A Synthetic Analog of the 3-Deoxy-D-Manno-2-Octulosonic

INFECrION AND IMMUNITY, Sept. 1993, p. 3616-3624 Vol. 61, No. 9 0019-9567/93/093616-09$02.00/0 Copyright X 1993, American Society for Microbiology A Synthetic Analog of the 3-Deoxy-D-manno-2-Octulosonic Acid Disaccharide Moiety of Rough-Type Endotoxins Does Not Bind to Mouse Peritoneal Macrophages and Human Monocytes ROBERT GIRARD,' THIERRY PEDRON,1 PAUL KOSMA,2 AND RICHARD CHABY3* Unite d'Immunophysiologie Moleculaire, URA-145 du Centre National de la Recherche Scientifique, Institut Pasteur, Paris Cedex 15,1 and Equipe "Endotoxines, " URA-1116 du Centre National de la Recherche Scientifique, Universite de Paris-Sud, 91405 Orsay,3 France, and Institut fur Chemie der Universitat fur Bodenkultur Wien, A-1180 Vienna, Austria2 Received 22 January 1993/Returned for modification 16 April 1993/Accepted 7 June 1993

Strong evidence supports the concept that lipid A is the main biologically active region of endotoxins and is recognized by specific binding sites of different cell types. However, receptors for carbohydrates are also present on mononuclear phagocytes, and it has been suggested that one of these lectin-like proteins may be specific for the 3-deoxy-D-manno-2-octolosonic acid (Kdo) residues of endotoxins. To reexamine this hypothesis, we prepared a "2'I-labeled conjugate consisting of a synthetic Kdo-2,4-Kdo disaccharide covalently linked to bovine serum albumin (125I-Kdo2-BSA). The Kdo disaccharide residues of this radiolabeled conjugate were fuly accessible to a monoclonal antibody which reacts specifically with this epitope. However, 1251-Kdo2-BSA did not exhibit any detectable specific binding on thioglycolate-elicited mouse peritoneal macrophages or on human monocytes. Furthermore, the specific binding of biotin-labeled lipopolysaccharide derivatives to mouse macrophages and human monocytes was not inhibited by a soluble synthetic Kdo-2,4-Kdo-polyacrylamide copolymer or by a synthetic glycolipid consisting of an a-Kdo residue glycosidically linked to 0-6 of allyl-4-0-phosphoryl-N-3-hydroxytetradecanoyl-1-D-glucosaminide. These results indicate that binding sites specific for Kdo are not present (or not accessible) on the surface of mouse macrophages and human monocytes.

Many deleterious effects associated with gram-negative gesting the presence of LPS-binding sites specific for the septicemia, including fever, hypotension, disseminated in- carbohydrate region of LPS (lectin-like receptors) have also travascular coagulation, and multisystem organ failure, are been reported. Parent (28) suggested the presence on rat due to mediators (25, 39) produced following the interaction hepatocytes of a receptor specific for the heptose unit(s) of the bacterial endotoxin (lipopolysaccharide [LPS]) with present in the core region of LPS. A lectin-like receptor is monocytes and macrophages (26). Stimulation of these also apparently involved in the interaction of Bordetella mononuclear phagocytes with LPS elicits a wide range of pertussis LPS with rabbit peritoneal macrophages (11), since responses, including the production of interleukin-1 (IL-1) the binding was inhibited by the polysaccharide fragment of (9), interleukin-6 (IL-6) (38), interleukin-8 (IL-8) (44), alpha/ the LPS and was not inhibited by lipid A. However, this beta interferon (15), tumor necrosis factor alpha (4, 6), putative lectin-like receptor was not detectable on rabbit prostaglandins (1), complement components (34), enzymes alveolar macrophages (11), and mouse peritoneal macro- (plasminogen activator) (37, 40), nitrous oxide (16), and phages have been shown to recognize exclusively and spe- reactive metabolites of oxygen (27). cifically the lipid A region of the same LPS (35). The Central to the mechanisms involved in these various presence on human monocytes of a lectin-like receptor for cellular responses is the question of specific receptors for the inner core region of the Salmonella minnesota R7 LPS LPS. In the last several years, a number of groups have has also been suggested by Couturier et al. (8). Recent reported the results of experiments designed to identify LPS experiments of Lei and Morrison (21) suggested that a minor receptors. At least four cell surface molecules of that type cell surface protein of 38 kDa, detected with a photoactivat- have been detected so far (41): the scavenger receptor (13, able LPS, might be specific for 3-deoxy-D-manno-2-octu- 14), the CD18 chain of integrins of the CD11/CD18 type (42), losonic acid (Kdo) residues, since the binding of the LPS the CD14 antigen (43), and a 73-kDa glycoprotein detected probe can be inhibited by the S. minnesota Re LPS (com- on mouse (22) and human (12) cells by labeling with a posed only of Kdo and lipid A) but cannot be inhibited by photoactivatable LPS probe. lipid A. In the present study, we reexamined the question of The lipid moiety (lipid A) of LPS is required for the the existence of a putative LPS receptor with binding interactions with all the binding sites mentioned above. specificity for Kdo by the use of a chemically defined ligand Three of these molecules (the scavenger receptor, the CD18 consisting of a synthetic Kdo disaccharide covalently cou- antigen, and the 73-kDa protein) have been reported to pled to bovine serum albumin (BSA). interact directly with lipid A, whereas the CD14 antigen interacts with complexes of lipid A and an acute-phase MATERIALS AND METHODS protein (LPS-binding protein) (43). However, results sug- Mice. Female BALB/c nul+ mice (8 to 16 weeks old) were from the Breeding Center of the Pasteur Institute (Paris, * Corresponding author. France). 3616 VOL. 61, 1993 ABSENCE OF RECEPTOR FOR SYNTHETIC Kdo DISACCHARIDE 3617

