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Vivo StudyMurine Macrophages, an In Vitro and InLipopolysaccharide to the Cell Surface of Lysosomal Accumulation and Recycling of

Jean-Pierre GorvelClaire Forestier, Edgardo Moreno, Javier Pizarro-Cerda and

http://www.jimmunol.org/content/162/11/67841999; 162:6784-6791; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Lysosomal Accumulation and Recycling of Lipopolysaccharideto the Cell Surface of Murine Macrophages, an In Vitro andIn Vivo Study1

Claire Forestier,* Edgardo Moreno,† Javier Pizarro-Cerda,* and Jean-Pierre Gorvel2*

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate ofBrucella abortusLPSin murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway ofB.abortusLPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitroafter LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages withB. abortusstrain19. After binding to the cell surface and internalization,Brucella LPS is routed from early endosomes to lysosomes with unusualslow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. Thisrecycling pathway is also observed for LPS released byBrucella S19 following in vitro infection. Indeed, by 72 h postinfection,bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward,LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters withthe O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved duringthe intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposingits immunogenic moiety toward the extracellular medium. The Journal of Immunology,1999, 162: 6784–6791.

L ipopolysaccharide, the most abundant component of theGram-negative bacteria outer membrane, is the main Ab-inducing Ag during bacterial infections and one of the

most biologically active molecules (1). LPS released in the bodyfluids by live or killed bacteria in the form of blebs or membranevesicles (2) is readily ingested by professional phagocytes follow-ing pinocytosis, receptor-mediated endocytosis, macropinocytosis,or phagocytosis (3–7). Alternatively, phagocytosed Gram-negativebacteria may release LPS within the intracellular milieu (8, 9).Enterobacterial LPS has been detected in phagosomes, endosomes,the Golgi complex, the nucleus, mitochondria, and cytoplasm (6,10–12). It has been shown that during the first 2 days after inges-tion, LPS is mainly found in the form of small, loosely packed,vacuoles that later seem to coalesce into larger and tightly packedcompartments (13). However, the characterization of the LPS-con-taining compartments during its intracellular routing in a time-dependent manner has not as yet been determined.

Due to their accumulation within macrophages, LPSs have beenregarded as slowly processable molecules (14–16). Macrophagesare able to deacylate and dephosphorylate the enterobacterial lipidA moiety (17–19), which is eventually exocytosed to the extracel-

lular medium (20). Other investigations have suggested that theO-chain and the core oligosaccharide of enterobacterial LPS arealso degraded within macrophages (13, 20) by mechanisms thathave not yet been elucidated. The slow processing of LPS withinphagocytic cells may be due to the absence of a molecular ma-chinery capable of efficiently digesting it. Alternatively, LPS maydirectly alter the constitutive intracellular trafficking of moleculeswithin cells perhaps in conjunction with its toxic effect. In thisrespect, the elucidation of the trafficking of LPS and the identifi-cation of LPS-containing compartments are necessary to under-stand better the pathophysiologic effects mediated by this moleculeand to resolve the fate and the intracellular processing of nonpro-tein Ags. This last aspect is relevant in the light of investigations,which have clearly demonstrated that microbial glycolipids may bepresented to T cells in the context of nonclassical histocompati-bility Ags (21, 22).

In this work, we detailed in a time-dependent manner the intra-cellular trafficking of the low endotoxicBrucella abortusLPS (23)in murine peritoneal macrophages. We studied the fate of LPSafter in vitro incubation and in vivo i.p. injection of purified LPS,and afterBrucella infection in vitro. The results show that LPSfollows the endocytic pathway described for proteins Ags, but withunusually slow kinetics. We observed thatBrucella LPS initiallyaccumulates within lysosomal compartments, followed by a grad-ual recycling to the plasma membrane, where it arranges as largeclusters. In contrast to enterobacterial LPS, no alteration in theimmunochemical properties ofBrucellaLPS was observed in spiteof the slow trafficking within lysosomes, suggesting that this mol-ecule resists intracellular degradation mechanisms.

Materials and MethodsMice and cells

Eight- to fifteen-week-old C3H/HeN female mice (Jackson ImmunoRe-search, West Grove, PA) were used in all experiments. Peritoneal murinemacrophages were obtained after cervical dislocation from normal mice orfrom mice injected i.p. with 0.4 ml ofB. abortusS19 LPS (1 mg/ml). Each

*Centre d’Immunologie de Marseille-Luminy, Parc Scientifique de Luminy, Case,Marseille, France; and†Programa de Investigacion en Enfermedades Tropicales, Uni-versidad Nacional, Heredia, Costa Rica

Received for publication November 30, 1998. Accepted for publication March17, 1999.

