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PathwaysMonocytes Via Two CD14-Dependent Bacterial Lipopolysaccharide Can Enter

MunfordRichard L. Kitchens, Ping-yuan Wang and Robert S.

http://www.jimmunol.org/content/161/10/55341998; 161:5534-5545; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/161/10/5534.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Bacterial Lipopolysaccharide Can Enter Monocytes Via TwoCD14-Dependent Pathways1

Richard L. Kitchens,2* Ping-yuan Wang,‡ and Robert S. Munford* †

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes.We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchoredCD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonicmedium obliterated coated pits and blocked125I-labeled transferrin internalization, but failed to inhibit CD14-mediated inter-nalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS movedprincipally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively littlemoved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS andBODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast,in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, amuch larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These resultssuggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginationsthat direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS entersvia coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediatedpathway. The Journal of Immunology,1998, 161: 5534–5545.

Sensitive immune recognition of Gram-negative bacterialLPS (LPS or endotoxin) requires CD14, a GPI3-anchoredprotein expressed by monocytes, macrophages, and neu-

trophils, and is enhanced by LPS-binding protein (LBP), a solubleserum protein (1–5). LBP binds LPS aggregates and rapidly trans-fers them to GPI-anchored CD14 (mCD14) or to soluble CD14(sCD14), which are thought to promote signal responses by facil-itating the interaction of LPS with as yet unidentified signalingmolecules (6–9). mCD14 may also internalize LPS monomers oraggregated LPS-LBP complexes (10–14). Although LPS internal-ization by phagocytes has generally been regarded as a disposalfunction (12), several recent studies have suggested that internal-ization may be required for LPS signaling (13, 15–17). Clarifica-tion of the signaling versus disposal functions of LPS internaliza-tion should be fostered by a more complete understanding of themembrane structures and biochemical mechanisms that mediatethe internalization process.

The most thoroughly characterized pathway for receptor-medi-ated endocytosis involves the entry of ligand-receptor complexesinto clathrin-coated pits, a process that largely involves transmem-brane proteins that have a specific cytoplasmic domain-targetingsignal (18, 19). Although GPI-anchored proteins lack coated pit-targeting signals, certain of these proteins have been shown toenter coated pits (20, 21) presumably by associating with anotherprotein. Some GPI-anchored proteins, when bound by anti-receptor Abs (22–24) or extracellular ligands (25), may also entercaveolae or similar noncoated invaginations. Caveolae, the best-characterized noncoated membrane invaginations, are thought tobe involved in potocytosis, transcytosis, and signal transduction(20, 26–28). Nonclathrin-mediated internalization pathways maybe taken by protein toxins, such as ricin (29, 30) or cholera toxin(31), that bind to membrane glycolipids, and by other proteins,such as IL-2 (32), that bind to transmembrane receptors lackingcoated pit-targeting signals. The biochemical and functional prop-erties of noncoated invaginations found in cells that do not expresscaveolin (e.g., resting monocyte/macrophages and lymphocytes)are not well understood, however, and it is unclear whether vesi-cles derived from these invaginations recycle or move to late en-dosomes and lysosomes.

Other noncoated structures may also mediate endocytosis inmacrophages. Nichols (33) noted that alveolar macrophages inter-nalize horseradish peroxidase into tubular invaginations of theplasma membrane (tubular pinosomes). Myers et al. (34) subse-quently showed that multivalentb-very low-density lipoprotein(b-VLDL) particles enter larger surface-connected tubules (sur-face tubules for entry into macrophages, or STEMs (35)) in murinemacrophages. Whereas tubular pinosomes could acquire acidphosphatase, presumably from fusion with lysosomes (33), the sur-face-connected tubules noted by Myers et al. were thought to de-tach from the surface and transport VLDL to perinuclear lyso-somes. More recently, Zhang et al. (36) have described even largersurface-connected compartments induced by aggregated LDL in

Departments of *Internal Medicine and†Microbiology, and‡Cell Regulation Grad-uate Program, University of Texas Southwestern Medical Center, Dallas, TX 75235

Received for publication April 16, 1998. Accepted for publication July 9, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grant AI18188 from the National Institute for Allergyand Infectious Diseases and Grant AR41940 from the National Institute for Arthritis,Musculoskeletal, and Skin Diseases.2 Address correspondence and reprint requests to Dr. Richard L. Kitchens, Universityof Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9113. E-mail address: RKITCH@mednet.swmed.edu3 Abbreviations used in this paper: GPI, glycosylphosphatidylinositol; BODIPY FL,BODIPY fluorescein; CHO, Chinese hamster ovary; DAg-LPS, partially disaggre-gated LPS; EM, electron microscope;125I-Tf, 125I-labeled human holo-transferrin;LBP, LPS-binding protein; LDL, low-density lipoprotein; LDLR, LDL receptor; LY,lucifer yellow; mCD14, membrane CD14; MFI, mean fluorescence intensity; sCD14,soluble CD14; SFM, serum-free medium; STEM, surface tubules for entry into mac-rophages; Tf, human holo-transferrin; VLDL, very low-density lipoprotein.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

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human monocyte/macrophages. Although the three reported sur-face-connected tubular structures differ in important ways, bothSTEMs and surface-connected compartments appear to take upmultivalent or highly aggregated ligands. We show in this studythat LPS molecules that bind GPI-anchored CD14 on monocyticTHP-1 cells are internalized predominantly by a nonclathrin-me-diated pathway that involves noncoated tubular membrane invagi-nations and intracellular tubular and vacuolar structures, while aminority of LPS molecules enter the cells via coated pits. Aggre-gation of LPS, which enhances both the rate and extent of LPSinternalization (14), accelerates its entry into the nonclathrin-me-diated pathway.

Materials and MethodsPlasmids

cDNA-encoding wild-type human CD14 (CD14-GPI) was a gift fromDouglas T. Golenbock (Boston University, Boston, MA). cDNA encodingthe transmembrane and cytoplasmic domains of the human LDLR (37) wasprovided by Steve Lacey (University of Texas Southwestern MedicalSchool, Dallas, TX). We generated a CD14-LDLR chimeric receptor byreplacing the C-terminal 21 amino acids of CD14 with the transmembraneand cytoplasmic domains of the LDLR (Fig. 1) by patch PCR. The CD14cDNA template was amplified using primer A (59-GGA ATT CAA GCTTAT GGA GCG CGC GTC CTG-39) and primer B (59-ACG CTA CTGGGC TTC TTC TCA CGT GCA CAG GCT GGG AC-39) to yield a prod-uct that contained a 59 HindIII restriction site. In a separate reaction, LDLRcDNA was amplified using primer C (59-GTC CCA GCC TGT GCA CGTGAG AAG AAG CCC AGT AGC GT-39) and primer D (59-GCT CTAGAT CAC GCC ACG TCA TCC TCC-39) to yield a product that containeda 39 XbaI site. The two isolated PCR products were then mixed and am-plified for five cycles without primers to generate a full CD14-LDLR tem-plate. The chimeric construct was then amplified with primers A and D andisolated on an agarose gel. The CD14-GPI and CD14-LDLR cDNAs werecloned intoHindIII and XbaI restriction sites in the pRc/RSV expressionvector (Invitrogen, San Diego, CA), and their structures were confirmed byautomated DNA sequencing.