Chemicals and reagents. Heparin and chloramine-T were aldehyde by the method of Cambiaso et al. (5). MAb El (12 from Sigma Chemical Co. (St. Louis, Mo.). Thioglycolate ,g) was incubated (1 h, 20°C) with the gel (column 30 by 2 broth was from Diagnostic Pasteur (Ville d'Avray, France). mm), and excess antibody was washed out by elution of the MSL was purchased from Eurobio Biotechnology (Les Ulis, column with 500 Rl of 0.1% Tween 20 in phosphate-buffered France). 1"I was from Amersham International (Bucking- saline (PBS). hamshire, United Kingdom). The streptavidin-fluorescein Radiolabeled ligands. After activation of LPS-Re and isothiocyanate (FITC) conjugate was from Becton Dickinson LPS-Sc by CNBr treatment (32), LPS-Re was coupled to (Mountain View, Calif.), and biotinyl-N-hydroxysuccinim- BSA and LPS-Sc was coupled to tyramine (24). BSA, ide was from Boehringer (Mannheim, Germany). Kdo2-BSA, the tyramine derivative of LPS-Sc, and the LPS and synthetic compounds containing Kdo. The LPS LPS-Re-BSA conjugate were then iodinated with "SI by the from Salmonella choleraesuis (group C2, smooth chemo- chloramine-T method (18) and dialyzed extensively against type, serotype 62,7,14) (LPS-Sc) was extracted by the phe- 0.15 M sodium chloride. The radiolabeled LPS-Sc was nol-water procedure and purified as described previously (7). separated from residual iodine by precipitation with ethanol The LPS from S. minnesota Re595 (LPS-Re) was from in the presence of a carrier protein (gelatin). To 1 volume of Sigma. The lipid A fragment of the B. pertussis LPS (LipA- the LPS-gelatin suspension (0.1 mg of LPS per ml; 5 mg of Bp) and the de-0-acylated (hydroxylamine-treated) derivative gelatin per ml), 5 volumes of ethanol was added. The mixture of LPS-Re (LPS-Re/OH) were prepared as described previ- was stored at -20°C for 30 min, and the precipitate was ously (36). Analysis by thin-layer chromatography (chloro- recovered by centrifugation (10 min, 900 x g). The pellet ammonia [100:80:20:4, containing the labeled endotoxin was suspended in PBS (1 form-methanol-water-concentrated ml, pH 7.4). The specific radioactivities of the radioiodinated vol/vol]) on silicic acid plates (Kiesel-gel 60 high-perfor- preparations of LPS-Sc, BSA, Kdo2-BSA, and LPS-Re-BSA mance thin-layer chromatography plates; Merck, Darm- (1251-LPS-Sc, 125I-BSA, 125I-Kdo2-BSA, and 125I-LPS-Re- stadt, Germany) indicated that these LPS derivatives were BSA) were 2.1, 2.9, 1.7, and 2.6 x 106 cpm/,ug, respectively. devoid of detectable contamination by native LPS. The Biotin-labeled LPS. Biotinyl-N-hydroxysuccinimide (150 glycolipid KM20a prepared by F.-I. Auzanneau (2) consists ,ul; 33 mg/ml in dimethylformamide) was added to a suspen- of an a-Kdo residue glycosidically linked to 0-6 of a sion of LPS-Re (2.8 mg in 1.1 ml of a 0.1 M sodium hydrogen glucosamine-derived lipid, allyl-2-deoxy-2-[(3R)-3-hydroxy- carbonate buffer, pH 9) (3). The mixture was incubated for tetradecanamido]-4-O-phosphoryl-o-D-glucopyranoside. The 150 min at 20°C with gentle rotation, and the suspension of acrylamide copolymer of the allylglycoside of a-Kdo-2,4-a- LPS-Re-biotin (LPS-Re-Biot) was dialyzed against PBS. Kdo (Kdo2-PA; 60 to 100 kDa; 0.34 ,umol of disaccharide per Mouse macrophages and human monocytes. Leukocytes mg; 15% Kdo2 [wt/wt]) was prepared as described previ- were isolated by centrifugation (120 min, 1,200 x g) of blood ously (19). Kdo2-BSA consisted of a cysteamine glycoside of of human volunteers (350 ml), collected on a citrate-phos- a-Kdo-2,4-a-Kdo disaccharide covalently linked to BSA by phate-glucose anticoagulant. The cells in the interphase the isothiocyanate