Vol. 61, No. 10INFECrION AND IMMUNITY, OCt. 1993, p. 4452-44610019-9567/93/104452-10$02.00/0Copyright X 1993, American Society for Microbiology

Neither CD14 nor Serum Is Absolutely Necessary forActivation of Mononuclear Phagocytes by

Bacterial LipopolysaccharideWILLIAM A. LYNN,'* YANNAN LIU,2 AND DOUGLAS T. GOLENBOCK2

Department ofIntemal Medicine, Division of Infectious Diseases, Maxwell Finland Laboratory for InfectiousDiseases, Boston City Hospital, Boston University School ofMedicine, Boston, Massachusetts 02118,2 and

Department ofInfectious Diseases, Hammersmith Hospital, London W12 ONN, England'Received 16 April 1993/Returned for modification 2 June 1993/Accepted 26 July 1993

The stimulation of mononuclear phagocytes by lipopolysaccharide (LPS) is facilitated by the binding ofcomplexes of LPS and LPS-binding protein to CD14. Although it is clear that CD14 is involved in LPS-inducedsignaling, other investigators have hypothesized the existence of additional signaling pathways in macrophages.We sought to determine whether CD14-independent pathways of monocyte activation might exist. Washedhuman mononuclear cells responded with reduced sensitivity to LPS in the absence of serum. Anti-CD14monoclonal antibody (MAb) inhibited the response to LPS in serum-free conditions, but this was easilyreversed at higher concentrations of LPS. We established a human monocytic cell line, designated SFM(derived from THP-1), in serum-free medium to examine LPS responses under defined conditions. Differen-tiation ofSFM cells with 1,25-dihydroxycholecalciferol promoted the expression ofabundant cell surface CD14.Differentiated SFM cells responded to LPS despite the complete absence of serum proteins for >20 generationsof growth. LPS stimulation of differentiated SFM cells was inhibited by anti-CD14 MAbs only when serum waspresent. In contrast to anti-CD14 MAb, the LPS antagonists lipid IVa and Rhodobacter sphaeroides lipid Ainhibited monocyte activation under serum-free conditions, implying that these compounds compete with LPSat a site distinct from CD14. Undifferentiated SFM cells (expressing minimal CD14) still responded to LPS inserum-free conditions, and anti-CD14 MAb had little inhibitory effect. The addition of purified LPS-bindingprotein or human serum promoted a CD14-dependent pathway of monocyte activation by LPS in these cells.We conclude that monocytes do not absolutely require serum proteins to be stimulated by LPS and thatCD14-independent LPS signaling pathways exist which are inhibitable by lipid IVa and R. sphaeroides lipid A.

Lipopolysaccharide (LPS or endotoxin), a constituent ofthe outer membrane of gram-negative bacterial cell walls, isimplicated in the pathogenesis of gram-negative bacterialseptic shock (23, 24, 27, 28). The structure of the lipid A coreof LPS is highly conserved between diverse species ofbacteria (26) and is the active inflammatory moiety of LPS(23, 27, 28). The activation of mononuclear phagocytes inresponse to lipid A plays a key role in the initiation of thesepsis syndrome (1). There is now considerable evidencethat LPS interacts with a number of specific cellular recog-nition proteins (4, 9, 10, 13, 17-19, 38, 39, 42), and althoughthe signaling function of each of these LPS-binding proteinsstill remains to be elucidated, it appears that the activation ofphagocytic granulocytes occurs as a result of a ligand-receptor interaction.The discovery of a serum protein, LPS-binding protein

(LBP), by Tobias and colleagues (29, 33-35) and the recentrecognition that LPS-LBP complexes are recognized byCD14 (2, 12, 14, 42) were the first clear evidence that LPSinduces signal transduction in phagocytic leukocytes byinteracting with a specific surface protein. CD14 is a 55-kDaglycosyl phosphatidylinositol-linked protein expressed onthe surface of monocytes, macrophages (11), and polymor-phonuclear leukocytes (PMN) (21, 40). Evidence that CD14participates in LPS-induced cellular signaling includes thedemonstration that some monoclonal antibodies (MAb) toCD14 inhibit the response of monocytes (12, 14, 29, 41, 42)

* Corresponding author.

and PMN (21, 40) to LPS. Furthermore, transfection ofhuman CD14 into the murine pre-B cell line 70Z/3 increasedthe sensitivity of these cells to complexes of LPS-LBP (16),and transfection of CD14 into Chinese hamster ovary (CHO)fibroblasts transforms the cells from LPS nonresponders intoLPS responders (43). Plasma proteins other than LBP,designated septin, have also been proposed as mediating theinteraction of LPS with CD14 (41). An additional complexityof LPS-induced cellular signaling is that CD14 is present inserum in a soluble form and may function as a solublesignaling molecule for nonphagocytic cell types (6).Although CD14 functions as a receptor for LPS-LBP

complexes and inhibition of LPS-LBP binding to CD14inhibits endotoxin-induced events, it is not known how cellsare signaled after binding occurs because glycosyl phos-phatidylinositol-linked proteins lack a cytoplasmic domainand any known signal transduction sequence motif. It is notknown yet whether CD14 is a single signaling receptor oracts in concert with other signaling proteins as part of areceptor complex. A separate issue is whether there existLPS recognition pathways which signal cells independentlyof CD14 and its putative signaling complex. Although hy-pothesized (28, 32, 37), the existence of such pathways hasnot been experimentally addressed.We have previously demonstrated that washed human

PMN were hyporesponsive to LPS in the absence of serum(21). Nevertheless, PMN were capable of responding to LPSunder serum-free conditions. The responses of washed PMNcould be inhibited by the addition of anti-CD14 MAb (21).Although these experiments suggested that LPS can directly

4452

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

SERUM AND CD14-INDEPENDENT PHAGOCYTE ACTIVATION BY LPS 4453

bind CD14 and activate phagocytes without the participationof serum proteins, we could not exclude the possibility thatsmall amounts of contaminating serum proteins were presentin our cellular preparations, or absorbed to the PMN sur-face, despite extensive washing.To elucidate the role of CD14 and serum accessory

proteins in LPS-induced signal transduction, a cell line wasestablished in a defined serum-free medium by using cellswhose expression of CD14 could be exogenously regulated.The THP-1 cell line, an LPS-sensitive monocytic cell line,was adapted to serum-free medium, washed, and grownserum free for at least 20 generations. These cells weredesignated SFM (or SFMIHS when the medium was supple-mented with 1% human serum [HS]); the serum-free mediumdid not include detectable LBP or septin (see Materials andMethods), and, thus, any observed responses to LPS couldnot be mediated by these serum factors. Under normalgrowth conditions, undifferentiated SFM cells do not ex-press detectable surface CD14 and will not release tumornecrosis factor alpha (TNF-a) or interleukin-13 (IL-1i) intothe extracellular medium in response to bacterial stimuli (9a)but will accumulate both TNF-a and IL-13 mRNA. Incontrast, after 48 h of treatment with 1,25-dihydroxychole-calciferol (vitamin D3; calcitriol) or phorbol 12-myristate13-acetate (PMA), SFM cells differentiate, express abundantsurface CD14, and release mature cytokine proteins inresponse to LPS. Therefore, by growing THP-1 cells in adefined serum-free medium which could be supplementedwith serum proteins when needed, the presence and levels ofthe known LPS signaling proteins (CD14, LBP, and septin)in our experimental system could be controlled. LPS respon-siveness was monitored by Northern (RNA) blotting forTNF-ot or IL-1,B mRNA in undifferentiated SFM cells or byassaying for the presence of released cytokines in 1,25-dihydroxycholecalciferol-differentiated cells.