Cells

THP-1 cells were obtained from D. Altieri (Scripps Research Institute, LaJolla, CA) and cultured, as previously described (6). For transfections, weused either bulk populations of the parental cells, or we obtained a singleclone by limiting dilution to minimize variation among the transfectants.To increase mCD14 expression above the virtually undetectable level seenin undifferentiated THP-1 cells (7), the cells were transfected with pRC/RSV containing either wild-type human CD14 (CD14-GPI) or the CD14-LDLR chimera (CD14-LDLR). Bulk populations of stably transformedcells were selected in 0.5 mg/ml G418. The cells expressing CD14 wereisolated using FACStarPLUS (Becton Dickinson Immunocytometry, SanJose, CA) and expanded in culture. Chinese hamster ovary (CHO) cells that

were stably transfected with recombinant human LPS-binding protein(rLBP) or empty vector control (pRc/RSV) were kindly provided by PeterTobias (Scripps Research Institute). The cells (CHO-rLBP or CHO-RSV)were cultured in serum-free medium (CHO-S-SFM II; Life Technologies,Grand Island, NY), and the media were tested and used as previouslydescribed (14). Human peripheral blood monocytes were isolated fromheparinized blood by centrifugation over Histopaque 1077 (Sigma, St.Louis, MO). The mononuclear cell fraction was harvested from the inter-face, washed in RPMI 1640 medium, and allowed to adhere to 22322-mm glass coverslips in six-well plates containing RPMI 1640 with 8.5%FCS and 10% autologous serum. After a 1–3-h incubation, the nonadherentcells were removed by aspiration, and the adherent cells were used in theexperiments.

Reagents

Purified recombinant human soluble CD141–356 (sCD14) was a generousgift of R. Thieringer (Merck, Rahway, NJ). Anti-DNP mAb HDP-1 wasgenerously provided by Drs. J. Goldstein and M. Brown (University ofTexas Southwestern Medical Center). Goat anti-mouse IgG (H1L) 10-nmgold conjugate and normal heat-inactivated goat serum were obtained fromBB International (Cardiff, U.K.) through Goldmark Biologics (Phillips-burg, NJ). Anti-CD14 mAb 26ic (IgG2b) was provided by D. Golenbock(Boston University). FITC-conjugated goat anti-mouse IgG (H1L) F(ab9)2was from Zymed Laboratories (South San Francisco, CA). Rabbit anti-fluorescein (Texas Red conjugate) and BODIPY FL-Escherichia coliwerefrom Molecular Probes (Eugene, OR). RPMI 1640, Cellgro Complete se-rum-free medium, and G418 were from Mediatech (Herndon, VA). Cy-tochalasins H and D were from Aldrich Chemical (Milwaukee, WI). PMA,dimethylamiloride, lucifer yellow (LY), proteinase K (fromTritirachiumalbum), cell culture-tested BSA, sucrose, PMSF, and 1,4-diazabicyclo(2,2,2) octane (DABCO) were from Sigma. Phosphatidylinositol-specificphospholipase C fromBacillus cereuswas from Boehringer Mannheim(Indianapolis, IN). Glutaraldehyde (25%), picric acid (2, 4, 6 trinitrophe-nol), osmium tetroxide (4%), tannic acid, uranyl acetate, propylene oxide,and EMbed-812 plastic-embedding medium were obtained from ElectronMicroscopy Sciences (Ft. Washington, PA).

Human holo-transferrin (Tf) was obtained from Sigma and radioiodi-nated by incubating 100mg in 0.1 ml of 50 mM sodium phosphate, pH 7.4,with 1 mCi Na125I over Iodogen (Pierce Chemical, Rockford, IL). The sp.act. was 3.43 106 cpm/mg Tf. BODIPY FL-Tf and Texas Red-X-Tf con-jugates were made using FluoReporter Protein Labeling Kits (F-10232 andF-6162, respectively; Molecular Probes), according to the manufacturer’sinstructions. The resulting molar ratios of dye to Tf were 1.8 for BODIPYand 7.2 for Texas Red.

LPS preparations

E. coli LCD25 [3H]LPS (1.53 106 dpm/mg) was biosynthetically labeledand isolated as previously described (38). For derivatization, unlabeledLCD25 LPS was obtained from List Biologic Laboratories (Campbell, CA)and repurified (39) to remove trace protein contamination. After repurifi-cation, contaminating protein could not be detected on silver-stained SDS-PAGE gels after loading 10mg of LPS per lane (not shown). Tracer quan-tities of [3H]LPS were added to aliquots of unlabeled LPS to determinerecovery after derivatization reactions. DNP-LPS was made by the methodof Rietschel et al. (40), except that 50mg of repurified LPS in 100ml of 1%triethylamine adjusted to pH 10.5 with boric acid was mixed with 200mlof 5% 1-fluoro-2,4-dinitrobenzene in ethanol. The DNP/LPS molar ratiowas 1.5, and the bioactivity of the DNP-LPS was equivalent to that ofunderivatized LPS, as determined by the threshold concentration of LPSrequired to stimulate IL-8 in THP-1 cells (7) (not shown). FITC-LPS wasprepared from repurified LCD25 LPS, as previously described (14). Themolar ratio of FITC/LPS was 0.36. BODIPY FL-LPS or Texas Red-X-LPSconjugates were prepared from the same repurified LPS using reagentsfrom the FluoReporter Labeling Kits (above). We mixed 100mg of LPSwith 500mg BODIPY FL or 80mg Texas Red-X succinimidyl esters in 200ml of a buffer containing 0.1 M sodium bicarbonate, 0.3% sodium deoxy-cholate (Sigma; Ultra pure), and 1 mM EDTA. The derivatized LPS wasdialyzed against 0.9% NaCl containing 10 mM Tris (Cl), pH 7.5, at 0–4°C.The BODIPY/LPS molar ratio was 0.14, and the Texas Red/LPS molarratio was 0.23.

Partially disaggregated LPS (DAg-LPS) and monomeric LPS-sCD14complexes were made as previously described (14).

LPS internalization assays

Internalization of [3H]LPS was measured by protease protection, as pre-viously described (14). To test the effect of hypertonic medium on LPS

FIGURE 1. Structure of the CD14-LDLR fusion construct. DNA en-coding the C-terminal 21 amino acids of CD14 (first line), which containthe putative GPI-anchor signal, was replaced with the DNA encoding theC-terminal 80 amino acids of the LDLR (lines 2–4), which contain thetransmembrane and cytoplasmic domains of the receptor (37), to generatethe CD14-LDLR chimera (line 5).

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internalization, the cells were preincubated in SFM or SFM containing 0.45M sucrose for 15 min at 37°C before adding LPS.

Internalization of FITC-LPS by nonadherent monocytes was measuredby flow cytometry by quenching surface-exposed FITC-LPS with rabbitantifluorescein IgG (Texas Red conjugate), as previously described (14).Briefly, serum-free PBMC (233 106 cells in 90ml SFM) were warmed to37°C for 15 min in the presence or absence of 0.45 M sucrose, 10mlFITC-LPS in CHO-rLBP, or CHO-RSV supernatant were then added tomake a final LPS concentration of 100–200 ng/ml, and the incubation wascontinued for an additional 10 min. The cells were washed with ice-coldPBS and analyzed by flow cytometry, as described (14). The mean fluo-rescence intensities (MFI) of the monocyte populations (gated by forwardand side angle light scatter) were analyzed.