method of Smith and Ginsburg (33). layer (40 ml) were suspended in PBS (200 ml) containing Briefly, disodium {3-deoxy-a-D-manno-2-octulopyranosy- heparin (20 U/ml) and centrifuged at room temperature (25 lonate-(2--4)-[3-(2-amino-ethylthio)]propyl 3-deoxy-a-D- min, 900 x g) on MSL (1.077 g/ml). The cells were washed manno-2-octulopyranosidonate} was prepared as reported four times with balanced salt solution (BSS) containing previously (17). A solution of the disaccharide (9.0 mg) in 0.1 heparin (20 U/ml) and once with BSS. The viability of the M aqueous sodium hydrogen carbonate (2 ml) was vigor- cells was evaluated by trypan blue exclusion. Peritoneal ously stirred with a solution of thiophosgene (3 ,ul) in exudates of five mice were harvested, 5 days after intraperi- chloroform (2 ml) for 3 h at room temperature. The organic toneal injection of 1.7 ml of thioglycolate broth, by perito- layers were removed, and the aqueous phase was washed neal washes with BSS (10 ml) containing 10 U of heparin per three times with 2-ml portions of chloroform. The solution ml. Human leukocytes (5 x 106 viable cells per well, was purged with nitrogen and added to a solution of BSA containing 19% CD14+ cells according to a fluorescence- (9.8 mg) in 0.1 M aqueous sodium hydrogen carbonate-0.3 M activated cell sorter [FACS] analysis) were incubated (1 h, sodium chloride (3 ml). The solution was kept at room 37°C) in RPMI 1640 buffered with 20 mM HEPES (N-2- temperature for 50 h, applied on a column of Sephadex G-25 hydroxyethylpiperazine-N'-2-ethanesulfonic acid) in plates (50 by 1.6 cm), and eluted with 0.01 M aqueous NaHCO3. with 24 wells. Mouse peritoneal exudate cells (containing Ninhydrin-positive fractions were pooled and dialyzed two 90% neutral red-positive macrophages) were incubated (18 times against water (2 liters). Lyophilization yielded the h, 37°C) in RPMI 1640 buffered with 20 mM HEPES and Kdo2-BSA conjugate (17 mg; 2.5 disaccharide units per containing 10% fetal calf serum (FCS). Nonadherent cells molecule of BSA) as an amorphous powder. The structures were removed by four washings (1 ml per well) with the of the natural (LPS-Re, LPS-Re/OH) and synthetic (KM20a, medium used in the adhesion step. Kdo2-BSA, Kdo2-PA) compounds containing Kdo used in Binding of LPS-Re-Biot to mouse peritoneal cells and hu- this study are presented in Fig. 1. man leukoctes. Thioglycolate-elicited mouse peritoneal Antibodies. Monoclonal antibody (MAb) El (a mouse cells (5 x 10 cells in 250 ,u of RPMI 1640 supplemented with immunoglobulin G3 [IgG3] antibody directed against the 10% FCS) were preincubated for 2 h at 4°C with inhibitors Kdo disaccharide present in LPSs of the Re chemotype) has (125 RI) in 1-ml polystyrene tubes. LPS-Re-Biot (125 ,ul; 50 been prepared and characterized previously (30). A rabbit ng/ml) was then added. After incubation for 18 h at 4°C with anti-mouse Ig was prepared by affinity chromatography of a gentle stirring, the cell suspension was analyzed by flow rabbit serum on a mouse IgG immunoabsorbent, as de- cytometry. Human leukocytes (5 x 105 cells in 250 1±l of scribed previously (10). The biotin-labeled mouse anti-hu- RPMI 1640 supplemented with 2% FCS) were preincubated man CD14 antibody (MY4-Biot) was from Coulter Immunol- for 1 h at 20°C with the inhibitors (125 pI) in 24-well culture ogy (Hialeah, Fla.). plates. The cells were then resuspended, incubated at 20°C MAb El immunoabsorbent. The rabbit anti-mouse IgG for 1 h with LPS-Re-Biot (125 RI; 50 ng/ml), washed, and was coupled to aminohexyl-Sepharose activated with glutar- analyzed by flow cytometry. 3618 GIRARD ET AL. INFECT. IMMUN.

