbenzi-dine (Kirkegaard and Perry Laboratories, Inc.). The reac-tion was stopped by the addition of 50 µl 1 N H3PO4/welland read using a microplate reader (MTP-32, CoronaElectric Co., Ltd.) at 450 nm.

Nitric oxide production

After incubating the RAW264.7 cells (1 x 106 cells/ml)with various concentrations of mAb and 10 ng/ml LPS for24 h, synthesis and release of NO were determined byassaying the culture supernatant for nitrite content.Briefly, 50 µl supernatant was reacted for 10 min at roomtemperature with an equal volume of Griess reagent (1%sulfanilamide, 0.1% naphthylethylene diaminedihydro-chloride, 2.5% phosphoric acid). The optical density wasmeasured at 550 nm (reference 630 nm). The nitrite con-tent was quantified by comparison with a standard curvegenerated with sodium nitrite in the range 0–100 µM.

Immunoprecipitation and immunoblotting

RAW264.7 (1 x 108 cells) were labeled with 100 µMbiotin-NHS (BioRad) in PBS for 30 min at 4°C, thenbiotin-labeled cell lysate was prepared by dissolving with

lysing buffer (pH 8.2) containing 20 mM Tris-HCl(Wako), 140 mM NaCl (Wako), 2 mM EDTA (Wako), 1%NP40 (Calbiochem), 5 mM iodoacetamide (Sigma), 1mM phenylmethylsulphonyl fluoride (Sigma), and 50 µM4-(2-aminoethyl) benzensulphonyl fluoride (Sigma).Lysates were incubated with 5 µg 4C1 or isotype controlantibody (4B12) for 2 h at 4°C, then precipitated by incu-bating with 50 µg Sepharose conjugated anti-rat IgG(Cappel) for 1 h. The precipitates were centrifuged andwashed 5 times with lysing buffer, then electrophoresedby SDS-PAGE. The proteins in 10% polyacrylamide gelwere electrotransfered to PVDF membrane. After block-ing the membrane with PBS containing 3% BSA, theblots were probed with streptavidin peroxidase and thereactive protein bands were visualized by an enhancedchemiluminescence method (ECL reagent; Amersham).Biotinylated SDS-PAGE standards (BioRad) were simul-taneously used to estimate molecular weights.

FITC-LPS binding assay

RAW264.7 (1 x 106 cells) was suspended with 1 ml RPMI1640 containing 10% FCS. Antibodies (0.5 µg/ml) wereadded followed by 1 µg/ml FITC-LPS (E. coli O111,Sigma) and the cell suspension was kept on ice for 18 h.After treatment, cells were suspended in PBS containing50% FCS, and centrifuged at 300 g for 5 min. The cell pel-let was resuspended with PBS containing 2% FCS.Binding of FITC-LPS to RAW264.7 was assessed usingflow cytometry.

RESULTS

Assessment of 4C1 reactive antigen on various cell types

The antigen specificity of 4C1 was assessed by flowcytometry after staining of various cell types with 4C1and anti-rat IgG F(ab)′2. As shown in Figure 1, neu-trophils and bone marrow-derived macrophages culturedwith L929 culture supernatant stained with antibody.However, undifferentiated bone marrow cells and spleno-cytes did not stain with 4C1. These results suggest thatantigens for 4C1 are only present on differentiatedmyeloidal cells such as macrophages and neutrophils.

Characterization of antigen for 4C1 by PI-PLCtreatment and immunoprecipitation

To determine whether the antigen recognized by 4C1 isglycosylphosphatidyl inositol (GPI)-anchored, severalcells were pretreated with phosphatidylinositol specificphopholipase C (PI-PLC). As shown in Table 1, PI-PLC