Internalization of BODIPY FL-LPS by THP-1 cells was measured byflow cytometry either by removing surface-exposed BODIPY-LPS withproteinase K or by quenching its fluorescence with trypan blue. The cells(6.3 3 105 cells in 90 ml SFM) were incubated with 100–200 ng/mlBODIPY-LPS in the presence of rLBP and washed with cold PBS, asdescribed above. Some aliquots of cells were stripped with ice-cold pro-teinase K, and other aliquots were resuspended in cold PBS. The unfixedcells were then analyzed by flow cytometry; then the cells were mixed withan equal volume of ice-cold 0.2% trypan blue in PBS for 1–2 min andreanalyzed. Data analysis was performed as described for FITC-LPS (14).

In all assays, the nonspecific (or non-CD14) binding of LPS to the cellswas determined by incubating the cells with labeled LPS in the absence ofrLBP or sCD14. Internalization of125I-Tf was measured by removing sur-face-exposed Tf by exposure to a low pH buffer, as previously described(41). Briefly, the cells were prepared in SFM as described above and in-cubated with 0.33mg/ml 125I-Tf for 1 h on ice. The cells were then warmedto 37°C for the indicated times and washed in ice-cold PBS, and surface-exposed125I-Tf was removed by washing the cells in ice-cold 0.2 M aceticacid in 0.5 M NaCl, pH 2.7, for 6 min. Radioactivity in acid supernatantsand cell lysates was measured by liquid scintillation, counted as described(14). Nonspecific binding was determined by adding 100mg/ml of unla-beled Tf. (SFM did not contain unlabeled Tf.)

Electron microscopy

DNP-LPS (500 ng LPS/ml SFM) was allowed to bind to nonadherentTHP-1 cells or adherent monocytes on glass coverslips for 1.5 min at 37°Cin the presence of CHO-rLBP or CHO-RSV supernatant. The cells werethen washed with ice-cold SFM and kept on ice for the following incuba-tions: 100ml of blocking medium (SFM containing 1% BSA, 5% normalheat-inactivated goat serum, and 5% human serum (heat inactivated) wasadded to the cells to block nonspecific and FcR-mediated binding of sub-sequently added Abs. The cells were then incubated for 30 min with 10mg/ml anti-DNP mAb HDP-1 in blocking medium, washed with SFM, andincubated for 30 min with goat anti-mouse IgG 10-nm gold conjugatediluted 1/10 in blocking medium. The THP-1 cells were gently washed byadding 10 ml cold SFM and centrifuged for 10 min at 523 g (adherentmonocytes were washed with 2 ml of cold SFM). The cells were mixedwith 100 ml of SFM and warmed for 0–2.5 min in a 37°C water bath toallow LPS internalization. The cells were placed on ice, washed twice withPBS, and fixed for 1 h in a solution containing 2% glutaraldehyde and 3mM picric acid in NaPi buffer (100 mM sodium phosphate, pH 7.4, con-taining 3 mM KCl and 3 mM MgCl2). Approximately 20 min after addingthe fixative, the adherent monocytes were scraped off the coverslips with arubber policeman, and the monocytes or THP-1 cells were centrifuged for1 min at 12,0003 g in a 1.5-ml microfuge tube to fix the cells together ina small pellet. The cells were postfixed for 1 h atroom temperature in 2%osmium tetroxide and 1.5% potassium ferrocyanide in NaPi buffer, fol-lowed by 0.05% tannic acid in NaPi buffer for 30 min. They were thenwashed with distilled water and dehydrated by exposure to increasing con-centrations of ethanol (30, 50, 70, 90, and 100%). The cells were stainedfor 1 h with 0.25% uranyl acetate in 70% ethanol during the dehydrationprocedure. The dehydrated cells were washed with propylene oxide andembedded in plastic (EMbed 812), according to the manufacturer’s proto-col. Thin (90 nm) and semithick (300 nm) sections were cut with a dia-mond knife and mounted on uncoated nickel or copper grids (200 mesh).The 90-nm thin sections were stained with uranyl acetate and lead citrate.The sections were viewed with a Jeol JEM-100SX electron microscope.

Laser confocal microscopy

THP-1 cells (33 105 cells in 40ml SFM) were placed on ice and mixedwith 5 ml BODIPY-Tf (30 mg/ml final concentration) for 1 h. A total of 5ml of Texas Red-LPS (complexed with rLBP or sCD14, as describedabove; 30–100 or 200 ng LPS/ml final concentration, respectively) wasadded, and the incubation was continued for an additional 15 min. The cells

were then warmed in a 37°C water bath for 0–5 min to allow internaliza-tion of the bound ligands. In some experiments, the cells were washed withcold SFM and reincubated at 37°C for 5–15 min. The cells were washedwith cold PBS, and surface-exposed LPS and Tf were removed by expos-ing the cells to 1 ml of ice-cold 0.02% proteinase K for 30 min (to removeLPS) (14), followed by 0.2 M acetic acid with 0.5 M NaCl, pH 2.7, for 6min (to remove Tf). The cells were fixed for 30 min in cold 4% parafor-maldehyde in NaPi buffer containing 0.5 mM PMSF, centrifuged ontopoly(L-lysine)-coated slides, and mounted in under No. 1 glass coverslips,as previously described (14). The cells were viewed with an MRC-1024laser confocal imaging system (Bio-Rad, Hercules, CA) using a363 ob-jective lens. Sequential optical sections (0.8mm) were collected digitallywith a resolution of 0.155mm per pixel. When only one fluorescent ligandwas applied to the cells, its fluorescence did not overlap detectably into thefluorescence channel used for the other fluorophore. Nonspecific bindingwas virtually undetectable when the cells were incubated with labeled LPSin the absence of rLBP or sCD14 or with labeled Tf in the presence of 3mg/ml unlabeled Tf.

Macropinocytosis and phagocytosis assays

PMA-stimulated pinocytosis (fluid-phase uptake) of LY (42) was measuredin THP-1 cells (6.33 105 cells/90ml SFM) after incubating the cells for10 min at 37°C in the presence or absence of the inhibitor, 300mM dim-ethylamiloride, or control medium containing an equivalent amount ofdimethlysulfoxide carrier. LY (0.5 mg/ml) was then added with or withoutPMA (100 nM), and the incubations were continued for an additional 30min. The cells were washed thoroughly with PBS, and cell-associated LYwas measured by flow cytometry (see above) using excitation and emissionwavelengths of 457 and 530 nm, respectively. The MFI of the THP-1 cellpopulations (gated by forward and side angle light scatter) were deter-mined, and the MFI of cells that had been exposed to LY without warmingto 37°C was subtracted. Phagocytosis of BODIPY FL-labeledE. coli byTHP-1 cells was measured by flow cytometry in the presence of trypanblue, essentially as described by Schiff et al. (43). Briefly, THP-1 cells inSFM were incubated in the presence or absence of 10mM cytochalasin Hor D for 30 min at 37°C, mixed with LBP-opsonized BODIPY-E. coli(BODIPY-E. coli was preincubated with CHO-rLBP supernatant for 30min at 37°C), and the incubation was continued for an additional 60 min.The MFI of gated THP-1 cell populations were measured before and aftermixing the cells with trypan blue.