FIG. 1. Structures of the compounds containing Kdo that were used as inhibitors.

Binding of radiolabeled ligands to human monocytes and inson Electronic Laboratories, Mountain View, Calif.) mouse macrophages. Human monocytes (adherent cells from equipped with an argon ion laser and coupled to a micro- 5 x 106 viable leukocytes) were incubated with radiolabeled computer system (9153 B; Hewlett-Packard). Cells were compounds in RPMI 1640 (150 ,ul) supplemented with BSA gated by uptake of propidium iodide to exclude dead cells (3.3 mg/ml). The binding of mouse macrophages to the (29) and by forward-angle light scatter to exclude small radiolabeled compounds was carried out in the same medium debris and large aggregates. Forward scatter and fluores- supplemented with 10% heat-inactivated FCS. Incubations cence histograms were generated with logarithmic amplifi- (2 h, 20°C) were done in triplicate in the presence and cation of light from single cells. Viable cells with a fluores- absence of a 10-fold excess of unlabeled ligand. After three cence intensity higher than the rapid (2 s) washings immersion in ice-cold 0.15 M autofluorescence level of by NaCl unlabeled cells were scored as fluorescent cells. solution, the bound material was solubilized with a solution (0.5 ml) of sodium dodecyl sulfate 1 mM ELISA. For enzyme-linked immunosorbent assays (ELI- (1%) containing 96-well EDTA, and the radioactivities were measured in a gamma SAs), Luxlon plates (CML-FRANCE, Nemours, counter (Kontron MR 480C). The specific binding was the France) were coated (20 h, 40C) with the antigen (100 ,ul of calculated difference of radioactivity measured in the ab- 1-mg/ml solutions in a pH 9.6 buffer consisting of 50 mM Tris sence and presence of a 10-fold excess of the homologous hydrochloride and 20 mM MgCl2). Residual binding sites of unlabeled compound. the plastic were saturated by incubation (1 h, 37°C) with a Flow cytometry analysis. Cells were incubated with LPS- solution of gelatin (0.25% in the same buffer). After washings, Re-Biot as described above. The binding of the biotin- the plates were incubated (2 h, 370C) with solutions of MAb labeled LPS was detected by incubation of the cell suspen- El. The plates were then washed, incubated (37°C, 90 min) sions (5 x 105 cells in 100 pul) for 20 min at 4°C with with peroxidase-labeled goat anti-mouse Ig reagent, washed FITC-labeled streptavidin (3 ,ul) as recommended by the again, and developed by incubation (15 min, 20°C) with supplier. The cell suspensions were then centrifuged through o-phenylenediamine (0.5 mg/ml) and H202 (0.025%) in 0.1 M FCS (50% in PBS), and the pellets were resuspended in 0.5 citrate buffer (pH 5.5). The reaction was stopped with a ml of PBS containing propidium iodide (0.1 ,ug/ml) to stain solution of 0.5% sodium sulfite in 2 N H2S04 (50 ,ul per well), dead cells. Immunofluorescence analysis (5,000 cells of each and the plates were scanned at 490 nm in a Dynatech MR 5000 population) was done on a FACS (FACScan; Becton Dick- spectrophotometer (Dynatech, Marnes-la-Coquette, France). VOL. 61, 1993 ABSENCE OF RECEPTOR FOR SYNTHETIC Kdo DISACCHARIDE 3619