In addition to being able to manipulate experimentalconditions to test the relative input of serum factors andCD14, we have also taken advantage of the availability oftwo lipid A-like molecules which are apparent competitiveantagonists of LPS (8, 15, 20, 21, 31). These inhibitorsinclude lipid IVa (the tetraacyl disaccharide precursor ofEschenichia coli lipid A; this compound is also known asprecursor Ia and synthetic lipid 406 or LA-14-PP) and lipid Aderived from Rhodobacter sphaeroides (RSLA; Fig. 1).The experiments presented herein demonstrate that nei-

ther LBP nor septin nor any other serum protein is abso-lutely necessary to activate cells with LPS and that LPS isonly minimally capable of interacting directly with CD14 inthe absence of serum proteins. Our findings suggest thatmonocytic cells can respond to LPS independently of CD14,and LPS activation of this CD14-independent signaling sys-tem is inhibited by lipid IVa and RSLA.

MATERIALS AND METHODS

Reagents. Unless otherwise stated, all chemical and im-munologic reagents were purchased from Sigma ChemicalCo. (St. Louis, Mo.). Phosphate-buffered saline (PBS) andRPMI 1640 were from MA Bioproducts (Walkersville, Md.).Ficoll-Hypaque, under the brand name Mono-Poly Resolv-ing Medium, was purchased from ICN Biomedicals (CostaMesa, Calif.).

Pipet tips were from VWR Scientific (Boston, Mass.).Plastic tissue culture centrifuge tubes and culture tubes werefrom Falcon (Becton Dickinson, San Jose, Calif.). SterileSarstedt vials (1.5 ml) were purchased from Sarstedt

HO

HOOO'-.COHo OH .o 0

lipid Amoiety

ReLPS

Lipid IVA R. sphaeroideslipid A

FIG. 1. Structure of ReLPS in comparison with those of the LPSinhibitors lipid IVa and RSLA. ReLPS represents the minimal LPSfound in gram-negative bacteria. ReLPS consists of a hexaacylatedglucosamine disaccharide 1,4'-bisphosphate known as lipid A(boxed), covalently linked to 2 molecules of 2-keto-3-deoxyoctu-losonic acid (26); lipid A constitutes the active moiety of LPS. LipidIVa is the tetraacyl 1,4'-bisphosphate diglucosamine precursor of E.coli lipid A. RSLA differs from the lipid A of E. coli by virtue of thenumber of acyl groups (five versus six), the chain lengths of the fattyacids, and the presence of a 3-keto moiety and a double bond.

(Princeton, N.J.). When necessary, glassware and plasticware were autoclaved at 115°C for 30 min and then renderedpyrogen free by baking overnight at 270°C and 125°C,respectively.PMA was stored at 0.3 mg/ml in dimethyl sulfoxide at

-70°C and diluted 1:10,000 in medium immediately prior touse. 1,25-Dihydroxycholecalciferol (Biomole, PlymouthMeeting, Pa.) was stored at 10 mM in ethanol at -20°C.Macrophage serum-free medium was from GIBCO (Grand

Island, N.Y.) and contains human serum albumin, trans-ferrin, and insulin but no other serum proteins. This mediumwas assayed for LBP (by P. Tobias, Scripps ResearchInstitute, La Jolla, Calif.) and septin (by S. D. Wright,Rockefeller University, New York, N.Y.), and neither pro-tein was detectable. Fetal calf serum (containing <6 pg ofLPS per ml) was from Hyclone (Logan, Utah). HS, used incell culture and stimulation steps, was derived from clottedwhole blood from healthy volunteers and heat inactivated at56°C for 45 min. Aliquots were frozen at -20°C for periodsup to 6 months until needed.

Purified Salmonella minnesota R595 LPS (ReLPS) andRSLA were the gifts of N. Qureshi and K. Takayama(University of Wisconsin, Madison). Synthetic lipid IVa waspurchased from ICN Biomedicals. All lipids were preparedas stock suspensions at 1 mg/ml in PBS and stored at -20°C.Prior to use, the suspensions were defrosted and sonicated in

VOL. 61, 1993

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

4454 LYNN ET AL.

a water bath sonicator (Laboratory Supplies, Inc., Hicks-ville, N.Y.) for at least 5 min. Heat-killed Staphylococcusaureus were prepared as described previously (8) and storedat 3 x 109 organisms per ml in sterile PBS at 4°C. Purifiedrabbit LBP was the gift of P. Tobias.Anti-CD14 MAbs included the following. Purified 3C10

(immunoglobulin G2b [IgG2b]) was the gift of S. D. Wright,My4 (IgG2b) was purchased from Coulter Electronics, Inc.(Hialeah, Fla.), and fluorescein isothiocyanate (FITC)-con-jugated Leu M3 (IgG2b) was from Becton Dickinson. Otherantibodies included unconjugated mouse myeloma IgG2bpurchased from Bionetics (Charleston, S.C.) and FITC-labeled goat anti-mouse IgG from Boehringer-Mannheim(Indianapolis, Ind.).

Soluble CD14 was isolated from the urine of a child withnephrotic syndrome (gift of Melanie Kim, Children's Hospi-tal, Boston, Mass.) by immunoaffinity purification (Pierce,Rockford, Ill.; Immunopure Ag/Ab kit number 2) with My4as described in the manufacturer's instructions.TNF-a and IL-113 ELISA. Reagents for the enzyme-linked

immunosorbent assay (ELISA) were as follows. Anti-humanTNF-ot MAb (no. 47) was the gift of Euan MacIntyre (MerckResearch Laboratories, Rahway, N.J.). Polyclonal rabbitanti-human TNF-a and recombinant human TNF-a werepurchased from Genzyme (Boston, Mass.), and goat anti-rabbit IgG-horseradish peroxidase was from Southern Bio-technology (Birmingham, Ala.). Anti-IL-1, MAb, poly-clonal rabbit anti-human IL-13, and recombinant humanIL-1, were the gift of Jayne Chin (Merck Research Labora-tories). The PBS-Tween wash solution, bovine serum albu-min (BSA) blocking solution, and TMB peroxidase solutionsA and B for the ELISAs were from Kirkegaard & PerryLaboratories (Gaithersburg, Md.). Both IL-11 and TNF-axassays were performed by using the same general protocol.ELISA plates (96 well) were prepared by coating the wellsovernight at 4°C with 100 ,ul of a murine-derived anti-humancytokine MAb in PBS (1 ,ug/ml) per well and blocking themfor 1 h at room temperature (RT) with 200 ,l of lx BSAblocking solution per well. After washing the wells threetimes with PBS-Tween wash solution, 100 ,1 of cytokinestandard (in the appropriate medium) or experimental super-natants was added to each well. After 1 h of incubation atRT, plates were washed three times with PBS-Tween washsolution, and 100 ,ul of rabbit polyclonal anti-human cytokineserum (1:1,000 [vol/vol] in PBS-Tween) per well was added.After an additional hour of incubation at RT, the plates werewashed three times, and 100 ,u of goat anti-rabbit IgG-horseradish peroxidase (1:4,000 [vol/vol]) was added for anadditional hour. The plates were developed by washing themthree times and adding 100 RI of freshly mixed TMB perox-idase solutions A and B (1:1 [vol/vol]) per well at RT. Thereaction was stopped after 4 to 12 min by the addition of 100RId of 1 M phosphoric acid per well. The optical density wasread at 450 nm by an automated ELISA plate reader. Thesensitivity of the assay was generally 10 to 30 pg of TNF-aper ml of supernatant.mRNA detection by Northern (RNA) slot blot analysis.