ResultsAgents that disrupt clathrin-coated pits do not blockCD14-mediated LPS internalization

We first tested whether agents that disrupt coated pits could blockCD14-mediated endocytosis of LPS. For these experiments, weused CD14-transfected THP-1 cells or normal human monocytes.Surface-exposed [3H]LPS was removed by incubating the cellswith ice-cold proteinase K (14), and the remaining protease-resis-tant, cell-associated LPS was considered to be internal. Sequestra-tion of LPS from proteinase K was strongly inhibited by agentsthat deplete intracellular ATP (14) or by keeping the cells on ice(see below).

Brief incubation of cells in hypertonic medium disrupts clathrin-coated pits in diverse cell types (44, 45). We found that exposureto hypertonic medium (0.45 M sucrose) eliminated coated pits onTHP-1 cells. Using thin-section (90-nm) electron microscopy, wefound 51 coated pits per millimeter (0.901 mm analyzed) of cellsurface on untreated cells, whereas no coated pits were found oncells that had been treated with hypertonic sucrose (0.482 mmanalyzed). In addition, we measured the receptor-mediated endo-cytosis of Tf, a protein that enters cells via coated pits (41), andfound that hypertonic sucrose treatment reduced the internalizationof cell-associated125I-Tf from 74 6 2% (SD, control cells,n 5 4)to 12 6 3% (treated cells,n 5 4) (Fig. 2). In contrast, CD14-dependent internalization of [3H]LPS aggregates and monomerswas only slightly inhibited under the same conditions (Fig. 3). Aswe have previously shown (14), monomeric [3H]LPS that binds tomCD14 was internalized more slowly than aggregated LPS, and aconsiderably smaller percentage of the total cell-associated LPS

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was internalized. This accelerating effect of LPS aggregation alsooccurred in the presence of hypertonic medium (Fig. 3,B andD).

Exposure of the cells to chlorpromazine, a partial inhibitor ofcoated pit function (46), decreased125I-Tf internalization by 30–50%, but had no effect on LPS internalization (not shown).

The kinetics of LPS internalization by human monocytes in sus-pension were similar to those found for THP-1 cells (not shown).The data in Table I show that in monocytes, as in THP-1 cells,

hypertonic medium did not inhibit the internalization of [3H]LPSby adherent cells or of FITC-LPS by cells in suspension (measuredby Ab quenching of surface-exposed FITC-LPS (14)). Electronmicroscope (EM) thin-section analysis of monocytes showed thatexposure to hypertonic medium reduced coated pits from 71 permm of cell surface (0.69 mm analyzed) to none (0.65 mmanalyzed).

CD14 that has the LDLR membrane anchor (CD14-LDLR)internalizes LPS more efficiently

To divert LPS into coated pits, we transfected THP-1 cells with achimeric CD14 receptor (CD14-LDLR) that contained the trans-membrane and cytoplasmic domains of the LDLR (Fig. 1). UnlikeCD14-GPI, CD14-LDLR was not released from the cell surface bytreatment with phosphatidylinositol-specific phospholipase C (notshown). CD14-GPI was also largely insoluble in Triton X-100,whereas CD14-LDLR was completely solubilized by this treat-ment (47) (not shown).

The rate and extent of LPS internalization were increased sig-nificantly in cells expressing CD14-LDLR (Fig. 4) compared withthose expressing CD14-GPI (Fig. 3), whether the LPS was in ag-gregated (Fig. 4B vs Fig. 3B) or monomeric form (Fig. 4D vs Fig.

FIGURE 2. Time course of Tf internalization by THP-1 cells: Effect ofhypertonic medium. THP-1 cells were incubated in SFM (f, M) or SFM1 0.45 M sucrose (Œ,‚) for 15 min and placed on ice, and125I-Tf wasallowed to bind to the cells (6.33 105 cells in 100ml) for 1 h in theabsence (filled symbols) or presence (open symbols) of excess unlabeledTf. The cells were then warmed to 37°C for the indicated times. Total,surface-bound (acid-releasable) and internalized (acid-resistant) Tf weremeasured as described inMaterials and Methods. Internalized125I-Tf isexpressed as total cpm (A) and percentage of total cell-associated Tf (B).

FIGURE 3. Time course of internalization of[3H]LPS aggregates or monomers by THP-1cells expressing CD14-GPI: Effect of hypertonicmedium. The cells were incubated in SFM (f,M) or SFM 10.45 M sucrose (Œ,‚) for 15 min,placed on ice for 45 min, incubated with[3H]LPS aggregates (100 ng/ml) (4.53 105 cellsin 100 ml) for 15 min in the presence of rLBP,and warmed to 37°C for the indicated times (Aand B). Cells (5.93 105 in 100 ml) were pre-pared in the same way and incubated with mo-nomeric [3H]LPS-sCD14 complexes (100 ng/ml) for the indicated times at 37°C (C and D).Internalization of nonspecific or non-CD14-bound LPS (absence of rLBP or sCD14) isshown by open symbols. Total, surface-bound(protease-sensitive) and internalized (protease-resistant) LPS were measured as described inMaterials and Methods. Internalized [3H]LPS isexpressed as total dpm (A andC) or the percent-age of total cell-associated LPS (B andD).

Table I. Internalization of LPS by normal human monocytes: effect ofhypertonic mediuma

FITC-LPS (MFI) [3H]LPS (dpm)

Control Hyp. Med. Control Hyp. Med.

Total 30.6 11.3 33366 82 18256 218(100%) (100%) (100%) (100%)

Internal 12.1 5.2 7596 83 7046 106(40%) (45%) (23%) (39%)

Surface 18.5 6.4 25776 45 11216 124(60%) (55%) (77%) (61%)

a PBMC were incubated in SFM with (Hyp. Med.) or without (Control) 0.45 Msucrose for 15 min at 37°C. FITC-LPS (200 ng/ml final concentration) was thenadded, and incubation was continued for 10 min. Internalized FITC-LPS was mea-sured in the gated monocyte population by flow cytometry after quenching surface-exposed LPS with rabbit antifluorescein IgG as described previously (14). [3H]LPSinternalization was measured in adherent monocytes in 24-well plates by removingsurface-exposed LPS with proteinase K as described previously (14).

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3D). Hypertonic treatment of cells expressing CD14-LDLR re-sulted in a sharp (50–75%) decrease in LPS internalization (Fig. 4,A andC). This decrease could be accounted for by a loss of LPSbinding due to the loss or sequestration of CD14 receptors, asdetermined by FACS analysis of cell surface CD14 (not shown).Unexpectedly, the rate of internalization of both aggregated andmonomeric [3H]LPS bound by the remaining CD14-LDLRs (Fig.4,B andD) was similar to that in the untreated cells, indicating thatCD14-LDLR can internalize LPS in the absence of coated pits. Toconfirm that coated pits were disrupted by hypertonic treatment ofthese cells, we measured the internalization of125I-Tf and [3H]LPSin aliquots of cells from the same experiment and found that Tfinternalization was strongly inhibited (not shown).

LPS can enter cells via noncoated structures of variedmorphology as well as via coated pits

To observe the morphology of endocytic structures that internal-ized LPS aggregates, we bound DNP-LPS to cell surface CD14 inthe presence of LBP and observed the internalization of gold par-ticles that were attached to the DNP-LPS by an anti-DNP mAb.