1.5 accessibility was analyzed by ELISA with an MAb (MAb El) which has been shown previously (30) to interact specifically with the Kdo disaccharide present in the rough-type LPS from S. minnesota Re595 (LPS-Re). We found (Fig. 2) E that the presence of biotin residues does not reduce the C 1.0 accessibility of the Kdo disaccharide in LPS-Re-Biot. In the 0 Kdo2-BSA conjugate, the Kdo disaccharide units are also fully accessible to the antibody. There was no detectable interaction between the antibody and the carrier. z The Kdo2-BSA conjugate was radiolabeled on its BSA 0.5 moiety with 125I. To determine if the radioactive material m0 0 (1_5I-Kdo2-BSA) still carries intact and accessible Kdo disac- mu) charide units, the absorption of this material (500 pg) on a column of MAb El immunoabsorbent was analyzed and compared with that of radiolabeled BSA. We found that a major fraction (72%) of the '"I-Kdo2-BSA preparation was 0 5 10 15 20 25 absorbed on the column, whereas most of the radiolabeled ANTIBODY El (ag/mI) BSA (93%) was not. MONOCLONAL Binding of radiolabeled ligands to mouse macrophages and FIG. 2. Binding of MAb El to ligands bearing a Kdo disaccha- monocytes. To analyze the interaction of LPS with ride. Plates were coated with BSA, LPS-Re, LPS-Re-Biot, LPS-Re- human BSA, and Kdo2-BSA. The binding of MAb El was determined by LPS receptors, tyramine was covalently coupled to the po- ELISA. Vertical bars represent standard deviations of the means of lysaccharide chain of a smooth-type LPS (from S. cholerae- triplicate determinations in one representative experiment. suis), and this LPS derivative was radiolabeled with 125I. Specific binding of this radiolabeled ligand on mouse peritoneal macrophages was observed (2.4 ng of LPS on 10 cells; RESULTS about 1.4 x 105 molecules per cell) (Fig. 3). On the other hand, the 125I-Kdo2-BSA conjugate did not bind specifically Accessibility of the Kdo disaccharide in Kdo2-BSA and to these cells. The interaction of the same ligands with human LPS-Re-Biot. A synthetic analog of the carbohydrate region monocytes was also analyzed. Again, specific binding of of Re-type LPSs (Kdo-a-2,4-Kdo disaccharide) (17) has been 1"I-LPS-Sc was observed, whereas the 1"I-KdO2-BSA con- covalently linked to BSA. A second probe containing the jugate did not exhibit any detectable specific binding (Fig. 4). Kdo disaccharide was obtained by coupling biotin to LPS- To demonstrate that a BSA carrier cannot hinder or inhibit Re. In order to use these compounds as ligands able to the binding of linked ligands to LPS receptors, an LPS-Re- interact with putative Kdo receptors, good accessibility of BSA conjugate and its radiolabeled derivative were prepared the Kdo disaccharide within the conjugate is required. This by the techniques previously used to obtain tyramine-sub-

TOTAL BINDING NONSPECIFIC BINDING SPECIFIC BINDING

10 10 E a. 8 8 0 0 6 6 C-

- 4 4 z a 2 2

0 0

0.1 1 10 0.1 1 10 0.1 1 10 RADIOLABELED LIGAND (pg/mI) FIG. 3. Binding of radiolabeled compounds to mouse macrophages. Thioglycolate-elicited mouse peritoneal macrophages (106 adherent cells) were incubated for 2 h at 20°C (in medium containing 3.3 mg of BSA per ml and 10% FCS) with various concentrations of radiolabeled compounds (15I-LPS-Sc, 125I-BSA, and 125I-Kdo2-BSA). Nonspecific binding was measured in the presence of a 10-fold excess of the homologous, unlabeled compound. Specific binding (C) was the calculated difference between total (A) and nonspecific (B) binding. Vertical bars represent standard deviations of the means of replicates in one representative experiment. 3620 GIRARD ET AL. INFECT. IMMUN.

TOTAL BINDING NONSPECIFIC BINDING SPECIFIC BINDING

80 80 0_ E 0 60 60 0

0 40 40 z

z 20 20

0 0

0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 12 RADIOLABELED LIGAND (Ag/mI) FIG. 4. Binding of radiolabeled compounds to human monocytes. Adherent cells (from 5 x 106 human leukocytes) were incubated for 2 h at 20°C (in medium containing 3.3 mg of BSA per ml) with various concentrations of radiolabeled compounds (1 I-LPS-Sc, 125I-BSA, and 125I-Kdo2-BSA). Specific binding was determined as described in the legend of Fig. 3. Vertical bars represent standard deviations of the means of replicates in one representative experiment. stituted LPS-Sc and 125I-LPS-Sc. An ELISA experiment and phosphorylethanolamine units). The Kdo disaccharide (Fig. 2) indicated that this LPS-Re-BSA conjugate was region of this modified LPS was fully accessible, as assessed recognized by MAb El as well as LPS-Re. The specific by its interaction with MAb El (Fig. 2). binding of I251-LPS-Re-BSA to human monocytes (Fig. 5A) The interaction of LPS-Re-Biot with mouse macrophages and to mouse macrophages (Fig. 5B) shows that covalent was analyzed by flow cytometry. Cells bearing bound LPS- linkage to BSA does not reduce the accessibility of the ligand Re-Biot were detected with FITC-labeled streptavidin. We to LPS receptors present on the two cell types. found (Fig. 6) that a large percentage (54.5%) of the macro- Interaction of LPS-Re-Biot with mouse macrophages. Bi- phages had fluorescence levels higher than the threshold of otin was coupled to the free amino groups reported to be autofluorescence (about 19 with the gain setting used). present in the lipid A region of LPS-Re (on 4-aminoarabinose Furthermore, the binding of high levels of LPS-Re-Biot