Plasmids containing full-length cDNA probes for humanTNF-c and F-actin were the gifts of Bradley Schwartz(University of Wisconsin/Madison, Madison). Human CD14cDNA was the gift of S. Law (Merck Research Laborato-ries), and the plasmid pKoNeo, which contained the full-length cDNA for neomycin phosphotransferase, was pur-chased from Stratagene (La Jolla, Calif.). Full-length cDNAinserts were excised by restriction endonuclease digestion,separated from plasmid DNA by agarose gel chromatogra-

phy, electroeluted in Tris-EDTA buffer, and precipitated in75% ethanol (3).

Full-length cDNA probes were labeled with a randomprimer DNA labeling kit from Boehringer-Mannheim Bio-chemicals with [a-32P]dCTP (New England Nuclear, Bos-ton, Mass.) as described in the manufacturer's instructions.Unincorporated [a-32P]dCTP was removed with a spin col-umn (Bio-Rad Laboratories, Richmond, Calif.), and purifiedcDNA probes contained s5% unincorporated 32p with aspecific activity of approximately 108 cpm/,ug. RadiolabeledcDNA probes were denatured by boiling for 5 min immedi-ately prior to use.

All buffers used for Northern blots were made upin autoclaved diethylpyrocarbonate (DEPC)-treated, doublydistilled H20 in baked glassware. Spotting buffer consistedof 6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate) plus 7.5% formamide, and hybridization buffer con-sisted of 5x Denhardt's solution, 50% formamide, 0.2%sodium dodecyl sulfate (SDS), Sx SSPE (lx SSPE is 0.18 MNaCl, 10 mM NaPO4, and 1 mM EDTA [pH 7.7]), and 200p,g of boiled salmon sperm DNA per ml.

Total cellular RNA was extracted from 5 x 106 to 10 x 106cells per condition by using a commercial reagent (Tri-reagent; Medical Research Center, Cincinnati, Ohio) asdescribed in the manufacturer's instructions. For each sam-ple point, 10 ,g of RNA was dissolved in 100 pl of deionizedformamide, mixed with 100 pl of spotting buffer, and appliedto a presoaked (5 min in H20 and 5 min in 2x SSC)Zeta-probe membrane through a slot blot apparatus (Bio-Rad) under gentle suction. After air drying, the blot wasbaked for 1 h at 80°C and prehybridized in 10 ml ofhybridization buffer for 4 h at 42°C. Radiolabeled cDNA (100ng per blot) was added, and the blots were hybridizedovernight. After hybridization, the blots were washed se-quentially as follows: 2x SSC plus 0.5% SDS for 15 min at22°C, 0.5x SSC plus 0.5% SDS for 15 min at 22°C; 0.1x SSCplus 0.5% SDS for 15 min at 22°C, and 0.1x SSC plus 0.5%SDS for 30 min at 65°C. The blots were autoradiographed at-70°C for 1 to 3 days. Where necessary, the autoradiographswere quantified by using a scanning densitometer (Chromo-scan 3; Joyce Loebl) to compare cytokine and F-actin signalsfor the same points.Measurement of cell surface CD14. Surface CD14 was

quantified by microfluorimetry with a FACScan flow cytom-eter (Becton Dickinson) utilizing both direct and indirectimmunofluorescent staining (21). Uninduced SFM cells orcells differentiated with 1,25-dihydroxycholecalciferol werewashed and resuspended at 107/ml in serum-free medium inthe presence or absence of 1% human serum. Fifty-microli-ter aliquots were pipetted on ice into polystyrene tubes (12by 75 mm), and 25 ,ul of unconjugated My4, 3C10, or IgG2bcontrol was added to achieve a final saturating concentrationof 10 p.g/ml. The cells were incubated at 4°C for 1 h, washedagain in serum-free medium, and resuspended in 50 RI ofFITC-labeled goat anti-mouse IgG (1:100 [vol/vol]). After anadditional hour on ice, the cells were washed and resus-pended in 0.5 ml of serum-free medium containing propidiumiodide (1 ptg/ml). In other experiments, direct immunofluo-rescent staining with FITC-conjugated Leu M3 (20 pl per50-pl cell suspension) was used to quantitate CD14 expres-sion. A total of 10,000 events (gated to exclude nonviablecells) were collected in the log mode and expressed ashistograms of relative fluorescence intensity. Immunopre-cipitation of cell surface CD14 was performed with [35S]me-thionine-labeled cells and the anti-CD14 MAb 3C10. West-ern blot analysis was performed with My4.

INFECT. IMMUN.

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

SERUM AND CD14-INDEPENDENT PHAGOCYTE ACTIVATION BY LPS 4455

TABLE 1. Characteristics of SFM cells when induced to differentiate with PMA or calcitriol for 48 72 h

SFM cells

Characteristic Induced with:Uninduced

PMA (30 ng/ml) Calcitriol (0.1 ,uM)

Growth and adherence SFM doubling time, 40 h; SFM/HS Growth arrest; cells become Growth slows slightly; cellsdoubling time, 34 h; nonadherent adherent to plastic and remain in suspension.under normal growth conditions; spread within minutespartially adherent when grown todensity of >106 cells per ml

CD14 expression Surface CD14 not detectable; small Moderate expression of Large amount of surfaceamount of CD14 mRNA can be immunofluorescent cell CD14 detectable; CD14detected under normal growth surface CD14 mRNA accumulationconditions enhanced by approximately

100-foldLPS response Responds by accumulating both TNF-a Responds by making cytokine Responds by making cytokine

and IL-1,B mRNA; no measurable mRNA and by releasing mRNA and by releasingamount of cytokine released under mature cytokine proteins mature cytokine proteinsnormal growth conditions (i.e., up to including TNF-a, IL-11, including TNF-a, IL-11,400,000 cells/ml). and IL-6; maximal response and IL-6.

to LPS observed at 48 hafter PMA induction

Cell preparations and cell culture conditions. (i) Prepara-tion of isolated human peripheral blood mononuclear cells.Human mononuclear cells from healthy volunteers wereisolated from heparinized undiluted whole blood (30 IU/ml)by layering at a ratio of 2:1 (vol/vol) over Ficoll-Hypaqueand centrifugation at 400 x g at RT for 30 min, thusseparating a PMN band from a mononuclear cell band and anerythrocyte pellet. The mononuclear cell fraction was re-moved and washed three times in serum-free medium.Under the described conditions, a suspension of 105 mono-nuclear cells was added to each well of a 24-well tissueculture dish and stimulated overnight at 37°C. Supernatantswere subsequently assayed in duplicate for TNF-a byELISA. All cell stimulation experiments were repeated on atleast three occasions.