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 grants from Institut National de la Sante´ et de laRecherche Medicale (INSERM; 4N004B) and institutional grants from INSERM andCentre National de la Recherche Scientifique. J.P.-C. is a recipient of a fellowshipfrom the Centre de la Recherche Scientifique (France), and C.F. from the Minister ofEducation and Research (France).2 Address correspondence and reprint requests to Dr. Jean-Pierre Gorvel, Centred’Immunologie de Marseille-Luminy, case 906, 13288 Marseille cedex 9, France.E-mail address: gorvel@ciml.univ-mrs.fr

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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killed mouse received an i.p. injection of 20 ml of sterile PBS/10% sucroseat 15°C. After injection, the abdomen of animal was massaged and theliquid was extracted from the peritoneal cavity. Pooled fluids were centri-fuged at 1200 rpm for 10 min at 4°C and the pellets were resuspended inDMEM (Life Technologies, Cergy-Pontoise, France) supplemented with10% FCS, 2 mM glutamine, 10 mM sodium pyruvate, 10 mM nonessentialamino acids, 10 mM HEPES, 100 U/ml penicillin, and 100mg/ml strep-tomycin (all from Life Technologies). A total of 23 105 peritoneal cellswas plated on 12-mm glass coverslips in 24-well tissue culture plasticdishes, at 106 cells/ml in 96-well plates (Costar, Cambridge, MA) or at 106

cells/35-mm dish (Nunc, Poly Labo) for immunofluorescence, ELISA, orimmunoblotting analysis, respectively. After incubation for 1 h at 37°Cunder 5% CO2, nonadherent cells were removed from the wells by aspi-ration, and the adherent macrophages were rinsed twice with PBS andincubated with fresh culture medium.

Bacteria and LPS preparations

The characteristics of smooth typeB. abortusS19 (Professional Biological,Denver, CO) have been previously described (24). Bacteria were grown at37°C in tryptic soy broth (TSB; Difco, Detroit, MI) to stationary phase, andaliquots were frozen at270°C in TSB-glycerol 30%. A log-phase culturebacteria was prepared by incubating a thawed aliquot (53 1010 CFU/ml)in TSB for 15–48 h at 37°C under agitation. Bacterial numbers were de-termined by comparing the OD at 600 nm with a standard curve.

The extraction and purification of LPS from smooth and roughB. abor-tushave been described elsewhere (25, 26). Briefly, smoothBrucellaLPSwas obtained from the phenolic phase of phenol-water extraction method(27). Purification of LPS (20 mg/ml LPS in 5 mM MgCl2/10 mM Tris-HCl,pH 7.5) was conducted by repeated digestion with nucleases and proteinaseK (Sigma, St. Louis, MO) for 24 h at 25°C. The LPS was then centrifugedat 100,0003 g for 6 h at 4°C, and the removal of phospholipids andornithine lipids was achieved with chloroform/methanol/water (1:8:0.8,v/v/v), followed by chloroform/methanol/7 M ammonia (65:25:4, v/v/v).LPS was then reextracted with phenol/water and recovered by centrifuga-tion at 100,0003 g for 6 h at 4°C. All LPS preparations were lyophilizedafter extensive dialysis and analyzed by standard procedures (26, 28). Forinternalization studies and injections in mice, LPS was dissolved in waterat the appropriate concentration with the aid of sonication and autoclavedbefore use.

Cell infection and LPS endocytosis

After removal of culture medium, macrophages grown on coverslips in24-well tissue plates were inoculated with 500ml of a bacterial suspensionin culture media at a rate of 50 bacteria/cell. Culture plates were centri-fuged for 10 min at 1000 rpm at room temperature, followed by incubationfor 20 min at 37°C under 5% CO2. Cells were washed five times withculture medium to remove nonadherent bacteria, and monolayers were fur-ther incubated with culture medium containing 50mg/ml of gentamicin(Sigma) to kill extracellular brucellae. In long-term experiments, this me-dium was replaced twice: at 1 h with medium containing 25mg/ml gen-tamicin, and at 24 h with medium supplemented with 5mg/ml gentamicin.

Macrophage layers grown on glass coverslips (for immunofluorescenceexperiments), 96-well plates (for ELISA), or 35-mm plastic dishes (forimmunoblotting analysis) were pulsed withB. abortusLPS. In each ex-periment, 15mg/ml of LPS was added for 1 h at4°C, followed by extensivewashings with culture media. Then macrophages were incubated in cellculture medium at 37°C for different time intervals (10 min to 48 h) andprocessed for immunofluorescence, ELISA, or immunoblotting analysis.