HUMAN MONOCYTES MOUSE MACROPHAGES

50 50

E 40 40

n C 30 30 o 20 z 20

miZ 10 10 0 0

0 2 4 6 8 10 0 1 2 3 4 5 1251-LPS-Re-BSA (.mg/ml) FIG. 5. Binding of radiolabeled LPS-Re-BSA. Mouse macrophages (B) and human monocytes (A) were incubated with various concentrations of 125I-LPS-Re-BSA as described in the legend of Fig. 3 and 4, respectively. Total, nonspecific, and specific binding were determined as described in the legend of Fig. 3. Vertical bars represent standard deviations of the means of triplicate determinations in one representative experiment. VOL. 61, 1993 ABSENCE OF RECEPTOR FOR SYNTHETIC Kdo DISACCHARIDE 3621

experiment was carried out (Fig. 7). We found that the soluble copolymer Kdo2-PA, which contains Kdo disaccharide units structurally identical to those present in LPS-Re (and which is a good inhibitor of the interaction of MAb El with LPS-Re) (30), was unable to inhibit the binding of LPS-Re-Biot to macrophages, even at a high dose (50 ,ug/ml). At that dose, the amount of Kdo2 in the added Kdo2-BSA was more than 600 times higher than that present in LPS-Re-Biot. The binding of LPS-Re-Biot was also not .0 inhibited with high doses of compound KM20a, an analog of

S~~~~~~~~~~~~~~~~~~~~~~S the hinge region between the carbohydrate and lipid moieties of LPS-Re. On the other hand, the binding of LPS-Re-Biot to macrophages was almost completely (97%) inhibited with LPS-Re, even at a low concentration (10 ,ug/ml). Removal of the ester-linked fatty acids from the lipid region of LPS-Re by treatment with hydroxylamine (compound LPS-Re/OH) induced a clear decrease of the inhibitory activity of the LPS. 1 10 100 Interaction of LPS-Re-Biot with human leukocytes. Human leukocytes were analyzed by flow cytometry (Fig. 8). We Fluorescence intensity found that 22.2% of the cells can be labeled with LPS-Re- Biot (Fig. 8B) and 16.7% can be labeled with anti-CD14-Biot FIG. 6. FACS analysis of binding of LPS-Re-Biot to mouse peritoneal cells. lhioglycolate-elicited peritoneal exudate cells were (Fig. 8D). The dots representing LPS+ cells and those incubated for 18 h at 4°C with medium (....), 50 ng of LPS-Re-Biot representing CD14+ cells appeared in the same area of the per ml ( ), and 50 ng of LPS-Re-Biot per ml after a preincubation forward scatter/side scatter plots (Fig. 8C and E, respective- of 2 h at 4°C with 50 1Lg of unlabeled LPS-Re per ml (- - - - ). After ly). These forward scatter and side scatter characteristics being stained with streptavidin-FITC, the cells were analyzed by were used to define a gate (dotted triangle in Fig. 8F) for flow cytometry. further inhibition experiments. The gated cell population (21.4% of the leukocytes) contained 76.8% LPS+ cells and 79.1% CD14+ cells. (fluorescence level higher than 38), exhibited by 28.1%o of the We next examined the ability of various compounds to cells, was completely inhibited by preincubation of the inhibit the binding of LPS-Re-Biot to the gated leukocyte macrophages for 2 h at 4°C with unlabeled LPS-Re (50 subpopulation (Fig. 9). The results were similar to those ,ug/ml) (Fig. 6). obtained with mouse macrophages. Smooth-type LPS (LPS- To identifyr the substructures of LPS-Re which are recog- Sc), rough-type LPS (LPS-Re), and lipid A (LipA-Bp) were nized by sites of an inhibition very efficient inhibitors of LPS binding. The inhibitory LPS-binding macrophages, activity of the de-O-acylated LPS-Re (LPS-Re/OH) was slightly lower. On the other hand, Kdo2-BSA, Kdo2-PA, and KM20a were unable to inhibit LPS binding. 100 DISCUSSION 80 - In this study we examined the interaction of the Kdo 60 region of endotoxin with mouse macrophages and human z 0 monocytes. Two chemically defined compounds were used for this purpose. Both are soluble macromolecules bearing 40 multiple units of a Kdo-a-2,4-Kdo disaccharide, which is z structurally and immunologically identical to the Kdo disac- 20 charide moiety of Re-type LPSs. One of these compounds, Kdo2-BSA, was labeled with 1"I and used as a ligand. The second compound, Kdo2-PA, was not labeled and was used 0 as an inhibitor of LPS binding. The interaction of the Kdo disaccharide with a putative INHIBITOR CONCENTRATION (pg/ml) receptor was mimicked by binding to MAb El. The interac- FIG. 7. Inhibition of binding of LPS-Re-Biot to mouse peritoneal tion of LPSs with MAb El is highly specific: it requires the cells. Thioglycolate-elicited peritoneal exudate cells from BALB/c presence of the two Kdo units of Kdo2 and the absence of nu/+ mice were preincubated (2 h, 4°C) with the inhibitor (LPS-Re, other sugar substituents (30). We found that MAb El inter- LPS-Re/OH, Kdo2-PA, or KM20a) and then incubated (18 h, 4°C) acted very efficiently with 1"I-Kdo2-BSA and Kdo2-BSA, with LPS-Re-Biot (50 ng/ml). The binding was detected by staining which were used as a ligand and a ligand inhibitor, respec- with streptavidin-FITC. Cells with a fluorescence intensity higher tively, in further experiments. Furthermore, we established than 38 were scored as LPS+ cells. In the absence of inhibitor, previously by ELISA inhibition that 28.1% of the peritoneal exudate cells were LPS'. The inhibition of (30) compounds Kdo2- the binding of LPS-Re-Biot to peritoneal exudate cells was esti- BSA and Kdo2-PA are more efficient than LPS-Re as inhib- mated by the decrease (percent) in the number of LPS' cells. itors of the binding of MAb El to LPS-Re, thus indicating Vertical bars represent standard deviations of the means of dupli- that Kdo2 is more accessible in these conjugates than in cate determinations in one representative experiment. native LPS-Re. These results indicate that the compounds 3622 GIRARD ET AL. INFECT. IMMUN.