No gold particles were found on sections of CD14-transfectedTHP-1 cells or monocytes that had been incubated with DNP-LPSin the absence of LBP or on THP-1 cells that were not transfectedwith CD14 (not shown), indicating that neither the DNP-LPS northe gold conjugates bound nonspecifically to the cells. In the pres-ence of LBP, cells that expressed GPI-anchored CD14 accumu-lated DNP-LPS in noncoated invaginations, tubules, and vacuolarstructures of various shapes and sizes (Fig. 5, A–G). The noncoatedinvaginations were usually tubular in structure (Fig. 5,A, B, C, andF), and the omega or flask-shape morphology that is characteristicof caveolae (20, 22, 48) was rarely seen. The tubular invaginationsthat contained LPS had an average diameter of 57 nm6 28 SD(n 5 22), and their lengths varied in the planes of the sections from74 to 850 nm. The diameters of these invaginations were muchmore variable that those of coated pits, which were 66 nm6 9 SD(n 5 10). LPS was also found in tubules (27–133 nm diameter) andelectron-lucent vacuoles (100–500 nm) that appeared to be intra-cellular (not connected to the plasma membrane in the plane ofsection). The fact that very few of these structures were labeledwhen the cells were not warmed to 37°C (Table II) indicates thatthey were either intracellular or connected to the surface by narrow

openings that restricted the movement of gold particles. The vacu-oles frequently had an irregular or convoluted structure, suggestingthat they were probably cross-sections of tubular inclusions, andsome of the vacuoles were found to be connected to the surface bytubules or narrow invaginations (Fig. 5,C andD). LPS was alsofound in coated pits and vesicles, but much less frequently than innoncoated structures.

In cells that expressed CD14-LDLR, relatively little LPS wasfound in coated pits before warming. After warming, most of thecoated pits that contained gold particles were found at the cellsurface (Fig. 5I), but coated pits were frequently found as exten-sions of tubular invaginations (Fig. 5H), internal tubules, and vacu-oles (Fig. 5K) in these cells.

The data in Table II show a quantitation of the EM data. Wecounted the gold particles associated with DNP-LPS and deter-mined the percentages of the total cell-associated gold particles(total LPS) contained in the various structures in cells expressingeither CD14-GPI or CD14-LDLR. The striking differences in thelocations of the LPS-Ab-gold complexes in coated versus non-coated structures in the two transfected cell lines suggest stronglythat the dominant localizing factor was the CD14 anchor, and thatthe presence of Abs or gold in the complexes was not determina-tive. In cells expressing CD14-GPI, only 0.6% (1 min) to 2.7%(2.5 min) of the gold was found in coated pits and vesicles, and10-fold more LPS-associated gold particles entered noncoated thancoated structures. In cells expressing CD14-LDLR, in contrast, wefound approximately 10% of the gold particles in coated pits orvesicles. Although in these cells a large fraction of the internalizedgold was also found in tubular invaginations, tubules, or vacuoles,at least 12% of the gold in these noncoated structures was in coatedpits that appeared to bud from them (Fig. 5K). In these cells, LPSmay thus enter coated pits either at the cell surface or after it isinternalized into tubular structures.

Peripheral blood monocytes internalized LPS into similar struc-tures (Fig. 6). In monocytes, we found LPS more frequently intubular invaginations and in apparently intracellular tubular andvacuolar structures, whereas relatively little LPS was found incoated pits and vesicles (Table II). In these cells, the coated pitsthat contained LPS were found in tubular invaginations and othertubular or vacuolar structures, and 3.5% of the gold particles inthese noncoated structures were in coated pits.

FIGURE 4. Time course of internalization of[3H]LPS aggregates or monomers by THP-1 cells ex-pressing CD14-LDLR: Effect of hypertonic medium.The cells were incubated in SFM (f, M) or SFM10.45 M sucrose (Œ, ‚) for 15 min, placed on ice for45 min, incubated with [3H]LPS aggregates (95 ng/ml) (4.2 3 105 cells in 100ml) for 15 min in thepresence of rLBP, and warmed to 37°C for the indi-cated times (A andB). Cells (5.33 105 in 100 ml)were prepared in the same way and incubated withmonomeric [3H]LPS-sCD14 complexes (83 ng/ml)for the indicated times at 37°C (C andD). Internal-ization of nonspecific or non-CD14-bound LPS (ab-sence of rLBP or sCD14) is shown by open symbols.Total, surface-bound (protease-sensitive) and inter-nalized (protease-resistant) LPS were measured asdescribed inMaterials and Methods. Internalized[3H]LPS is expressed as total dpm (A andC) or thepercentage of total cell-associated LPS (B andD).

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Some internalized LPS may be accessible to small extracellularmolecules, but not proteinase K

Surface-connected tubules have been shown to sequesterb-VLDLin murine macrophages (34, 35). Before complete internalization,the b-VLDL is poorly accessible to Abs, whereas it is readilyaccessible to trypan blue, a low m.w. fluorescence quencher. Asshown in Figs. 5 and 6, we found internalized LPS in surface-connected tubular structures. Accordingly, we tested whether cell-associated BODIPY FL-LPS was more accessible to trypan blue(Mr 5 961) than to proteinase K (Mr 5 28,900). As shown in TableIII, the ability of proteinase K or trypan blue to remove or quenchBODIPY-LPS that was bound to mCD14 on the cell surface at0–4°C was equivalent. However, after warming the cells to 37°C,22–28% of the BODIPY FL-LPS that was not removed by pro-teinase K was accessible to trypan blue (Table III). These findingsare consistent with the conclusion that, likeb-VLDL, LPS passesthrough intracellular structures that, while open to the surface, arerelatively inaccessible to large extracellular probes; these struc-tures are most likely the tubular invaginations visualized usingelectron microscopy. In each cell line, the effect of hypertonic

medium on LPS internalization was similar whether surface-ex-posed LPS was detected using proteinase K or trypan blue (datanot shown).

GPI-anchored CD14 directs LPS to an endocytic pathwaydifferent from that taken by Tf

We next asked whether CD14-GPI cells internalize LPS into en-dosomes that also contain Tf, a well-established marker for earlysorting endosomes that accumulate the contents of coated pits andcoated vesicles (35). We bound Texas Red-DAg-LPS andBODIPY-Tf to THP-1 cells that expressed either CD14-GPI orCD14-LDLR, warmed the cells to 37°C for 3 or 5 min, and viewedthe cells with a laser confocal microscope. In cells that were notrewarmed, both ligands uniformly stained the surfaces of receptor-positive cells without punctate focal accumulations (not shown).Focal accumulations of both LPS and Tf appeared after warmingthe cells to 37°C, and these accumulations increased in intensityand apparent size over time. We removed the surface-exposed li-gands so that the locations of the internalized ligands could be

FIGURE 5. Electron-microscope analysis of LPS internalization by THP-1 cells. DNP-LPS (500 ng/ml) was allowed to bind to mCD14 on THP-1 cellstransfected with CD14-GPI (A–G) or CD14-LDLR (H–K) in the presence of rLBP. While kept on ice, the cells were incubated with mouse anti-DNP (mAbHDP-1), followed by 10-nm gold-conjugated goat anti-mouse IgG. The washed cells were warmed to 37°C for 1–2.5 min to begin internalization, washedwith cold buffer, fixed, and embedded in plastic, as described inMaterials and Methods. Thin (90 nm) (B, D, E, G, H–J) or semithick (300 nm) (A, C, F)sections were analyzed by transmission-electron microscopy. The bar inA 5 100 nm. Arrowheads (G, H, I, K) point to coated pits.