Antibodies

Murine mAb against O-chain C/Y epitope (Baps C/Y) was produced andcharacterized, as previously described (29). Cow polyclonal serum againstB. abortusS-LPS3 was described in earlier works (29). Briefly, the serumwas collected from aB. abortus-infected cow and was shown to reactstrongly against two O-chain epitopes (C/Y and A) from S-LPS, coreepitopes from isolated R-LPS, and isolated lipid A epitopes. At the dilutionused, this serum does not react against core or lipid A when LPS moleculeis complete (core and lipid A epitopes are hidden due to the presence of

O-chain). Rabbit polyclonal antisera against R-LPS were produced by im-munizing rabbits with a purified preparation ofB. abortus45/20 R-LPS(30). This antiserum reacts against core epitopes from the R-LPS, but doesnot react against the O-chain, core epitope from S-LPS, and lipid A (29).Rabbit IgGs anti-cow Ig and Baps C/Y mAb were linked to horseradishperoxidase, as previously described (31). At the dilution used, none of thepolyclonal or mAbs used demonstrated reactivity against LPS-untreatedmacrophages. Rabbit polyclonal IgGs anti-cation-independent mannose-6-phosphate receptor (anti-CI-M6PR; Dr. B. Hoflack, Institut Pasteur deLille, Lille, France); rabbit anti-cathepsin D (Dr. S. Kornfeld, WashingtonUniversity School of Medicine, St. Louis, MO); and rabbit anti-giantin (Dr.H. P. Hauri, University of Basel, Basel, Switzerland) were used for im-munofluorescence experiments. Secondary Abs used were: goat Texas red-conjugated anti-cow IgG and donkey FITC-conjugate anti-rabbit IgG(Jackson ImmunoResearch). Rabbit IgG-peroxidase anti-mouse Ig and goatIgG-peroxidase anti-rabbit Ig conjugates were used (Sigma) as secondaryAbs for Western blotting and ELISA.

Confocal microscopy

For fluorescent confocal microscopy, macrophages grown in coverslipswere fixed at room temperature with 3.7% paraformaldehyde in PBS, pH7.4, for 20 min, followed by 10-min incubation with 0.1 M glycine tosaturate aldehyde groups and with 0.1% saponin for cell permeabilization.Then cells were incubated with a PBS solution containing 0.1% sapo-nin/5% mouse serum/5% horse serum for 20 min to block unspecific bind-ing. Primary Abs diluted in the same buffer were added to the cells for 30min. After extensive washings with 0.05% saponin in PBS, macrophageswere incubated for 30 min with secondary fluorescent Abs. Coverslips withadherent macrophages were washed, mounted in Mowiol (Hoechst,Frankurt, Germany), and viewed under a Leica TCS 4D confocal micro-scope (Leica Lasertechnik, Heildeberg, Germany). In all immunofluores-cence experiments, LPS was revealed using the antiserum from infectedcow, followed by anti-cow IgG Ab conjugated to fluorescein.

Western blotting

After internalization of B. abortus LPS, macrophages were lysed inPBS/1% Nonidet P-40 detergent for 30 min, and treated with 1 mg/ml ofproteinase K (Sigma) at 37°C for 30 min. Lysates were run in 12% SDS-PAGE and electrophoretically transferred onto Immobilon-P membranes(Millipore, Bedford, MA). Membranes were blocked in PBS/5% milk/0.05% Tween-20 (Sigma) for 2 h. Primary and secondary Abs were suc-cessively added in this buffer, each being left for 2 h before staining withthe enhanced chemoluminescence system (ECL; Amersham, Courtaboeuf,France). As primary anti-LPS Abs, Baps C/Y, cow, and rabbit antiserawere used to detect the different epitopes of LPS in macrophage cell ly-sates.

Dot-blot analysis

Peritoneal liquids from LPS-inoculated mice were recovered by injecting 5ml of PBS/10% sucrose in peritoneal cavity; the liquid was extracted andcentrifuged to eliminate macrophages. Supernatants were filtered through a0.45-mm cutoff membrane (Gelman Sciences, Ann Arbor, MI) to eliminatecellular debris. A total of 100ml of supernatant was applied to Immo-bilon-P membrane (Millipore) in a dot-blot manifold (Bio-Rad, Hercules,CA). The membrane was removed from the manifold and blocked with0.2% BSA for 1 h before adding the peroxidase-conjugated anti-O-chainAb (Baps C/Y). Detection has been done with the enhanced chemolumi-nescence system.