(ii) Establishment of the SFM cell line. The human promon-omyelocytic cell line THP-1 (36) was from the AmericanType Culture Collection (Rockville, Md.). The cells werecultured in macrophage serum-free medium (SFM cells) ormacrophage serum-free medium plus 1% HS serum(SFMIHS cells), both supplemented with ciprofloxacin (10p.g/ml) or penicillin (100 IU/ml) and streptomycin (50 p.g/ml).To be certain that no serum proteins were carried over fromprevious growth conditions, the cells were washed (after aperiod of weaning from serum-containing medium) and thengrown for at least 20 generations (and usually 250 genera-tions) prior to experimentation. The characteristics of theSFM cell line are summarized in Table 1.

(Mif) CHO cells cotransfected with human CD14 and pKoNeo.A CHO cell line transfected with CD14 (designated DG4)was established in collaboration with R. A. Zoeller (BostonUniversity, Boston, Mass.) by calcium phosphate precipita-tion (22) and were used as a source of control mRNA forCD14 Northern blots. CHO cells transfected with the controlplasmid pKoNeo had no detectable CD14 (data not shown).

(iv) SFM cell stimulation experiments. For cytokine releaseexperiments, we induced SFM cells for 36 to 48 h with1,25-dihydroxycholecalciferol (0.1 ,uM) or PMA (30 ng/ml).Immediately prior to experimentation, cells were washedtwice, to remove conditioned medium, and resuspended inserum-free medium or serum-free medium plus 1% HS at 106cells per ml. An 80-,ul portion of the cell suspension was then

added to each well of a 96-well dish, and 10 ,ul of a 10-foldconcentrate of control myeloma protein, anti-CD14 MAb, orLPS inhibitor, followed by 10 pA of a 10-fold dispersion ofReLPS, was added. The plate was incubated overnight at37°C, and the supernatant was assayed for TNF-at or IL-11by ELISA (for IL-13 measurement, the cells were subjectedto a brief freeze-thaw to release intracellular IL-1,B). Forexperiments with undifferentiated SFM or SFM/HS cells, 5x 106 uninduced cells, followed by blocking MAb or inhib-itors and ReLPS, was added to polypropylene tubes on ice.The tubes were incubated at 37°C in a shaking water bath for1 (TNF-a) or 3 (IL-11) h, returned to ice, and RNA extractedas described above.

RESULTS

Isolated human peripheral blood mononuclear cells appar-ently washed free of serum proteins respond to LPS by aCD14-dependent pathway. Human mononuclear cells thathad been washed three times by low-speed centrifugation inserum-free medium were then stimulated overnight withReLPS in the presence or absence of 1% autologous HS. Thewashed mononuclear cells were 10- to 30-fold less sensitiveto ReLPS in the absence of serum (data not shown). Withconcentrations of LPS which induced similar amounts ofTNF-a release, the anti-CD14 MAb 3C10 inhibited LPS inboth the presence and absence of serum (Fig. 2). In both thepresence and absence of serum, increasing concentrations ofLPS could fully overcome the inhibition by anti-CD14 MAb(Fig. 2B and data not shown). Similar results were obtainedwith anti-CD14 MAb My4 and when measuring IL-1-.

Characterization of the SFM cell line: expression of CD14.When SFM or SFM/HS cells were assayed during mid-loggrowth, we were consistently unable to detect surface im-munofluorescent CD14 on undifferentiated SFM or SFM/HScells by flow microfluorimetry with several anti-CD14 MAb(Fig. 3A). The sensitivity of detection of surface proteins byflow microfluorimetry with FITC-labeled antibodies is be-tween 500 and 3,000 molecules per cell (30). Immunoprecip-itation of 35S-labeled monocytes and Western blot (immuno-blot) analysis, by using MAb My4, also failed to detect cell

VOL. 61, 1993

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

4456 LYNN ET AL.

180

_ 160

l 140

f 120

zM 100-80

@ 60co

40-

- 20

oJ

B10.0

8.0

6.0

4.0

2.0

T LPS, 10ng/ml

+LPS

O NoLPS

LPS, 100 ng/mI

No 3C10 + 3C10 No 3C10 +3C10+ 1% Serum Serum-free

IgG2b, 10 pg(mI

* 3C10, 10 tgml

0.0 --

o1 100 01 1 o2 i03 04

[LPS], pg/mlFIG. 2. LPS-induced TNF-a release from washed, serum-free

mononuclear cells is inhibited with the anti-CD14 MAb 3C10. (A)Mononuclear cells were prepared from fresh heparinized blood bycentrifugation over Ficoll-Hypaque. The cells were washed threetimes in serum-free medium and resuspended at 105 cells per ml inserum-free medium supplemented with 1% autologous HS or an

equal volume of PBS. Each cell suspension was divided into twoaliquots on ice, and the anti-CD14 MAb 3C10 or myeloma proteincontrol (final concentration, 10 p.g/ml) was added. A 0.9-ml portionof the cell suspension, followed by 100 ,ul of a sonicated dispersionof ReLPS at 0, 0.1, 10, and 100 ng/ml, was then added to each wellof a 24-well tissue culture dish. The plate was incubated overnight at37°C, and each supernatant was assayed for released TNF-a induplicate. Values shown are taken from wells with approximately90% of maximal response for each condition and represent meanTNF-a concentrations; error bars represent the ranges of duplicatewells. (B) Human mononuclear cells were resuspended in RPMI1640-10% FCS at 106 cells per ml. Aliquots (180 p,l), followed by 10p1 of a 20x solution of MAb 3C10 (final concentration, 20 p,g/ml) orIgG2b control antibody (20 pg/ml), were pipetted into each well of a96-well tissue culture dish, and then 10 Ill of a 20-fold dispersion ofReLPS was added to each well. After overnight culture at 37'C in5% CO2, the supematants were harvested and assayed for TNF-a asdescribed in Materials and Methods. Results shown are from a

single representative experiment, and the same pattern of inhibitionwas seen on several occasions.

surface CD14 in undifferentiated SFM and SFM/HS cells(data not shown).

In contrast, when differentiated for 48 h with 1,25-dihy-droxycholecalciferol (Fig. 3A) or PMA (not shown), therewas an approximately 100-fold increase in the immunofluo-rescent signal compared with that of uninduced cells, indi-cating abundant expression of cell surface CD14. Similarresults were obtained for SFM cells irrespective of thepresence of added HS during growth and staining. Exposureto LPS (up to 3 jig/ml) did not affect surface expression ofCD14 in SFM or SFM/HS cells (data not shown). Further-more, when LPS was incubated with 1,25-dihydroxychole-calciferol-treated SFM or SFM/HS cells, the ability of MAb3C10, My4, and Leu M3 to label CD14 as assessed byimmunofluorescent staining was unimpaired (data notshown).Northern analysis of slot-blotted RNA revealed an ex-

tremely small, yet consistently detectable, amount of CD14mRNA (Fig. 3B) in undifferentiated SFM and SFM/HS cells.Induction with 1,25-dihydroxycholecalciferol greatly in-creased the CD14 mRNA in both SFM and SFM/HS cells(Fig. 3B). Thus, undifferentiated SFM cells express smallamounts of CD14 mRNA when compared with differentiatedcells and the expression of surface protein is below thedetection ability of flow microfluorimetry. These observa-tions suggest that the actual expression of surface CD14molecules on the undifferentiated SFM cells is extremelylow.