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clearly evaluated. In cells that expressed CD14-GPI, Texas Red-LPSand BODIPY-Tf (Fig. 7) (or BODIPY-LPS and Texas Red-Tf; notshown) accumulated predominantly in different locations after 3 min

of internalization (Fig. 7, A–C). After 5 min, LPS and Tf partiallycolocalized (Fig. 7, G–I), but extended incubation (up to 20 min) didnot increase the coincidence of the two ligands (not shown). Incontrast, in cells expressing CD14-LDLR, Texas Red-LPS andBODIPY-Tf nearly always accumulated in the same locations after 5min at 37°C (Fig. 7, J–L). At earlier (3 min) time points (Fig. 7, D–F),however, there were differences in the locations of LPS and Tf; thisfinding is consistent with the conclusion that some of the LPS inter-nalized by CD14-LDLR cells can be internalized rapidly into non-coated structures before it enters coated pits (see Table II).

FIGURE 6. Electron-microscope analysis of LPS internalization bynormal human monocytes. DNP-LPS (500 ng/ml) was allowed to bind tomCD14 on adherent monocytes on glass coverslips in the presence ofrLBP. Gold particles were attached to surface-bound DNP-LPS, as de-scribed in Fig. 5, and the cells were warmed to 37°C for 2.5 min. Repre-sentative semithick (300 nm) plastic sections are shown. In theright panel,the opening of the tubular invagination to the surface is not shown. The bar(upper left)5 100 nm.

Table II. Quantitation of immunogold localization of DNP-LPS by thin section electron microscopy (Figs. 5 and 6)a

Expt. Cells Time (min)TotalGold

Surface(mm)

Goldpermm

Coated Structures Noncoated Structures

Pits Vesicles Total InvaginationTubules and

vacuolesSmall

vesicles Total

1 CD14-GPI 0 3033 650 4.7 0 0 0 5 71 0 76(0.2%) (2.4%) (2.6%)

1 1536 389 3.9 7 1 8 28 142 3 173(0.5%) (0.1%) (0.6%) (1.8%) (9.2%) (0.2%) (11.3%)

2.5 1841 570 3.2 41 10 51 20 444 102 566(2.2%) (0.5%) (2.7%) (1.1%) (24.1%) (5.5%) (30.7%)

CD14-LDLR 0 377 350 1.08 15 3 18 0 0 0 0(4.0%) (0.8%) (4.8%)

1 335 302 1.11 26b 19 45 7 17 1 25(7.8%) (5.7%) (13.4%) (2.1%) (5.1%) (0.3%) (7.4%)

2.5 478 363 1.32 42b 28 70 22 98 7 127(8.8%) (5.9%) (14.6%) (4.6%) (20.5%) (1.5%) (26.5%)

2 CD14-GPI 0 1024 217 4.7 0 0 0 0 0 0 01 2461 381 6.4 18 0 18 57 396 0 453

(0.7%) (0.7%) (2.3%) (16.1%) (18.4%)CD14-LDLR 0 672 255 2.6 14 0 14 4 2 0 6

(2.1%) (2.1%) (0.6%) (0.3%) (0.9%)1 2642 619 4.3 204b 52 256 83 118 0 201

(7.7%) (2.0%) (9.7%) (3.1%) (4.5%) (7.6%)

3 Monocytes 2.5 3781 875 4.3 90c 37 127 214 2315 31 2560(2%) (1%) (3%) (6%) (61%) (1%) (68%)

a Thin sections (90 nm) of THP-1 cells or adherent human monocytes were randomly photographed and the numbers of gold particles were counted on randomizedcoded prints. Data from three independent experiments are shown. The table shows the time that the cells were warmed to 37°C, the total number of cell-associated goldparticles counted in each experimental group, the total length of cell surface analyzed, and the number of gold particles/mm of cell surface. The remaining columnsindicate the number of gold particles and the percent of the total cell-associated particles found in each type of coated or non-coated structure. Small noncoated vesicleswere ,100 nm in diameter.

b Seven (Expt. 1, 1 min), 6 (Expt. 1, 2.5 min), and 28 (Expt. 2, 1 min) of these gold particles were found in coated pits that were in noncoated invaginations, tubules, orvacuoles. Overall, 27% of the coated pit-associated gold in these cells was in these structures.

c All of the coated pit-associated gold in these cells was in noncoated invaginations, tubules, or vacuoles.

Table III. Internalization of BODIPY/LPS by THP-1 cells expressingCD14-GPI or CD14-LDLR: comparison of accessibility to trypan blueor proteinase Ka

Timeat 37°C(min)

TotalMFI

% Internal BODIPY/LPS% Difference,

Mean (95% C.I.)P.K. T.B.

CD14-GPI 0b 23.46 5 4.56 0.6 4.36 0.8CD14-GPI 5 40.46 20 266 2 206 4 22 (12.8–30.0)

10 36.76 15 276 1 206 4 24 (15.8–31.7)CD14-LDLR 5 18.96 4.1 696 9 496 2 28 (19.4–38.0)

10 15.06 6.7 686 2 546 8 21 (10.5–31.6)

a BODIPY FL/LPS (100–200 ng/ml) was mixed with rLBP and allowed to bindto the cells for 5-10 min at 37°C or for 15 min to cells that were kept on ice. The MFIof specifically bound LPS was determined by flow cytometry as described inMate-rials and Methods. Internalized LPS is expressed as the percent of the total specificcell-associated LPS (Total MFI) that was not removed by proteinase K (P.K.) or notquenched by trypan blue (T.B.). The differences between values derived from the T.B.method and those from the P.K. method were determined on cells within each ex-periment by the formula: % difference5 [(P.K.%2 T.B.%)/P.K.%]3 100. The 95%confidence intervals (C.I.) are shown;n 5 4–14 measurements per comparison.

b Fifteen minutes on ice.

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These observations were made using LPS aggregates (DAg-LPS). When we incubated cells expressing CD14-GPI withmonomeric Texas Red-LPS for 5 min at 37°C, the intracellularLPS was diffusely localized along the cell periphery (Fig. 8A).The location of internalized LPS monomers did not correspond tothe location of Tf-containing vesicles (Fig. 8B). In contrast, in cellsthat expressed CD14-LDLR, monomeric LPS accumulated in dis-

crete foci that usually overlapped with accumulations of Tf (Fig. 8,C andD).

Effect of inhibitors of macropinocytosis and phagocytosis onCD14-dependent LPS internalization

We next asked whether nonclathrin-mediated LPS internalizationinvolves macropinocytosis or phagocytosis. The data presented

FIGURE 7. Confocal microscope analysis of internalized LPS and Tf. Texas Red-LPS (100 ng/ml)1 rLBP and BODIPY FL-Tf (30mg/ml) wereallowed to bind to THP-1 cells transfected with CD14-GPI (A–CandG–I) or CD14-LDLR (D–F andJ–L) at 0–4°C. The cells were then warmed to 37°Cfor 3 min (A–CandG–I) or 5 min (D–F andJ–L) to allow internalization. The cells were chilled; surface-exposed ligands were removed with proteinaseK (for LPS), followed by acid buffer (for Tf); and optical sections (0.8mm) were analyzed by two-color laser confocal microscopy. Each panel containsimages of representative cells from two experiments. Red (LPS) and green (Tf) images are superimposed inC, F, I, andL.