U 2 U) 101 c L 0 1 '. .j ._ - , -, .C 1. !'1~-W )o. m0,CD 1 . IO 200 400 600 900 1000 CD Forward Scatter

I 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 B Inhibtor Concentration &g/ml) 00I FIG. 9. Inhibition of binding of LPS-Re-Biot to a gated subpop- U 2 ulation of human leukocytes. Human leukocytes were preincubated 0 4) U) 1 3 (1 h, 200C) with the inhibitor (LPS-Sc, LPS-Re, LipA-Bp, LPS-Re/ .. , I :- ead...6.- N:4 11 1 I -: '. .0. 0 Z-11, OH, Kdo2-BSA, Kdo2-PA, or KM20a) and then incubated (1 h, E n l0o !0..!- 41 20°C) with LPS-Re-Biot (50 ng/ml). Binding was detected by staining ,. !. z I, with streptavidin-FITC. The fluorescence of a cell subpopulation, 1 0 .1 fVl. gated as defined in Fig. 8F, was analyzed by FACS. Cells with a I 4 200 400 600 900 100 I (00 10 10I 02 I10 10 fluorescence intensity higher than 64 (threshold of specific binding) Fluorescence Forward Scatter were scored as LPS' cells. In the absence of inhibitor, 37% ± 1% of (LPS-Re-Blot) the gated cells were LPS+. The inhibition of the binding of LPS-Re- I Biot was estimated by the decrease (percent) of the number of LPS+

4 cells. Vertical bars represent standard deviations of the means of I 0~~ 10! duplicate determinations in one representative experiment. l 4) 0011 0 receptors or steric hindrance) since 125I-LPS-Re-BSA exhibited specific binding with both cell types (Fig. 5). These .0 .0 1*o l0 I E 00 .' F. results clearly show that receptors for the Kdo disaccharide zI 1 oo are not present on mouse macrophages and human mono- I cytes. This conclusion is strengthened by the results ob- I illi 2 3 111.9 0 200 400 600 00 1000 10 10 10 10 10 tained in experiments examining inhibition of binding of Fluorescence Forward Scatter biotin-labeled LPS-Re. In these experiments, we used an- (anti-CD 14-Blot) other soluble inhibitor, Kdo2-PA, consisting of Kdo disaccharide coupled to another carrier, polyacrylamide. We 4 found that Kdo2-PA was unable to inhibit the binding of ~1o3 F LPS-Re-Biot to mouse peritoneal cells (Fig. 7) and to human 0 10 leukocytes (Fig. 9). The binding was also not inhibited with 4)O 2 compound KM20a, a structural analog of the hinge region 401 101 between the Kdo and lipid A domains of endotoxins. On the lal other hand, the binding was completely inhibited with unlabeled LPS-Re, and the inhibition decreased slightly after 0 CAO deacylation of LPS-Re with hydroxylamine (Fig. 7 and 9). These results suggest that LPS receptors on mouse macro- 0 200 400 00 800 1O00 Forward Scatter phages and human monocytes interact exclusively with the lipid A domain of LPS Re and that the ester-linked fatty FIG. 8. Determination of optimal gating parameters for FACS acids of lipid A are not critically involved in this interaction. analysis of human monocytes. Spot intensities of dot plot A are The absence of Kdo on proportional to cell frequencies. Bold dots in plots C and E receptors phagocytic cells is correspond to the fluorescent subpopulations (black areas of histo- consistent with our previous finding that the polysaccharide grams B and D) of LPS+ and CD14+ cells, respectively. The triangle region of the B. pernussis LPS, as well as a Kdo monosac- in plot F represents the subpopulation of human leukocytes (gated charide covalently coupled to BSA, did not bind to macro- according to its forward scatter/side scatter characteristics) selected phages (35). Our previous observation that several synthetic for inhibition experiments. Kdo derivatives were unable to induce IL-1 production by mouse macrophages and human monocytes (20) also supports our present findings. Referring to experiments with human neutrophils and with the human promonocyte cell