Characterization of 1,25-dihydroxycholecalciferol-inducedSFM cells (CD14+): the presence of serum proteins increasesthe sensitivity ofSFM cells to LPS. To establish that growth inserum-free medium did not fundamentally alter the pheno-type of SFM cells with respect to LPS responsiveness, westimulated 1,25-dihydroxycholecalciferol-differentiated cells(CD14+) with LPS and compared the responsiveness ofthese cells to those grown and stimulated in the presence ofserum. The SFM cells were approximately 30-fold lesssensitive to LPS (with respect to IL-1,B and TNF-ao release)in comparison with SFM cells stimulated in the presence ofserum (data not shown). Nevertheless, 1,25-dihydroxychole-calciferol-treated SFM cells could be stimulated to releaseTNF-a, in response to LPS, despite the complete absence ofserum proteins for >20 generations of growth (Fig. 4). Therelease of TNF-a by SFM cells in response to heat-killed S.aureus was not affected by these culture conditions (data notshown). Because the growth conditions of the cells werestringently serum-free, it is reasonable to conclude that theability of macrophages to respond to LPS was not dependenton the presence of serum proteins although these proteinsclearly facilitated LPS-induced cellular activation. Similarresults were obtained when the supernatants were assayedfor IL-10 rather than TNF-ax (data not shown).Comparison of the effects of anti-CD14 MAb and lipid A

analog or substructure inhibitors on CD14+ SFM cells revealsa CD14-independent mechanism ofLPS inhibition by lipid IVaand RSLA. Because the known serum LPS accessory pro-teins have been shown to mediate LPS binding to CD14, wehypothesized that cellular activation under serum-free con-ditions might be occurring primarily through a CD14-inde-pendent pathway. When 1,25-dihydroxycholecalciferol-treated SFM cells (CD14+) were stimulated in the presenceof human serum, LPS-induced activation could be abrogatedsubstantially by coincubating cells in the presence of theanti-CD14 MAb My4 (Fig. 4A). However, LPS stimulationunder serum-free conditions could not be inhibited by MAbto CD14 (Fig. 4B). Identical results were obtained with MAb

A

'b

E-

INFECT. IMMUN.

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

SERUM AND CD14-INDEPENDENT PHAGOCYTE ACTIVATION BY LPS 4457

No -IgG2b, 11i

(,I- i).

/], .')

I A

Uiii;iduced SFNILIS

. =i-' I F IT1 - I 1 T r

lit 11 iC 1-4

IgG2b

Vlitaiiiri D3Iiiduced SFNMIIS

- -- MIY4

Relative Intensity Of Fluorescence

Bl

Co

lb C,L

4am

1 2 3 4 5

3C10 (data not shown). Anti-CD14 MAb did not impair theresponse to heat-killed S. aureus (data not shown).

In contrast to the results obtained with MAb My4 and3C10, the LPS inhibitor RSLA inhibited the effects of LPSon differentiated SFM cells both in the presence (Fig. SA)and absence (Fig. 5B) of serum. In serum-free conditions,equal concentrations of RSLA and LPS resulted in approx-imately 50% inhibition of the response; a 10-fold excess ofRSLA produced at least a 10- to 30-fold shift in the dose-response curve to LPS (Fig. SB). Similar results were seenwith lipid IVa (data not shown). The differences in serum

dependence between RSLA and lipid IVa compared withspecific anti-CD14 MAb suggest that these molecules com-pete with LPS at a common receptor site that is differentfrom the site recognized by the MAb, i.e., a protein that isdistinct from CD14.Comparison of CD14-dependent and independent pathways

of LPS stimulation in uninduced (CD14-) SFM cells. To

FIG. 3. (A) Detectable cell surface CD14 on SFM/HS cells ispresent only when the cells are induced to differentiate. UninducedSFM/HS cells and SFM/HS cells treated with 0.1 ,uM 1,25-dihy-droxycholecalciferol for 48 h were stained for CD14 expression byindirect immunofluorescence, and FACScan analysis was carriedout as detailed in Materials and Methods. The histograms on the leftshow IgG2b and My4 (10 pg/ml) staining of uninduced SFM/HScells, and the histograms on the right show IgG2b and My4 stainingof 1,25-dihydroxycholecalciferol-induced cells. (B) CD14 mRNA inSFM and SFMIHS cells is minimally detectable in undifferentiatedcells and is greatly induced by 1,25-dihydroxycholecalciferol. Totalcellular RNA was extracted from 5 x 106 cells per point and probedfor CD14 mRNA as described in Materials and Methods. Lanes: 1and 2, 10 p,g of RNA from SFM cells; 3 and 4, 10 p,g of RNA fromSFM/HS cells; 2 and 4, cells were induced for 48 h with 1,25-dihydroxycholecalciferol (D3); 5, control RNA from clone DG4(top, 0.1 pLg of total RNA; bottom, 1 p,g of total RNA), a transfectedCHO fibroblast cell line expressing high levels of human CD14.Probing for F-actin gave an equal signal for lanes 1 to 4 (not shown).

assess the ability of monocytic cells to respond indepen-dently of CD14, undifferentiated SFM cells were exposed toLPS in the absence or presence of serum and assayed for theaccumulation of TNF-a mRNA by Northern slot blot hybrid-ization (Fig. 6). Although less responsive to LPS in theabsence of serum, as little as 1 ng of ReLPS per mlconsistently induced a two- to threefold increase in TNF-amRNA in undifferentiated SFM cells (Fig. 6A, lane 1, andFig. 6B, top). This was despite the complete absence ofserum-derived LPS accessory proteins for several months ofcell growth and the expression of extremely low amounts ofcell surface CD14. The addition of anti-CD14 blocking MAb

A-2

1

i ii3

VOL. 61, 1993

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

4458 LYNN ET AL.

*~- IgG2b.t.**.- +My4

10 100 1000

A87000 -

i 6000-.