ELISA assays

After LPS internalization, macrophages were fixed with 3.7% paraformal-dehyde in PBS, then incubated with PBS/10% mouse serum/0.1% saponinto block unspecific sites and to permeabilize the cells. Parallel cultureswere not permeabilized to measure LPS exclusively on the cell surface.Cells were then incubated with 100ml of the antiserum from infected cowdiluted in PBS/5% normal mouse serum, with or without the addition ofsaponin for nonpermeabilized and permeabilized samples, respectively.After 45-min incubation, cells were washed extensively with PBS, and 100ml of horseradish peroxidase-conjugated anti-bovine Ig was added for 45min at room temperature. After washings, 100ml of tetramethylbenzidinesubstrate (Sigma) was added to the cells. After 30-min incubation, theenzymatic reaction was stopped by adding 10ml of 0.5 M H2SO4, and thecolor reaction was read at 450 nm OD. To estimate at which LPS dose theOD corresponded, various fixed amounts of LPS were deposited on anImmobilon-P membrane in a dot-blot manifold, as described above. Eachspot was cut off, and the membrane was then deposited in distinct wells on

3 Abbreviations used in this paper: S-LPS, smooth LPS; R-LPS, rough LPS; CI-M6PR, cation-independent mannose-6-phosphate receptor.4 C. Forestier, E. Moreno, A. Phalipon, P. Sansonetti, and J.-P. Gorvel. Interactions ofBrucella LPS and MHC class II molecules.Submitted for publication.5 W. Beatty, S. Meresse, J. Davoust, P. Gounon, P. Sansonnetti, and J. P. Gorvel.Trafficking of ShigellaLPS in polarized intestinal epithelial cells.J. Cell Biol. Inpress.

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24-well plates. The amount of LPS was revealed by ELISA assays (de-scribed above) using peroxidase-conjugated anti-O-chain Ab (Baps C/Y).

ResultsLPS is delivered with slow kinetics to lysosomes, where itaccumulates

To define the internalization pathway ofBrucella LPS in macro-phages, we compared, by indirect immunofluorescence, the intra-cellular distribution of LPS with several markers of endocyticcompartments and the Golgi apparatus. Macrophages were initiallyincubated with LPS for 1 h at4°C, followed by different periods ofchase at 37°C. After 10 min, a punctate LPS staining pattern, lo-cated intracellularly at the cell periphery, colocalized with FITC-transferrin, indicating that LPS had reached the early endosomalnetwork (Fig. 1,A and B). From early endosomes, internalizedmaterial can be recycled back to the plasma membrane or directedto more acidic intracellular compartments, such as late endosomesand eventually lysosomes (32, 33). From 15 min onward, LPSlocalized mostly within FITC-transferrin-negative vacuoles, sug-gesting a transient and rapid passage of LPS through early endo-cytic organelles (Fig. 1,C and D). After 90 min, LPS becameconcentrated in intracellular compartments positive for the CI-M6PR (Fig. 1,E andF). This marker has been described as cyclingbetween the Golgi apparatus and late endosomes and, dependingon cell type, it accumulates either in thetrans-Golgi network or in

late endosomes (34). Since no colocalization was observed withgiantin, a specific marker of the Golgi apparatus (Fig. 1,G andH),we concluded that LPS was transferred from early to late endo-somes. From late endosomes, LPS took about 5 h toreach cathep-sin D-positive lysosomal compartments (Fig. 2,Ba andBb). Fromthis time up to 24 h, LPS steadily accumulated within the lyso-somes (Fig. 2,Bc and Bd), demonstrating that LPS is not elimi-nated from the host cell. The slow kinetics of LPS traffickingseems to be independent of the activation state of macrophages,since in macrophages that were pretreated with IFN-g for 48 hbefore LPS addition, LPS reached the lysosomes after 5-h inter-nalization (data not shown).

LPS escapes from lysosomes and recycles to the plasmamembrane

The persistence of LPS in macrophages prompted us to investigatethe fate of LPS at late time points. At 48 h, LPS was still presentin macrophages. Although a minor proportion of LPS-containingvesicles remained positive for the lysosomal marker cathepsin D,the majority of LPS was found within intracellular nonlysosomalcompartments (Fig. 2,Be and Bf) and at the plasma membraneforming large clusters (Fig. 3B). These results suggest that, afterlysosomal accumulation, LPS escaped lysosomes and migrated tothe cell surface. We also estimated the relative amounts of cellsurface-bound versus intracellular LPS by ELISA (Fig. 3A). Even

FIGURE 1. Intracellular trafficking ofBrucella LPS in murine perito-neal macrophages. Cells were incubated with 15mg/ml of LPS in cellculture medium for 1 h at 4°C, then washed and further incubated forvarious times at 37°C. After paraformaldehyde fixation, macrophages wereprocessed for double immunofluorescence. LPS was detected by indirectimmunofluorescence using the antiserum fromBrucella-infected cow. Af-ter 10 min of internalization at 37°C, LPS (A) colocalized with transferrin-FITC (B) and, at 60 min, LPS (C) was no longer associated with trans-ferrin-FITC (D). At 90 min, LPS (E, G) was found in a compartmentpositive for the CI-M6PR (F) and negative for giantin, a specific marker ofthe Golgi apparatus (H). Bar 5 10 mm.