5I-Kdo2-BSA, Kdo2-BSA, and Kdo2-PA represent well- line THP-1, Lynn and Golenbock (23) also suggested that the adapted tools to search for putative Kdo receptors. Kdo2 domain is not necessary for LPS inhibition on the basis Mouse peritoneal macrophages were able to bind radiola- of observations that lipid IVa and Kdo2 IVa are both beled LPS in a specific way but did not bind radiolabeled effective inhibitors of LPS-induced CD11b/CD18 expres- Kdo2-BSA (Fig. 3). Human monocytes were also unable to sion. bind '"I-Kdo2-BSA (Fig. 4). The inability of Kdo2-BSA to On the other hand, our results conflict with recent prelim- bind to mouse macrophages and human monocytes was not inary observations (21) suggesting that a minor protein due to some inhibitory effect of BSA (blocking of LPS component (38 kDa) on the cell surface of mouse macro- VOL. 61, 1993 ABSENCE OF RECEPTOR FOR SYNTHETIC Kdo DISACCHARIDE 3623 phages and pre-B cells binds LPS and that the binding can be macrophages. I. Binding characteristics. J. Immunol. 128:1950- inhibited by LPS-Re and not by lipid A. It remains possible, 1954. however, that the determinant recognized by the 38-kDa 12. Halling, J. L., D. R. Hamill, M.-G. Lei, and D. C. Morrison. protein is present in the lipid domain of LPS-Re and is 1992. Identification and characterization of lipopolysaccharide- binding proteins on human peripheral blood cell populations. destroyed (or that its conformation is modified) during the Infect. Immun. 60:845-852. acid treatment used for the cleavage of the glycosidic bond 13. Hampton, R. Y., D. T. Golenbock, M. Penman, M. Krieger, and of Kdo required for the preparation of lipid A. C. R. H. Raetz. 1991. Recognition and plasma clearance of It must be pointed out that the absence of Kdo receptors endotoxin by scavenger receptors. Nature (London) 325:342- on monocytes/macrophages does not mean that the Kdo 344. units of endotoxins have no indirect effects on the binding of 14. Hampton, R. Y., D. T. Golenbock, and C. R. H. Raetz. 1988. LPS to its receptors. The vicinity of the Kdo disaccharide Lipid A binding sites in membrane of macrophage tumor cells. may favor one of the thermodynamically possible conforma- J. Biol. Chem. 263:14802-14807. tions of the A and thus allow a A receptor 15. Havell, E. A., and G. L. Spitalny. 1983. Endotoxin-induced lipid region lipid interferon synthesis in macrophage cultures. J. Reticuloendo- to interact more efficiently with this lipid. Therefore, selec- thel. Soc. 33:369-380. tive attacks on the Kdo units of LPS by chemical treatments 16. Hibbs, J. B. J., R. R. Taintor, Z. Vavrin, and E. M. Rachlin. or MAbs may modulate the interaction of lipid A with its 1988. Nitric oxide: a cytotoxic activated macrophage effector receptor. This was actually observed in a previous study (31) molecule. Biochem. Biophys. Res. Commun. 157:87-94. that showed that MAb El inhibits the binding of LPS-Re- 17. Holst, O., L. Brade, P. Kosma, and H. Brade. 1991. Structure, Biot to mouse macrophages. serological specificity, and synthesis of artificial glycoconju- It is noteworthy that our results do not preclude the gates representing the genus-specific lipopolysaccharide epitope existence of lectin-like LPS receptors that recognize sugar of Chlamydia spp. J. Bacteriol. 173:1862-1866. residues distinct from Kdo. The existence of such lectins 18. Hunter, R. 1970. Standardization of the chloramine T method of protein iodination. Proc. Soc. Exp. Biol. Med. 133:989-992. that may interact with the polysaccharide domain of some, 19. Kosma, P., J. Gass, G. Schulz, R. Christian, and F. M. Unger. but not all, endotoxins remains an attractive hypothesis. 1987. Artificial antigens. Synthesis of polyacrylamide copoly- mers containing 3-deoxy-D-manno-2-octulopyranosylonic acid ACKNOWLEDGMENTS (KDO) residues. Carbohydr. Res. 167:39-54. 20. Lasfargues, A., A. Ledur, D. Charon, L. Szabo, and R. Chaby. 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