5000 _

Z 4000-

PC 3000-

w 2000 -

X 1000-

01

no added RSLA. + RSLA, 1 jg/mi

-h-- + RSLA, 10 ,ug/mI

10 100 1000

[LPS], ng/ml

B 600-JS500-

S50 1IgG2b400 ii****. +My4

pz 300

Y 200Y

100

0 -II, .. ..... _K0 10 100 1000 10000

[LPS], ng/mlFIG. 4. Anti-CD14 MAb My4 inhibits LPS only in the presence

of HS. SFM/HS (A) and SFM (B) cells were induced to expressCD14 with 1,25-dihydroxycholecalciferol for 48 h and resuspendedin the appropriate fresh medium at 106 cells per ml. Cells were platedin 96-well dishes (160 pl per well), a 20-pul portion ofMy4 (100 p,g/ml)or IgG2b was added immediately, and the addition of an equalvolume of a 20-fold dispersion of LPS in PBS or PBS alone followed.Cells were placed in a 5% CO2, 37°C incubator overnight, and thesupernatants were assayed for released TNF-a by ELISA. Theresults are representative of three separate experiments.

had only a slight (maximum of 20 to 30%) inhibitory effect inserum-free conditions (Fig. 6A, lane 2, and Fig. 6B, top). Incontrast, SFM cells stimulated both in the presence of 1 ,ugof LBP per ml or 1% HS were readily activated by LPS, aneffect which was largely inhibited with anti-CD14 MAb (Fig.6A, lanes 3 to 6, and Fig. 6B, middle and bottom). Theinhibition by MAb My4, in the presence of serum or LBP,was overcome by high concentrations of LPS. Attempts tosubstitute for serum with immunoaffinity-purified solublehuman CD14 (1 ,ug/ml) were not successful (data not shown).Similar results were obtained if IL-1lB mRNA was measuredafter a 3-h incubation with LPS and when 3C10 was used(data not shown). The induction of TNF-at mRNA in re-sponse to heat-killed S. aureus was unaffected by anti-CD14MAb.

[LPS], ng/ml

B 450

400

9 350

300

Z 250

PCs 200

c 150a.)

p 100

50

0

0

° no added RSLA-.....RSLA, 1 g/ml---a--l RSLA, 10 pg/mI

/04

i~'II !S-I ----

100 1000 10000

[LPS], ng/mlFIG. 5. The LPS inhibitor RSLA inhibits LPS in both the pres-

ence and absence of HS. SFM/HS (A) and SFM (B) cells weredifferentiated with 1,25-dihydroxycholecalciferol and plated in 96-well dishes as described in the legend to Fig. 4. A 20-pl portion of a10-fold suspension of RSLA was then added immediately, and theaddition of a 10-fold suspension of ReLPS to achieve the indicatedfinal concentrations of ligand and inhibitor followed. Plates wereplaced in a 5% CO2 atmosphere at 37°C overnight. Supernatantswere then assayed for released TNF-a by ELISA. The results of acompetition study performed in the presence (A) and absence (B) ofserum are shown. Each point represents the mean of three separatedeterminations ± the standard deviation. The results are represen-tative of three separate experiments.

DISCUSSION

The discovery that CD14 could function as a receptor forLPS-LBP complexes (42) and initiate signal transductionwas a fundamental breakthrough in understanding how LPSactivates macrophages and PMN. Even before the role ofCD14 was first described as an LPS signaling molecule,transmembrane forms of CD14 were sought unsuccessfully(5, 11). Thus, the glycosyl phosphatidylinositol-linked pro-tein, lacking an intracellular domain, is the only form of theprotein known to exist. Previous investigators have hypoth-esized that CD14 might act as a receptor complex with otherproteins and that alternative pathways of cell activation by

A6A1pi5000-

v 4000

zE 3000

cu 2000

X4 1000

INFECT. IMMUN.

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

SERUM AND CD14-INDEPENDENT PHAGOCYTE ACITIVATION BY LPS 4459

A LPSJ, ng/'mI-p..

1 SFM

2 SFM

3 SFM LBP

4 SFM LBP

5 SFM 1%HS

6 SFM 1%°.o HS

0 1 102 104 My4

- - 4~ -

- -_ -

-If"~.,'W_ 4 .m -

L

Lane 1 2 3 HKSAFIG. 6. Undifferentiated SFM cells can respond to LPS in the

complete absence of serum proteins. Purified LBP or 1% HSenhances a CD14-dependent pathway despite very low levels ofCD14 expression on undifferentiated SFM cells. (A) A total of 5 x106 SFM cells (rows 1 and 2), SFM cells incubated with purifiedrecombinant LBP (1 pg/ml, rows 3 and 4), and SFM/HS cells (rows5 and 6) were incubated with the indicated final concentrations ofLPS for 1 h at 37°C in a shaking water bath. Total cellular RNA wasextracted and loaded per slot as described in Materials and Meth-ods. The boxed slots represent SFM/HS cells stimulated with a1:100 dilution of heat-killed S. aureus (HKSA). In row 3, lane 2, itappears that slightly less TNF-a mRNA accumulated in the pres-ence of LBP; this was not seen in other experiments under identicalconditions. The experiment shown is representative of four similarindividual experiments. (B) The Northern blot was quantified with ascanning densitometer. The results are expressed as the ratio ofTNF-a mRNA/F-actin mRNA for each point. (Top) SFM cells(panel A, rows 1 and 2); (middle) SFM cells plus 1 Fg of LBP per ml(panel A, rows 3 and 4); (bottom) SFM cells plus 1% HS (panel A,rows 5 and 6).

B 2.2-

.8- SFMN +MN-* 1.6cc; 1.4

..* 1.2cu 1.0

0.8Z 0.6

0.4 -

0.2 -

I

0.0

0

A4

1J-

I

t- - ......... 3 .......~~~~~

100 101:000

[LPS], ng/ml

El SFNIM+LBP

0coCu

C)

zEt

0.40.20.0

El SFM+LBP+My4

0 1 100 10000

[LPS], ng/ml

LPS may be present in human cells (28, 32, 37). Data fromKitchens et al. suggested that at least two proteins areinvolved in CD14-dependent signaling of THP-1 cells (14),namely, CD14 and a lipid IVa-inhibitable element which hasnot yet been defined. This latter protein may constitute a

portion of the hypothesized CD14 receptor complex. Thedemonstration that transfected CD14 greatly enhanced LPSresponsiveness in the already LPS-responsive murineB-lymphoma cell line 70713 is evidence that a cell lineutilizing two or more LPS receptors can be geneticallyengineered (16). However, it cannot be determined fromthese studies if the transfected CD14 signals in collaborationwith the previously existing signaling system or represents a

completely separate activation pathway. In addition, whiletransfected murine lymphocytes are a model for LPS signal-ing in human macrophages, it is conceivable that macro-phages may utilize different signaling pathways. Thus, thisreport provides the first direct evidence that human mono-

cytic cells can be stimulated independently of CD14 andserum proteins.

In this report, we present several lines of evidence whichdemonstrate that a simple model of LPS signaling in macro-phages involving CD14 or a CD14 receptor complex as thesole signaling protein is likely to be incomplete. AlthoughLPS was capable of stimulating washed monocytes, and thisresponse could be abrogated by anti-CD14 MAb, it could notbe concluded on the basis of these experiments that LPS was

capable of stimulating via CD14 without the help of serum

accessory proteins. Serum proteins, such as LBP, may have

2.2 -

2.0- E SF.NIHS.3 SFMIHS+My4

1.6

r 1.41l.2-1.0

Z08 -

X

F- 0.6 -

0.4-0.2 -

0.01* 0 1 100 HKSA

[LPS], ng/ml

remained adsorbed to the mononuclear cell surface and werestill capable of mediating LPS-CD14 interactions.These unexplained results encouraged us to establish the

SFM cell line, where the levels and expression of LBP, otherserum factors, and CD14 could be controlled. Our resultswith the SFM cell lines lead us to conclude that mononuclearcells can be stimulated with LPS in the complete absence ofserum LPS-binding proteins and that such activation primar-ily occurs independently of CD14 receptor binding. Thus, wehave provided direct experimental evidence of a non-CD14-dependent LPS signaling pathway. Our observations that the

VOL. 61, 1993

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

4460 LYNN ET AL.

LPS inhibitors lipid IVa and RSLA were not dependent uponthe presence of serum to antagonize LPS, in contrast toanti-CD14 MAb, is strong evidence for the existence of analternate LPS receptor(s).