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above make it unlikely that LPS internalization requires macropi-nocytosis, since fluid-phase uptake of LPS is quantitatively trivialcompared with CD14-mediated uptake (Fig. 3, compare uptakewith or without LBP), and LPS stimulation of THP-1 cells in thepresence of LBP does not greatly enhance fluid-phase uptake (notshown). Moreover, in contrast to previous studies that used higherconcentrations of LPS (49, 50), our partially disaggregated LPSwas not visible as LPS bilayers by thin-section EM analysis (Figs.5 and 6), suggesting that these LPS aggregates are probably toosmall to require engulfment by the membrane “zipper” mechanismof phagocytosis. Nevertheless, the cellular mechanisms that areessential for these processes (e.g., membrane ruffling and actinpolymerization) may be important for nonclathrin-dependent LPSinternalization. To test this hypothesis, we first treated THP-1 cellswith dimethylamiloride, a potent inhibitor of membrane rufflingand macropinocytosis (42, 51), and found that the drug had virtu-ally no inhibitory effect on [3H]LPS internalization, whereas itstrongly inhibited PMA-stimulated uptake of LY (Fig. 9). Thesedata suggest that membrane ruffling is not responsible for the for-mation of noncoated invaginations that internalize LPS. We alsotreated the cells with inhibitors of actin polymerization and phago-cytosis, cytochalasins H and D, and found that whereas CD14-dependent phagocytosis of BODIPY-E.coli (43) was almost com-pletely inhibited by these drugs, CD14-dependent internalizationof [3H]LPS aggregates was only partially inhibited (Fig. 9). Wealso found that cytochalasin D partially inhibited BODIPY-LPSinternalization, as measured by quenching surface LPS with trypanblue (seeMaterials and Methods). In three experiments, the inter-nalized fraction of BODIPY-LPS in cytochalasin-treated cellswas 686 13% SD (n 5 6) of that of untreated control cells.(Control cells internalized 226 3% of the total cell-associatedLPS, MFI 5 42 6 8, in 10 min). These data suggest thatwhile actin polymerization may have a role in LPS internalization,the endocytic mechanism appears to be distinct from that ofphagocytosis.

Impact of the CD14-dependent internalization pathway on LPSdeacylation and signaling

Although previous studies showed that LPS signaling occurs nor-mally in cells that express CD14, regardless of the structure of its

FIGURE 8. Confocal microscope analysis of internalized monomeric LPS and Tf. BODIPY-Tf was allowed to bind to THP-1 cells transfected withCD14-GPI (A,B) or CD14-LDLR (C, D) at 0–4°C; Texas Red-LPS (200 ng/ml, in monomeric sCD14 complexes) was added; and the cells were warmedto 37°C for 5 min to allow internalization. The cells were processed and analyzed as described in Fig. 7. Representative images are shown in grayscalefor Texas Red-LPS (A andC) and BODIPY-Tf (B andD). Focal accumulations of ligands were in the same positions inC andD (note cells c-e), whereasthe ligands did not colocalize inA andB (note cells a and b).

FIGURE 9. Effects of inhibitors of macropinocytosis and phagocytosison LPS internalization by THP-1 cells. THP-1 CD14-GPI cells (4–63 105

cells in 100ml SFM) were preincubated for 10 min at 37°C with controlmedium or 300mM dimethylamiloride (DM-amiloride) or for 30 min withcontrol medium or 10mM cytochalasin H (Cyto H) or D (Cyto D).[3H]LPS aggregates (100 ng/ml) were added with rLBP, and the incuba-tions were continued for 5 or 10 min. Total, surface-bound (protease-sen-sitive) and internalized (protease-resistant) LPS were measured as de-scribed in Materials and Methods. Internalized LPS is shown as thepercentage6 SD (n 5 4–8) of internalized LPS found in uninhibitedcontrol cells. (Control cells internalized 306 4% (n 5 8) of the totalcell-associated LPS, 74826 702 dpm.) Macropinocytosis was measuredby flow cytometry after adding 0.5 mg/ml LY and 100 nM PMA andcontinuing the incubations for 30 min. (MFI of LY in PMA-stimulatedcells was 2036 14 (n 5 4); uptake by unstimulated cells was approxi-mately twofold lower.) Phagocytosis was measured by flow cytometry afteradding BODIPY-E. coliin the presence of rLBP for 60 min. (MFI ofinternalized BODIPY-E. coliin control cells was 2856 7 (n 5 2).)

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membrane anchor (52, 53), none of the anchors used for thosestudies contained specific targeting signals. In this study, we testedwhether an anchor that has a coated pit-targeting signal (i.e.,CD14-LDLR) would alter the ability of the cells to respond toLPS. As noted above, however, CD14-LDLR does not target LPSexclusively to coated pits: it often directs LPS to coated pits thatare attached to noncoated invaginations. Moreover, CD14-GPIcould also direct some LPS to coated pits. Although similar LPSdose responses for nuclear factor-kB translocation and IL-8 pro-duction were found in cells that expressed equivalent amounts ofeither CD14-GPI or CD14-LDLR (not shown), the lack of local-ization specificity frustrates interpretation of these results. On theother hand, we found that cells that internalize LPS via CD14-GPIor CD14-LDLR perform LPS deacylation at closely similar rates,suggesting that both clathrin-coated and noncoated structures cantarget LPS to the endosomes, where deacylation is presumed tooccur (54) (not shown).

DiscussionWe used three complementary strategies to study the initial stepsin LPS internalization by monocytic cells. The kinetics of LPSinternalization were quantitated by measuring the disappearance ofthe ligand from the cell surface, which we defined operationally asthe loss of accessibility to extracellular proteinase K or trypanblue. Disruption of coated pits with hypertonic media was used todetermine whether LPS endocytosis was predominantly clathrinmediated. The movement of LPS-Ab-gold complexes into the cellwas then tracked using thin- and thick-section transmission elec-tron microscopy. Finally, laser confocal microscopy was used tostudy the uptake of fluorescent Tf and LPS into intracellular ves-icles. In each of these approaches, we compared LPS internaliza-tion in cells that express GPI-anchored CD14 with that in cellsexpressing CD14 modified so as to favor cell entry via clathrin-coated pits (CD14-LDLR).

We also limited our analysis to LPS that bound CD14, the majorreceptor for LPS on phagocytes, by using relatively low concen-trations of LPS and by binding LPS to cells using LBP or sCD14.Our approach therefore differed substantially from previous ultra-structural analyses of LPS internalization, which used muchgreater concentrations of LPS and/or LBP- and sCD14-freeconditions (49, 50, 55–57).