FIGURE 2. Transient accumulation ofBrucellaLPS in lysosomes. InA,macrophages untreated with LPS were stained with the antiserum from aB.abortus-infected cow (Aa) and with anti-cathepsin D Ab (Ab). In B, mac-rophages were incubated with LPS as in Fig. 1. Times of chase were from1–48 h. LPS, detected with the antiserum fromBrucella-infected cow, isshown inBa, Bc, andBe, and cathepsin D inBb, Bd, andBf. After 5 h at37°C, LPS (Ba) reached lysosomes positive for cathepsin D (Bb) and ac-cumulated in this compartment even after 24 h (BcandBd). At 48 h, LPS(Be) was mainly found outside lysosomes (Bf). Bar5 10 mm.

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though the majority of LPS was rapidly internalized after 10 min,as shown in Fig. 1,A andB, after 2 h, 25% of total LPS still residedat the plasma membrane (Fig. 3A). Complete internalization oc-curred by 24 h. Then LPS gradually reached the macrophage cellsurface, and at 48 h, most of the LPS was detected by anti-O-chainAbs, at the external leaflet of the plasma membrane (Fig. 3A). Weevaluated the total amount of macrophage-associated LPS to be 1ng of LPS/106 cells (see ELISA assays inMaterials and Methods).In addition, the total amount of LPS associated with macrophagesdid not vary significantly during the in vitro chase periods, and wenever detected exocytosed LPS in the cell culture supernatant bydot-blot analysis (not shown). Altogether, these data support theidea that LPS is not eliminated by macrophages.

BrucellaLPS remains associated with peritoneal macrophagesfor several months

To investigate the fate of LPS in vivo, we injected C3H/HeN micei.p. with 400 mg of Brucella LPS. The kinetics of LPS removalfrom the peritoneal cavity was followed at various times after LPSinjection by dot-blot analysis. Complete clearance of LPS from the

peritoneal fluids occurred between 17 and 20 days (not shown),and inoculated LPS internalized by resident macrophages re-mained associated with these cells for at least 3 mo after injection(Fig. 4). Similarly to what was observed in cell cultures after 2days, LPS aggregates forming large clusters, distributed inside andoutside lysosomal compartments as well as at the plasma mem-brane (Figs. 4 and 3D). This unique cellular distribution patternpersisted up to 90 days postinjection (not shown). Peritoneal mac-rophages from LPS-injected mice, harvested at 60 days postinjec-tion, were then cultured in vitro in the absence of LPS. Fig. 3Cshows that LPS started to recycle from lysosomal and nonlysoso-mal compartments to the plasma membrane after 48 h. The ma-jority of LPS molecules reached the macrophage cell surface after5 days (Fig. 3C), confirming the results shown in Fig. 3A.

The immunochemical properties ofBrucellaLPS are unalteredduring its intracellular trafficking

As shown in Fig. 5,A and B, nativeBrucella S-LPS displays acharacteristic m.w. heterogeneity related to the different lengths ofthe O- chain, core oligosaccharide, and lipid A (35), whereas R-LPS appears as a lower m.w. band (Fig. 5C). After internalizationand trafficking within macrophages, LPS did not show any signif-icant SDS-PAGE profile variation or altered antigenic reactivityagainst a collection of monoclonal anti-O-chain Abs (Fig. 5A) aswell as polyclonal Abs from aB. abortus-infected cow, and wasshown to react strongly against two O-chain epitopes (C/Y and A)from S-LPS, core epitopes from isolated R-LPS, and isolated lipidA epitopes (Fig. 5B), suggesting that LPS was not cleaved duringits intracellular trafficking in peritoneal macrophages. In addition,we never detected the appearance of R-LPS (Fig. 5C), indicatingthat the O-chain moiety was not cleaved from S-LPS. By compar-ing the signal intensity corresponding to cell-associated LPS withthat of purified LPS, the amount of cell-associated LPS was esti-mated to 1 ng/106 cells in agreement with ELISA measurements.