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treatment markedly reduced the binding of 4C1 to themacrophage cell line RAW264.7, bone marrow-derivedmacrophages or neutrophils. These results strongly sug-gest that the 4C1 antigen is GPI-anchored. In order tocharacterize the 4C1 antigen further, RAW264.7 cellswere labeled with biotin-NHS in PBS for 30 min at 4°C;then, biotin-labeled cell lysate was prepared by dissolvingwith detergent. Samples immunoprecipitated with 4C1 orisotype control antibody from the lysate were analyzed bySDS-PAGE and immunoblotted using streptavidin perox-idase as a probe. A positive signal having a molecularweight around 55 kDa was detected in the 4C1-treatmentlane, suggesting the 4C1 antigen is close to the molecularweight of CD14 (Fig. 2A) . Further characterization wasdone by immunoprecipitation and immunoblotting usingan anti-CD14 mAb. Immunoprecipitation of cell lysatewith 4C1 was electrophoresed and blotted on a PVDFmembrane. The cellular proteins on the membrane wereprobed with a commercially available anti-CD14 mAb

(rmC5-3).14 As shown in Figure 2B, 4C1 immunoprecipi-tates were visualized by probing with rmC5-3 and werearound 55 kDa in size. No bands were detected in the iso-type matched mAb lane. These results strongly indicatethat 4C1 binds to the CD14 antigen.

Immunochemical staining of CD14 transfectant by 4C1

To confirm that the antigen for 4C1 mAb is murine CD14,we prepared a CD14 transfectant using COS7 cells and anexpression vector constructed from pcDNA3 and PCR-

142 Adachi et al.

Fig. 1. 4C1 staining of various cells. The cells were stained with 4C1(solid) or isotype-matched control mAb (open), and FITC-conjugated goatanti-rat IgG F(ab)′2. Similar results were observed in two independentexperiments.

Table 1 Effect of treatment with PI-PLC on binding of 4C1 to various cells

Mean fluorescence intensityControl Ab 4C1

PI-PLC treatmentCells – + – +

RAW264.7 5.8 6.9 49.8 8.5Bone marrow macrophages 7.6 6.7 31.9 7.2Granulocytes 9.2 7.1 77.7 45.6

106 cells were pretreated with PI-PLC (0.125 U/ml) for 30 min at 37°C in RPMI 1640 containing 10% FCS. After washingwith cold PBS containing 2% FCS, the cells were stained with 4C1 and FITC-conjugated anti-rat IgG F(ab)′2 for 30 min onice. Fluorescence intensity of 104 cells was measured by flow cytometry.

Fig. 2. Immunoprecipitation of cellular antigen with 4C1. (A) RAW264.7cell lysate labeled with biotin was immunoprecipitated with isotype-matched control mAb (lane 2), or 4C1 (lane 3) and anti-rat IgG-Sepharose.The precipitation was electrophoresed by SDS-PAGE and transferred ontoPVDF membrane. The antigen was visualized by probing withstreptavidine-peroxidase and enhanced chemiluminescence reagents. Lane1: biotinylated standard molecular weight marker. (B) RAW264.7 celllysate was immunoprecipitated with isotype-matched control mAb (lane1), rmC5-3 (lane 2) or 4C1 (lane 3) and anti-rat IgG-Sepharose. Theprecipitation was electrophoresed by SDS-PAGE and transferred to PVDFmembrane. The antigen was visualized by probing with anti-mCD14 mAb(rmC5-3) , peroxidase-conjugated goat anti-rat IgG and enhancedchemiluminescence reagents. Similar results were observed in twoindependent experiments.

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amplified murine CD14 cDNA. After transfection with avector containing mouse CD14 cDNA, 5.5% of a cell pop-ulation expressed murine CD14 as determined by flowcytometry (data not shown). Staining of the CD14 trans-fectant was further determined by fluorescence micro-scopy. As shown in Figure 3, COS7 cells transfected withthe CD14 vector showed positive signals in 4C1-staining,while control transfectants did not. This result stronglysupports that an antigen of 4C1 mAb is mouse CD14.

4C1 inhibition of cytokine and NO production inducedby LPS

To examine whether 4C1 influences the response of macro-phages to LPS, 4C1 was added to the culture of RAW264.7,and production of cytokines and nitric oxide (NO) in the

supernatants was determined by ELISA and Griessreagents, respectively. Supernatants from the culture con-taining LPS plus control antibody exhibited increased lev-els of TNFα, IL-6, and NO. However, addition of 4C1potently suppressed LPS-induced release cytokines and NO(Fig. 4). It was confirmed that 4C1 did not affect cell viabil-ity by assessment of lactate dehydrogenase release andlatex beads phagocytosis (data not shown). These resultsdemonstrate that the engagement of CD14 by 4C1 caninhibit the stimulation of murine macrophages by LPS.