Lipid IVa has been shown to bind to LBP in vitro (34), andit has been suggested that the lipid A-like LPS antagonistsRSLA and lipid IVa may act by competing with LPS forbinding to LBP. Our results indicate that RSLA, lipid IVa,and, presumably, other lipid A substructure inhibitors suchas deacylated LPS (8, 14, 21, 25) and (2-keto-3-deoxyoctu-losonic acid)2-IVa (8, 21) can inhibit LPS activation ofmonocytes without interacting with LBP or septin. Theseobservations are consistent with those reported by Kitchenset al. (14) and indicate that this class of LPS inhibitorscompetes with LPS at a site which is neither LBP, septin,nor CD14.

Finally, experiments performed with undifferentiatedSFM cells (in which cell surface CD14 could not be detectedby flow microfluorimetry and Western blot analyses butwhich produce small amounts of CD14 mRNA) demon-strated that these cells could nevertheless be stimulated inthe complete absence of serum. When serum or LBP was

present, a CD14-dependent pathway of activation predomi-nated at low concentrations of LPS. This could not beexplained by the recruitment of soluble CD14 from serum

since the CD14-dependent enhancement of LPS response

was also seen when pure LBP was added to the serum (andCD14)-free medium. This result is presumably due to a

potent effect of extremely low numbers of CD14 moleculeson the surface of these cells. In contrast, at higher concen-

trations of LPS, in the presence of blocking anti-CD14 MAband in the absence of serum accessory proteins, a CD14-independent pathway of LPS stimulation appears to predom-inate.Although the sensitivity of SFM cells to LPS was in-

creased by LBP or serum, SFM cells were still responsive toas little as 1 ng of LPS per ml in completely serum-freeconditions (Fig. 6). Non-CD14 pathways of phagocytic acti-vation may be important at local sites of infection where theconcentration of LPS is very high. We have calculated thatconcentrations of up to 10 ,ug of LPS per ml occur with fecalsoiling of the peritoneum (on the basis of calculations of LPSdensity described by Galloway and Raetz [7]), and similarlyhigh concentrations of LPS are likely to exist in abscesscavities. Therefore, although the CD14-LBP pathway ofmonocyte activation is likely to predominate for circulatingleukocytes, a CD14-independent pathway of cell stimulationat other sites of LPS exposure may be physiologicallyrelevant.The goal of defining LPS receptors on macrophages and

the LPS signal transduction pathway has been elusive.Perhaps the most important contribution of the discovery ofCD14 as an LPS signaling protein is that it provides directevidence that a signaling system exists. The delineation ofthe various signaling receptors is important because theelucidation of the precise cellular events underlying LPSstimulation of phagocytes will increase our understanding ofthe pathogenesis of gram-negative sepsis. Ultimately, theselines of inquiry may suggest new therapeutic options for thiscommon and still highly lethal disorder.

ACKNOWLEDGMENTS

This work was supported by NIH grants R29-GM47127 andPO1-AI33087 (D.T.G.). W.A.L. was the recipient of a travelingfellowship from Glaxo.

REFERENCES1. Bone, R. C. 1991. The pathogenesis of sepsis. Ann. Intern. Med.

115:457-469.2. Couturier, C. N., N. Haeffner-Cavaillon, L. Weiss, E. Fischer,

and M. D. Katzatchkine. 1990. Induction of cell associated IL-1through stimulation of the adhesion-promoting proteins LFA-1(CD11a/CD18) and CR3 (CD11b/CD18) of human monocytes.Eur. J. Immunol. 5:999-1006.

3. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basicmethods in molecular biology, p. 388. Elsevier Science Publish-ing Co., Inc., Cambridge.

4. Dziarski, R. 1991. Peptidoglycan and lipopolysaccharide bind tothe same binding site on lymphocytes. J. Biol. Chem. 266:4719-4725.

5. Ferrero, E., C. L. Hsieh, U. Francke, and S. M. Goyert. 1990.CD14 is a member of the family of leucine-rich proteins and isencoded by a gene syngenic with multiple receptor genes. J.Immunol. 145:331-336.

6. Frey, E. A., D. S. Miller, T. G. Jahr, A. Sundan, V. Bazil, T.Espevik, B. B. Finlay, and S. D. Wright. 1992. Soluble CD14participates in the response of cells to LPS. J. Exp. Med.176:1665-1671.

7. Galloway, S. M., and C. R. Raetz. 1990. A mutant of Escherichiacoli defective in the first step of endotoxin biosynthesis. J. Biol.Chem. 265:63946402.

8. Golenbock, D. T., R. Y. Hampton, N. Qureshi, K. Takayama,and C. R. H. Raetz. 1991. Lipid A-like molecules that antago-nize the effects of endotoxins on human monocytes. J. Biol.Chem. 266:19490-19498.

9. Golenbock, D. T., R. Y. Hampton, C. R. H. Raetz, and S. D.Wright. 1990. Human phagocytes have multiple lipid A-bindingsites. Infect. Immun. 58:4069-4075.

9a.Golenbock, D. T., and W. A. Lynn. Unpublished data.10. Hampton, R. Y., D. T. Golenbock, M. Penman, M. Krieger, and

C. R. H. Raetz. 1991. Recognition and plasma clearance ofendotoxin by scavenger receptors. Nature (London) 352:342-344.

11. Haziot, A., S. Chen, E. Ferrero, M. G. Low, R. Silber, and S. M.Goyert. 1988. The monocyte differentiation antigen, CD14, isanchored to the cell membrane by a phosphatidylinositol link-age. J. Immunol. 141:547-552.

12. Heumann, D., P. Gallay, C. Barras, P. Zaech, R. J. Ulevitch,P. S. Tobias, M. P. Glauser, and J. D. Baumgartner. 1992.Control of lipopolysaccharide (LPS) binding and LPS-inducedtumor necrosis factor secretion in human peripheral bloodmonocytes. J. Immunol. 148:3505-3512.

13. Kirkland, T. N., G. D. Virca, T. Kuus-Reichel, F. K. Multer,S. Y. Kim, R. J. Ulevitch, and P. S. Tobias. 1990. Identificationof lipopolysaccharide-binding proteins in 70Z/3 cells by photo-affinity cross-linking. J. Biol. Chem. 256:9520-9525.

14. Kitchens, R. L., R. J. Ulevitch, and R. S. Munford. 1992.Lipopolysaccharide (LPS) partial structures inhibit responses toLPS in a human macrophage cell line without inhibiting LPSuptake by a CD14-mediated pathway. J. Exp. Med. 176:485-494.