Intracellular LPS released from digested bacteria is detected atthe plasma membrane

We have shown that the attenuatedB. abortusS19 strain is de-graded within lysosomes of phagocytes (36). To explore the fate ofLPS released intracellularly after the degradation of phagocytosedattenuated S19 bacteria in lysosomes, we followed the intracellular

FIGURE 3. Recycling of Brucella LPS to the cell surface of macro-phages. InA andB, peritoneal macrophages were harvested from normalmice, plated for 2 h (53 105 cells/ml), then incubated withBrucellaLPS(15 mg/ml) for 1 h at4°C. In C andD, macrophages were harvested fromLPS-injected mice, at 60 days postinjection. After several washings, cellswere incubated at 37°C and fixed. One-half of the samples were perme-abilized with saponin. Quantification of intracellular versus cell surface-bound LPS was analyzed by ELISA assay (A, C) using the antiserum froma B. abortus-infected cow. Nonpermeabilized cells allowed the quantifica-tion of cell surface-associated LPS (f), whereas permeabilization gave themeasurements of total LPS (intracellular1 surface-bound) (E). Theamount of intracellular LPS (L) was calculated by the subtraction of thevalues obtained with nonpermeabilized samples from those obtained withpermeabilized samples. Immunofluorescence (B,D) shows the distributionof Brucella LPS in permeabilized (upper panel) and nonpermeabilizedmacrophages (lower panel), following 48 h (B) or 2 h (C) of chase in vitro.f, Surface-bound LPS;L, total LPS;E, intracellular LPS.

FIGURE 4. Distribution of Brucella LPS in macrophages after perito-neal injection of LPS. Mice were i.p. injected withBrucellaLPS (400mg).Peritoneal macrophages were recovered after 6 or 60 days and processedfor double immunofluorescence. LPS (upper panels), revealed by theantiserum from aBrucella-infected cow, colocalizes partially even after60 days with cathepsin D-positive compartments (lower panels).Bar 5 10 mm.

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trafficking of S19Brucella LPS within macrophages at varioustimes postinfection. Forty-eight hours after inoculation, most intactbacteria were found to be within compartments positive for thelysosomal marker cathepsin D (Fig. 6A). At 72 h, most bacteriawere degraded within lysosomes, and bacterial debris containingLPS were found within cathepsin D-positive vesicles, scatteredthroughout the cytoplasm (Fig. 6A), indicating that while bacteriaunderwent enzymatic degradation in the cathepsin D-positive com-partment, the epitopic structure of LPS resisted the intracellulardegradation processes. From 72 h onward, LPS was gradually de-tected at the macrophage plasma membrane (Fig. 6B). Finally, at6 days postinfection, almost all infected cells exposed high levelsof LPS at their plasma membrane (Fig. 6B), indicating that a con-siderable proportion of glycolipids released by the degraded bac-teria followed the recycling pathway described above.

DiscussionMacrophages are specialized cells for capturing, processing, andpresenting exogenous Ags to T lymphocytes. They are also theprincipal target for many intracellular Gram-negative bacteria andtheir endotoxins. The biological action of LPS on macrophages has

been studied extensively (37). Among the less well-characterizedevents following LPS interaction with macrophages are the intra-cellular trafficking and processing of internalized LPS. A compre-hension of these steps is critical for the understanding of the bio-logical activity and immunogenicity of this nonpeptidic Ag.

In this study, we demonstrate that, in conditions that favor re-ceptor-mediated endocytosis in vitro (38), LPS is rapidly internal-ized in early endosomes and transported, with slow kinetics, to lateendosomes and then to lysosomes, where it accumulates. Later,LPS is found in cathepsin D-negative compartments before itreaches the cell surface, where it forms large clusters. In contrastto other reports, we did not detectBrucellaLPS either in the Golgiapparatus, nucleus, mitochondria, nor free in the cytoplasm (6, 10).This discrepancy may be explained by the different experimentalprocedures, including the use of different target cells and highdoses of cytotoxic LPS. Despite the fact thatBrucellaLPS inducesmany biological effects, characteristics of so-called typical LPSmolecules, it possesses very low toxicity (23), which favors long-term studies in cultured cells and in animals (39). In general, pre-vious studies were performed by using continuous enterobacterialLPS incubation at 37°C, which may allow receptor-mediated up-take, macropinocytosis, and fluid-phase endocytosis (6, 10). Re-cent studies from Kitchens et al. demonstrated that the route andkinetics of LPS trafficking depend upon its internalization pathway(CD14 dependent or independent) and its aggregation state, re-spectively (40, 41), thus offering an explanation for the divergenceof the results obtained between different studies.