Effect of 4C1 on the binding of LPS to murinemacrophages

As described above, we have shown that LPS-induciblemonokine production and NO production are inhibited

New anti-murine CD14 mAb 143

Fig. 3. Staining of CD14 transfectant with TRITC-labeled 4C1 mAb. COS7 cells were transfected with an expression vector pcDNA3 (Control) orpmCD14neo (CD14) by electroporation. The cells maintained in DMEM containing 10% FCS for 48 h were stained with TRITC-labeled 4C1. Differenceinterference contrast (DIC) and fluorescence images were incorporated by confocal laser fluorescence microscopy. Similar results were observed in twoindependent transfection experiments.

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by 4C1, indicating that surface 4C1 antigen may beinvolved in the binding of LPS to cells. Therefore, weanalyzed the effect of 4C1 on binding of FITC-LPS tomouse macrophages. RAW264.7 cells were treated withincreasing concentrations of anti-CD14 mAb for 30 minat 4°C, then the cells were further incubated with FITC-LPS for 18 h at 4°C. As shown in Figure 5, binding ofFITC-LPS to cells was reduced by addition of 4C1 mAb.

Another anti-CD14 mAb (rmC5-3) had no effect on theLPS-binding. These results suggest that the 4C1 epitopeis involved in, or proximal to, the LPS binding site, andthat 4C1 inhibition of macrophage activation by LPS isvia blockade of LPS binding.

4C1 and rmC5-3 have different binding sites

To assess whether an epitope for 4C1 is close to or thesame as the rmC5-3 epitope, FACS analysis was per-formed using FITC-labeled 4C1 and rmC5-3. As shownin Figure 6A, the binding of FITC-4C1 to RAW 264.7cells was not inhibited by pretreatment with unlabeledrmC5-3 mAb. Similarly, cell staining with FITC-rmC5-3was not inhibited by prestaining of cells with unlabeled4C1 (Fig. 6B). These results suggest that these mAbs donot share the same or proximal epitopes.

144 Adachi et al.

Fig. 4. Effect of 4C1 on LPS-mediated cytokine and nitrite synthesis inmacrophages. RAW264.7 cells were stimulated with 10 ng/ml LPS in thepresence of various concentrations of isotype-matched control mAb or4C1. After incubation for 12 h (TNFα, A and nitrite, C) or 24 h (IL-6, B),cytokine or nitrite concentrations in the culture supernatant weredetermined by ELISA or by Griess reagent, respectively. Nil correspondsto a cell culture without antibodies. One representative result out of threeperformed is given. An inhibitory effect of 4C1 was observed similarly inthree independent experiments. Statistical analysis was done by Student’st-test. *P < 0.05, **P < 0.01.

Fig. 5. Effect of pretreatment of RAW264.7 cells with 4C1 on the bindingof FITC-LPS. Cells were incubated with 25 µg/ml isotype-matched controlmAb, 4C1, or rmC5-3 at 4°C for 18 h in the presence of 5 µg/ml FITC-LPS. Binding of FITC-LPS to the cells was determined by flow cytometry.The solid histograms show FITC-LPS staining, open histogram showscontrol staining in the absence of FITC-LPS. Representative results wereexpressed out of four independent experiments.

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DISCUSSION

CD14 is generally accepted as a LPS receptor that isinvolved in endotoxin shock.2 CD14 recognizes theLPS–LBP complex and reportedly transfers activationsignals by collaborating with transmembrane receptorssuch as toll-like receptors.15,16 CD14 triggers host-defensereactions even in the presence of very low concentrationsof LPS.17 It has been recently reported that there is aCD14-mediated signal transduction system that activatesphagocytes in response to peptideglycans9 from Gram-positive bacteria and mycobacterial lipoarabinomannan7