Internalization of CD14-bound LPS occurs predominantlythrough a nonclathrin-mediated pathway

Three lines of evidence support the conclusion that the predomi-nant pathway of LPS internalization mediated by GPI-anchoredCD14 is nonclathrin mediated: 1) LPS internalization was rela-tively insensitive to the effects of hypertonic medium (Fig. 3),which destroyed coated pits and strongly inhibited Tf internaliza-tion (Fig. 2). 2) EM analysis showed that gold particles attached toDNP-LPS accumulated rapidly (1 min) in noncoated invaginationsand intracellular vesicles in THP-1 cells (Table II and Fig. 5) andmonocytes (Fig. 6), whereas at least 10-fold less DNP-LPS wasfound in clathrin-coated pits and vesicles. 3) Confocal microscopeanalysis of internalized Texas Red-LPS and BODIPY-Tf showedthat these ligands accumulated in different intracellular locationsimmediately (3 min) after internalization by CD14-GPI cells (Fig.7, A–C), and that lack of colocalization persisted to a significantextent for many minutes thereafter (Fig. 7, G–I).

Entry into coated pits is an alternative pathway for LPSinternalization

EM analysis of cells expressing CD14-GPI showed that a smallpercentage of cell-associated DNP-LPS entered coated pits (Fig.

5G and Table II). This observation is consistent with the apparentcolocalization of some intracellular Texas Red-LPS withBODIPY-Tf (Fig. 7, G–I). Intracellular accumulations of LPS andTf occurred mostly in different locations after 3 min of internal-ization, but appeared to converge partially by 5 min. Colocaliza-tion of the LPS- and Tf-containing compartments was never socomplete in these cells as in cells in which LPS was bound toCD14-LDLR.

Although the bulk of the LPS entered cells expressing CD14-GPI by a nonclathrin pathway, the failure of fluorescent LPS andTf to colocalize completely in intracellular foci was somewhatsurprising in view of previous studies of nonclathrin-mediated en-docytosis (31, 58, 59). For example, Hansen et al. (58) showed thatin potassium-depleted HEp-2 cells, Con A-gold is taken up bynonclathrin-mediated endocytosis and moves to early endosomesthat contain Tf receptors, although the internalized Con A-gold isultimately excluded from late endosomes and lysosomes. Perhapsthe noncoated invaginations that internalize LPS turn over moreslowly than coated pits and coated vesicles (34), or they may failto fuse efficiently with early endosomes. Whether endosomes fromthe clathrin-mediated pathway fuse with those from nonclathrin-mediated pathways may differ in various cell types (35).

We produced the CD14-LDLR chimeric receptor to directCD14-bound LPS into coated pits. As shown in the electron-mi-croscope images (Fig. 5,H andJ, and Table II), however, LDLR-CD14 cells also internalize LPS via nonclathrin structures. Mono-cyte/macrophages, like hepatocytes and many epithelial cells (60),evidently do not restrict the membrane location of LDLR to coatedpits. Indeed,b-VLDL, which binds to LDL (apoE/B) receptors(61), also moves into noncoated tubular invaginations in macro-phages (34). Treatment of CD14-LDLR cells with hypertonic su-crose blocked Tf internalization and diminished the number of cellsurface CD14 receptors without significantly diminishing the rateof LPS internalization (Fig. 4). This observation suggests thatthese cells can divert LPS almost entirely into noncoated struc-tures, in keeping with previous observations that inhibition ofcoated pit function may up-regulate nonclathrin-mediated endocy-tosis in other cell types (62).

We also found that noncoated tubular invaginations, tubules,and vacuoles can contain coated pits (Fig. 5,H andK). In THP-1cells expressing CD14-LDLR (and to a lesser extent in humanmonocytes), the immunogold-LPS found in tubular invaginationswas often in, or near, these coated structures. In these cells, there-fore, the LDLR anchor may target CD14 to coated pits that existeither on the cell surface or within surface-connected membraneinvaginations. This phenomenon may account for the observationsthat, in cells expressing CD14-LDLR, 1) similar amounts of im-munogold-LPS were found in coated and noncoated structures(Table II), yet 2) after internalization, Texas Red-LPS overlappedsubstantially with BODIPY-Tf, even at early time points (Fig. 7).Presumably, coated vesicles derived from different membranes(plasma membrane and noncoated tubular invaginations or vesi-cles) fuse with early endosomes.

Nonclathrin-mediated endocytosis of LPS occurs in tubularinvaginations and vesicles

The results of the EM analysis of LPS internalization (Fig. 5, TableII) suggest that nonclathrin-mediated endocytosis of LPS aggre-gates occurs via tubular invaginations of the plasma membrane.The diameters of the tubular invaginations (576 28 nm) weresimilar to those of coated pits (666 9 nm) and those reported for

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caveolae (50–80 nm (27)). Morphologically, the tubular invagi-nations resemble the tubular pinosomes, noted in alveolar macro-phages, that take up horseradish peroxidase and contain acid phos-phatase (33). They also resemble the surface-connected tubules(STEMs), described in murine macrophages, that internalize andpartially process largeb-VLDL particles (34, 35, 63), althoughSTEMs are significantly larger in diameter (;250 nm). The rolesof noncoated vesicles in LPS signaling and intracellular pro-cessing and their relationship to low-density, lipid-enrichedmembrane microdomains that bind CD14-bound LPS (64) areunder investigation.

The kinetics of LPS internalization by cells expressing eitherCD14-GPI or CD14-LDLR is strongly influenced by the LPS ag-gregation state. Regardless of the CD14 anchor, aggregationgreatly accelerates LPS movement into noncoated structuresand/or the endosomes derived from them. Monomeric LPS entersthese structures much more slowly in cells that express CD14-GPI,so that much of the cell-associated LPS remains on the surfaceover time. In cells that express CD14-LDLR, however, monomericLPS that binds CD14 is targeted to coated pits, so that it disappearsmore rapidly from the cell surface into Tf-containing endosomes.This formulation is consistent with the reported pattern ofb-VLDL internalization by macrophages, since the presence ofmultiple apolipoprotein E molecules in largeb-VLDL particlesalso promotes movement into surface-connected tubules ratherthan coated pits (34, 63, 65). In keeping with these results, theinternalization-accelerating effect of LPS aggregation occurredeven in cells that had no functional coated pits (Figs. 3 and 4).

Conclusions

While some LPS internalization is mediated by clathrin-coatedpits, most occurs via nonclathrin-coated membrane invaginationsand tubules. Aggregation promotes entry by accelerating uptakevia the noncoated pathway. Like the plant-derived protein toxin,ricin (29), LPS thus has a complex pattern of cell entry, and itseems reasonable to expect similarly complex pathways of intra-cellular movement. Moreover, the fate of the LPS studied by var-ious techniques may be different from that of a much smaller pop-ulation of LPS molecules that has important or different biologicconsequences. Understanding the role of internalization in LPSsignaling centers on this issue; it is entirely possible that the “bulk”LPS, followed by virtue of its radioactivity or a visual tag, does notinclude a small population of molecules that triggers cellular re-sponses. Another possibility, given the pleiotropic nature of re-sponses to LPS, is that different cellular reactions are initiated bythe LPS that finds its way into different surface domains or intra-cellular compartments. Sorting out these possibilities will be a ma-jor challenge for future research.

AcknowledgmentsWe thank Drs. Richard Anderson, Stephen Lacey, Michael Roth, and LeonEidels for helpful advice; Stephen Lacey for providing the low-densitylipoprotein receptor cDNA; and Rolf Thieringer for providing solubleCD14.

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