It has been proposed that the kinetics of intracellular traffickingof different substances is independent of the nature of the ingestedmolecule in many respects (42–44). However, several studies havedemonstrated that LPS follows the endocytic pathway with slowkinetics (11) (Beatty et al., submitted). In agreement with these, weobserved a substantial delay in the intracellular transit of LPS fromearly endosomes to lysosomes, in comparison with proteins suchas BSA-FITC, which is rapidly transported from early endosomesto lysosomes within 60 min after internalization and remains lo-cated in perinuclear lysosomes until its degradation (45). The slowkinetics ofBrucellaLPS traffic considerably differs fromBrucellaorganisms. In the case of virulent bacteria, it is known that bacteriaevade lysosomes and replicate within the endoplasmic reticulum.For nonvirulentBrucella, the bacteria are found within phago-somes that rapidly fuse with lysosomes, where degradation occurs12 h postinfection (36). In the latter case,Brucella LPS is conse-quently released from the outer membrane of the killed bacteriaand remains within lysosomes for at least 140 h. This last obser-vation is in agreement with several investigations that have dem-onstrated the appearance and retention of free LPS within hostcells after phagocytosis of different bacteria (8, 9, 13, 46). It is thenpossible that LPS remains in a nonfunctional lysosomal compart-ment in which resident proteins, such as cathepsin D and LAMP(lysosome-associated membrane protein), have been degraded orremoved. In favor of this is the finding that indigestible polysty-rene particles are contained within nonfunctional lysosomes char-acterized by the absence of hydrolases (44). In addition, it may bethat LPS prevents the acquisition of newly synthesized hydrolasesarriving from Golgi-derived vesicles, the consequence of whichwould be the generation of hydrolase-defective lysosomes (47).Therefore, the retention process and coalescence of LPS in largevacuolar aggregates within macrophages seem to be a property ofthis complex molecule and independent of the bacteria from whichit originates.

The clustering of LPS molecules and the formation of discretepatches on the macrophage plasma membrane are consistent with

FIGURE 5. Brucella LPS analysis by immunoblotting. Cells were in-cubated withBrucella S-LPS (15mg/ml) at 4°C for 1 h, washed, andfurther incubated with cell culture medium at 37°C for 0.5, 2, 5, 24, 30, and48 h. Then macrophages were lysed and the lysates were loaded on 12%SDS-polyacrylamide gels, and after transfer to Immobilon membranes,LPS was revealed inA using the Baps C/Y peroxidase-conjugated mAbthat recognizes specifically C/Y sugar epitopes of the O-chain moiety. InB,LPS was revealed using a serum from aB. abortus-infected cow that rec-ognizes the O-chain C/Y most abundant epitope and the A epitope ofS-LPS, the outer core epitope exposed in isolated R-LPS and the backbonedisaccharide of the isolated lipid A. InD, the presence of both R-LPS wastested by using a rabbit antiserum againstB. abortus45/20 R-LPS recog-nizing specifically the core moiety of R-LPS. A total of 10 ng of purifiedS-LPS (S) (AandB) and 100 ng of purified R-LPS (R) was deposited andrevealed, as described above.

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studies describing the preferential association of LPS with partic-ular classes of membrane lipids (48) at specific membrane domains(5, 49). Based on recent experiments conducted in our laboratoryin which the membrane fluidity ofBrucella LPS-treated cells hasbeen measured (unpublished results), and experiments demonstrat-ing the direct (50, 51) or LPS binding protein (LBP)-mediated (52,53) intercalation of LPS into bilayers, we propose thatBrucellaLPS is inserted into the cell membranes by its lipid A moiety,exposing its immunogenic O-chain moiety toward the extracellularmedium. The existence and the fate of these LPS large clusters inthe plasma membrane remain unknown. Although it was found atthe cell surface, no freeBrucellaLPS was detected in supernatantsof cell cultures, as demonstrated by the ELISA and colocalizationexperiments, respectively, suggesting that there is no exocytosis ofLPS from macrophages. The fact that LPS disappeared from peri-toneal fluids of mice only 3 wk after its injection suggests thatphagocytosis of high doses of LPS by peritoneal cells may be thelimiting factor for its clearance in vivo. The presence of conspic-uous large aggregates in peritoneal macrophages after 6 and 60days highlights the remarkable stability of this molecule.