in addition to LPS. Numerous monoclonal antibodies tovarious epitopes of human CD14 have been created andfor this, and other diverse reasons, most CD14 studieshave been performed on human CD14.18 Since there isonly a single commercially available murine CD14 anti-body,19 it is difficult to study physiological roles of CD14in the murine system except for the use of geneticallydeficient CD14 knockout mice20,21 or CD14 transgenicmice.22,23 Therefore, we created a hybridoma that pro-duces a monoclonal antibody (4C1). 4C1 significantlyinhibits LPS-mediated cell activation. 4C1 reacted withRAW264.7 cells, as well as neutrophils and bone marrow-derived macrophages cultured with L929-conditionedmedium. 4C1 binding to lymphocytes and undifferenti-ated bone marrow cells was negligible. Expression ofCD14 on promyelocytic and monocytic cell lines HL-60and THP-1 increases after differentiation induced by

phorbol myristate acetate or activated vitamin D3.24 This

evidence seemed to be similar to our results, i.e. that 4C1recognizes fully differentiated myeloid cells. Peritonealgranulocytes induced by intraperitoneal injection ofcasein also reacted strongly with 4C1 antibody.Polymorphonuclear leukocytes increase expression ofCD14 when activated by pro-inflammatory cytokines,3

although it is constitutively expressed in lower levels. Aphenomenon similar to human neutrophils might beapparent even in a mouse by inducing inflammation.

CD14 is expressed on the cell surface as a GPI-anchored glycoprotein.1 GPI-anchored glycoproteins canbe cleaved by enzymatic digestion with PI-PLC.24

Reactivity of macrophages with 4C1 decreased remark-ably after processing with PI-PLC. This suggests that the4C1 epitope is GPI-anchored. Further, since the antigenimmunoprecipiated with 4C1 could be detected byanother mouse CD14 monoclonal antibody, 4C1 probablyrecognizes murine CD14. Evidence that 4C1 binds toCD14 was obtained by immunochemical staining of aCD14 transfectant. COS7 cells transfected with a vectorcontaining murine CD14 cDNA sequence showed posi-tive signals in 4C1-staining, while control transfectantsdid not.

The binding epitope for LPS–LBP complex has beensuggested to be amino acid residues 39–44,25 57, and64,26 on the N-terminal moiety.27 The epitope for rmC5-3has been shown to be near the C-terminal of murineCD14.19 The exact binding site for LPS is unclear in

New anti-murine CD14 mAb 145

Fig. 6. Effect of pretreatment of RAW264.7 cells with 4C1 on the binding of FITC-4C1 or FITC-rmC-5. Cells were pre-incubated with 50 µg/ml isotype-matched control mAb, 4C1, or rmC5-3 at 4°C for 30 min. After incubation, cells were stained with 0.5 µg/ml FITC-4C1 or FITC-rmC5-3. Binding ofFITC-4C1 (A) or FITC-rmC5-3 (B) to the cells was determined by flow cytometry. The solid histograms show staining with FITC-4C1 or FITC-rmC5-3,open histogram shows control staining in the absence of FITC-mAbs. Similar results were observed in three independent experiments.

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murine CD14. However, it is not likely to be near the C-terminal, since rmC5-3 only barely inhibits binding ofFITC-LPS to RAW264.7 cells. However, binding of 4C1to RAW264.7 cells resulted in inhibition of LPS-bindingand macrophage activities induced by LPS. Pretreatmentof macrophages with rmC5-3 did not reduce 4C1 binding.These results suggest that 4C1 does not recognize a C-ter-minal moiety on murine CD14. This monoclonal antibodyshould be useful for examining CD14-related pathways.

ACKNOWLEDGEMENTS

The authors deeply appreciate the technical assistance ofMiss Ai Takizawa and Messrs Keisuke Moriya, DaiSakuma and Tadashi Morino. The authors also thank DrKazuhiro Tamura for allowing us to use the electropora-tion system. This study was supported by a Grant-in-Aid for Scientific Research (C, 10672058) from theMinistry of Education, Science, Sports and Culture.

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2. Ulevitch RJ, Mathison JC, Schumann RR, Tobias PS. A newmodel of macrophage stimulation by bacteriallipopolysaccharide. J Trauma 1990; 30: S189–S192.

3. Wright SD, Ramos RA, Hermanowski-Vosatka A, Rockwell P,Detmers PA. Activation of the adhesive capacity of CR3 onneutrophils by endotoxin: dependence on lipopolysaccharidebinding protein and CD14. J Exp Med 1991; 173: 1281–1286.

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