15. Kovach, N. L., E. Yee, R. S. Munford, C. R. H. Raetz, and J. H.Harlan. 1990. Lipid IVA inhibits synthesis and release of tumornecrosis factor induced by lipopolysaccharide in human wholeblood ex vivo. J. Exp. Med. 172:77-84.

16. Lee, J. D., K. Kato, P. S. Tobias, T. N. Kirkland, and R. J.Ulevitch. 1992. Transfection of CD14 into 70Z/3 cells dramati-cally enhances the sensitivity to complexes of lipopolysaccha-ride (LPS) and LPS binding protein. J. Exp. Med. 175:1697-1705.

17. Lei, M., S. A. Stimpson, and D. C. Morrison. 1991. Specificendotoxic lipopolysaccharide-binding receptors on murine sple-nocytes. III. Binding specificity and characterization. J. Immu-nol. 147:1925-1932.

18. Lei, M. G., and D. C. Morrison. 1988. Specific endotoxiclipopolysaccharide-binding proteins on murine splenocytes. I.Detection of lipopolysaccharide-binding sites on splenocytesand splenocyte subpopulations. J. Immunol. 141:996-1005.

19. Lei, M. G., and D. C. Morrison. 1988. Specific endotoxic

INFEcr. IMMUN.

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

SERUM AND CD14-INDEPENDENT PHAGOCYTE ACTIVATION BY LPS 4461

lipopolysaccharide-binding proteins on murine splenocytes. II.Membrane localization and binding characteristics. J. Immunol.141:1006-1011.

20. Loppnow, H., H. Brade, I. Durrbaum, C. A. Dinarello, S.Kusumoto, E. T. Rietschel, and H. D. Flad. 1989. IL-1 inductioncapacity of defined lipopolysaccharide partial structures. J.Immunol. 142:3229-3238.

21. Lynn, W. A., C. R H. Raetz, N. Qureshi, and D. T. GolenbocL1991. LPS induced stimulation of CD11b/CD18 expression onneutrophils: evidence of specific receptor-based response andinhibition by lipid A-based antagonists. J. Immunol. 147:3072-3079.

22. McClelland, A., M. E. Kamarck, and F. H. Ruddle. 1987.Molecular cloning of receptor genes by transfection. MethodsEnzymol. 147:280-291.

23. Morrison, D. C., and J. L. Ryan. 1987. Endotoxins and diseasemechanisms. Annu. Rev. Med. 38:417-432.

24. Parillo, J. E., M. M. Parker, C. Natanson, A. F. Suifredini, R. L.Danner, R. E. Cunnion, and F. P. Ognibene. 1990. Septic shockin humans. Advances in the understanding of pathogenesis,cardiovascular dysfunction, and therapy. Ann. Intern. Med.113:227-241.

25. Pohlman, T. H., R. S. Munford, and J. M. Harlan. 1987.Deacylated lipopolysaccharide inhibits neutrophil adherence toendothelium induced by lipopolysaccharide in vitro. J. Exp.Med. 165:1393-1402.

26. Raetz, C. R. H. 1990. Biochemistry of endotoxins. Annu. Rev.Biochem. 59:129-170.

27. Raetz, C. R. H., K. A. Brozek, T. Clementz, J. D. Coleman,S. M. Galloway, D. T. Golenbock, and R. Y. Hampton. 1988.Gram-negative endotoxin: a biologically active lipid. ColdSpring Harbor Symp. Quant. Biol. 53:973-982.

28. Rietschel, E. T., and H. Brade. 1992. Bacterial endotoxins. Sci.Am. 267:26-31.

29. Schumann, R. R., S. R. Leong, G. W. Flaggs, P. W. Gray, S. D.Wright, J. C. Mathison, P. S. Tobias, and R. J. Ulevitch. 1990.Structure and function of lipopolysaccharide binding protein.Science 249:1429-1431.

30. Shapiro, H. M. 1988. Practical flow cytometry, p. 176. Alan R.Liss, Inc., New York.

31. Takayama, K., N. Qureshi, B. Beutler, and T. N. Kirkland. 1989.Diphosphoryl lipid A from Rhodopseudomonas sphaeroidesATCC 17023 blocks induction of cachectin in macrophages bylipopolysaccharide. Infect. Immun. 57:1336-1338.

32. Tobias, P. S., J. Mathison, D. Mintz, J. D. Lee, V. Kravchenko,K. Kato, J. Pugin, and R. J. Ulevitch. 1992. Participation oflipopolysaccharide-binding protein in lipopolysaccharide-de-pendent macrophage activation. Am. J. Respir. Cell. Mol. Biol.7:239-245.

33. Tobias, P. S., J. C. Mathison, and R. J. Ulevitch. 1988. A familyof lipopolysaccharide binding proteins involved in responses togram-negative sepsis. J. Biol. Chem. 263:13479-13481.

34. Tobias, P. S., K. Soldau, and R. J. Ulevitch. 1989. Identificationof a lipid A binding site in the acute phase reactant lipopolysac-charide binding protein. J. Biol. Chem. 264:10867-10871.

35. Tobias, P. S., K. Soldau, and R J. Ulevitch. 1986. Isolation of alipopolysaccharide-binding acute phase reactant from rabbitserum. J. Exp. Med. 164:777-793.

36. Tsuchiya, S., M. Yamabe, Y. Yamaguchi, Y. Kobayashi, T.Konno, and K. Tada. 1980. Establishment and characterizationof a human acute monocytic leukemia cell line (THP-1). Int. J.Cancer 26:171-176.

37. Wright, S. D. 1991. Multiple receptors for endotoxin. Curr.Opin. Immunol. 3:83-90.

38. Wright, S. D., P. A. Detmers, Y. Aida, R. Adomowski, D. C.Anderson, Z. Chad, L. G. Kabbash, and M. S. Pabst. 1990.CD18 deficient cells respond to lipopolysaccharide in vitro. J.Immunol. 144:2566-2571.

39. Wright, S. D., and M. T. C. Jong. 1986. Adhesion-promotingreceptors on human macrophages recognize Escherichia coli bybinding to lipopolysaccharide. J. Exp. Med. 164:1876-1888.

40. Wright, S. D., R. A. Ramos, A. H. Hermanowski-Vosatka, P.Rockwell, and P. A. Detmers. 1991. Activation of the adhesivecapacity of CR3 on neutrophils by endotoxin: dependence onlipopolysaccharide binding protein and CD14. J. Exp. Med.173:1281-1286.

41. Wright, S. D., R. A. Ramos, M. Patel, and D. S. Miller. 1992.Septin: a factor in plasma that opsonizes lipopolysaccharide-bearing particles for recognition by CD14 on phagocytes. J.Exp. Med. 176:719-727.

42. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, andJ. C. Mathison. 1990. CD14, a receptor for complexes oflipopolysaccharide (LPS) and LPS-binding protein. Science249:1431-1433.

43. Zoeller, R. A., Y. Liu, F. H. Millham, M. W. Freeman, and D. T.Golenbock. Surface expression of human CD14 in chinesehamster ovary fibroblasts transfers macrophage-like responsive-ness to bacterial endotoxin. J. Biol. Chem., in press.

VOL. 61, 1993

on March 26, 2021 by guest

http://iai.asm.org/

Dow

nloaded from