After storage in lysosomes, LPS escapes these compartmentsand appears on the plasma membrane of macrophages, in agree-ment with the results previously obtained by Korn et al. (54). Al-though the mechanism by which LPS recycles from intracellularcompartments to the plasma membrane remains to be character-ized, it is likely that LPS-containing vesicles ensure the transportof LPS from intracellular compartments to the plasma membraneby specific target mechanisms. It may be that the insertion of thelipid A into the membrane of the LPS-containing compartmentrecruits constitutive recycling molecules, promoting its own trans-portation to the cell surface. Recycling pathways have been par-tially characterized for complex membrane lipids or exogenousprotein Ags. For instance, ceramide-containing sphingolipids havebeen shown to be translocated from the Golgi apparatus to theplasma membrane by a vesicle-mediated process (55–57). Despite

the fact that LPS possesses structural and functional similaritieswith sphingolipids such as ganglosides and cerebrosides (58, 59),LPS does not recycle by the same mechanism since we never de-tected this molecule in the Golgi apparatus. It has also been dem-onstrated that processed proteic Ags recycle from late endocyticcompartments to the cell surface by transiting within specializedMHC class II (MHC-II)-enriched compartments, where peptidesassociate with Ag-presenting molecules (60). It is possible thatBrucellaLPS, which is also found in late endocytic compartments,recruits and modifies MHC-II products within lysosomes, as sug-gested by previous experiments in which this molecule was foundto interact and induce the formation of SDS-resistant forms (Cforms) of MHC-II proteins in B lymphocytes (61) (Forestier et al.,submitted). The coalescence of LPS in discrete patches at theplasma membrane may be the result of a combination of the in-trinsic properties of LPS, which promotes its own arrangement ascrystal layers in the outer leaflet of membranes (62), and the trans-port of LPS to specific plasma membrane domains by carriermolecules.

Macrophages are capable of processing enterobacterial LPS bya slow mechanism that may involve a combination of oxidativeand enzymatic hydrolytic procedures (18–20). These cells modifythe chemical structure of any of the three enterobacterial LPS moi-eties (lipid A, core, and O-chain) and, as a consequence, alter theelectrophoretic mobility of this molecule and its reactivity againstspecific Abs (13). For instance, deacylation and dephosphorylationof LPS result in reduced amounts of lipid A fatty acids, less bind-ing of SDS, and slower migration in SDS-PAGE (13, 46, 63).Oxidation and hydrolysis of core sugars diminish the imunoreac-tivity against core determinants and result in free lipid A detectedwith specific Abs (13, 35, 64). Commonly, the resulted free O-chain is unable to migrate in SDS-PAGE due to the absence ofSDS binding sites (28, 35). Removal of sugar substituents or re-peating units from the O-chain causes faster aberrant migration inSDS-PAGE and reduced reactivity to Abs (13, 65). In spite of its

FIGURE 6. Distribution ofBrucellaLPS in macro-phages followingBrucellaS19 infection. Macrophageswere infected withB. abortusstrain 19 for 10 min,washed, incubated for various times at 37°C, then pro-cessed for double immunofluorescence. Infected mac-rophages were permeabilized with saponin (A) and la-beled with both anti-cathepsin D Ab andB. abortus-infected cow serum, which detects either the LPSassociated to bacteria or LPS liberated from degradedbacteria. InB, macrophages were not permeabilized tomonitor the presence of surface-bound LPS at differentpostinfection times (48, 72, and 120 h).

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long residence inside lysosomes,Brucella LPS seems to remainuncleaved. This observation is based on the unaltered migrationpattern in SDS-PAGE of theBrucella LPS extractable from mac-rophages, its consistent reactivity against monoclonal and poly-clonal Abs, and the absence of detectable free core or lipid Adeterminants. The resistance ofBrucellaLPS to macrophage pro-cessing may simply reflect the absence of a cellular machinerycapable of oxidizing and hydrolyzing this complex molecule. In-deed, the O-chain, the core oligosaccharide, and the lipid A dis-sacharide backbone ofBrucella LPS are respectively more resis-tant to oxidation, acid hydrolysis (35, 66), and deacylation (26)than enterobacterial LPS. In conclusion, the unique chemical struc-ture ofBrucellaLPS may not be prone to regular processing mech-anisms described in the case of enterobacterial LPS.

Finally, our results indicate that LPS is retained in an immuno-logically detectable form in murine peritoneal macrophages forlong periods of time, then recycles and associates as clusters at theplasma membrane without detectable modification. The long per-manence of LPS within cells of individuals may result at least inseveral pathologic effects, such as reactive arthritis or hypersensi-tivity reactions, developed after acute and chronic bacterial infec-tions (67–69). The presence of LPS at the plasma membrane ofAPC may lead to the activation of T lymphocytes, as it has beenshown for other glycolipids (70–75). The recycling phenomenommay explain how LPS activates some T lymphocytes, as recentlyshown by others (75–78).

AcknowledgmentsWe thank J. Ewbank, I. Moryion, and S. Meresse for critically reading themanuscript.

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