INFECTION AND IMMUNITY, JUlY 1992, p. 2769-2776 0019-9567/92/072769-08$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 60, No. 7

Legionella pneumophila Lipopolysaccharide Activates the Classical Complement Pathway CLIFFORD S. MINTZ,1* DUANE R. SCHULTZ,2 PATRICIA I. ARNOLD,2 AND WILLIAM JOHNSON3 Department of Microbiology and Immunology' and Department of Medicine,2 University of Miami School of Medicine, Miami, Florida 33101, and Department of Microbiology, University of Iowa, Iowa City, Iowa 522423 Received 29 January 1992/Accepted 9 April 1992

Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37°C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes. Legionella pneumophila is a gram-negative facultative intracellular bacterium that causes the human respiratory illness known as Legionnaires' disease (34). The pathogenicity of L. pneumophila is attributed to its ability to enter and multiply within alveolar macrophages and monocytes (8). L. pneumophila evades the antimicrobial defenses of mononuclear phagocytes because phagosomes that contain legionellae fail to fuse with lysosomes (7). L. pneumophila can be taken up by mononuclear phagocytes in the absence of opsonizing antibody or other serum components (14). However, legionellae incubated in normal human serum (NHS) are phagocytosed at a much greater rate by monocytes (9, 10). Recently, Payne and Horwitz (20) showed that opsonic complement component C3 and phagocyte complement receptors CR1 and CR3 promoted the uptake of L. pneumophila by human monocytes and alveolar macrophages. Interestingly, several laboratories have shown that the presence of bound C3 or immune antibody on the L. pneumophila cell surface does not interfere with intracellular multiplication (9, 20, 29). Several groups have demonstrated that incubation of L. pneumophila in NHS results in the activation of complement with the subsequent deposition of C3 on the surface of the organism (20, 29). However, the bacterial ligand(s) responsible for activation of complement by L. pneumophila is still uncertain. Lipopolysaccharide (LPS) produced by a variety of gram-negative bacteria can activate complement by either the classical or the alternative pathway or both (16, 17). This prompted us to assess the effects of L. pneumophila LPS on the classical and alternative complement pathways in NHS. We found that incubation of LPS in NHS mainly resulted in the activation of the classical pathway. Classical pathway activation by LPS was dependent on antibody since activation did not occur in an immunoglobulin (Ig)-deficient serum *

Corresponding author.

obtained from an agammaglobulinemic (AG) patient. Subsequently, natural antibodies of the IgM class were purified from NHS and shown to be responsible for classical pathway activation in the presence of LPS.

MATERIALS AND METHODS Isolation and purification of LPS. LPS was prepared from L. pneumophila Philadelphia-1 by EDTA extraction according to the methods of Otten et al. (19). LPS was also isolated from strain Philadelphia-1 by using affinity chromatography. In this procedure, crude LPS fractions were isolated as previously described (12). Further purification was accomplished by affinity chromatography with the monoclonal antibody 1E6 (15). The 1E6 antibody was dissolved in 0.1 M NaHCO3 containing 0.5 M NaCl (pH 8.3) (coupling buffer) at a concentration of 8 mg/ml. CNBr-activated Sepharose 4B (Pharmacia LKB, Piscataway, N.J.) was suspended in coupling buffer and added to the ligand at a ratio of 1 part activated Sepharose 4B to 2 parts ligand solution and incubated overnight at 4°C. The gel was removed by centrifugation at 5,000 x g for 15 min and suspended in coupling buffer containing 0.2 M glycine (pH 8.0). The mixture was incubated for 2 h at room temperature and washed twice with coupling buffer, followed by two washes with 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl and two washes with coupling buffer. The washed gel was poured into a column (1.0 by 5 cm), and 1 ml of crude LPS (20 mg/ml in 0.14 M NaCl) was applied to the column. The column was washed with 4 volumes of 0.14 M NaCl (pH 7.5), and the LPS was eluted with 10 mM Na3PO4 (pH 12.0) containing 10% dioxane. Fractions containing LPS were dialyzed against distilled water and lyophilized. The purity of the LPS preparations was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then by silver staining (19). Because LPS preparations frequently contain trace 2769

2770

MINTZ ET AL.

amounts of outer membrane proteins, we determined by an enzyme-linked immunosorbent assay (ELISA) the amount of major outer membrane protein (MOMP) contained in our LPS samples. In these experiments, different amounts of purified MOMP (0.097 to 12.5 jig) prepared by the methods of Ehret and Ruckdeschel (3) were blotted onto nitrocellulose by using a slot blot apparatus (Bethesda Research Laboratories, Bethesda, Md.). LPS (6.15 to 100 Vig) was blotted onto nitrocellulose directly beneath the MOMP samples. The blots containing both MOMP and LPS were exposed to MOMP-specific monoclonal antibody 4H10 for 2 h at room temperature, washed several times with buffer, and then incubated with horseradish peroxidase-conjugated goat anti-mouse Ig (Cappel, West Chester, Pa.) for 1 h at room temperature. Monoclonal antibody 4H10 was generated against purified MOMP in W. Johnson's laboratory and specifically binds to MOMP in Western blot (immunoblot) experiments (27). Color was developed by immersing the blots in development solution which contained 0.05% 4-chloro-1-naphthol (Sigma Chemical Co., St. Louis, Mo.) and 0.015% hydrogen peroxide. The amount of MOMP found in the LPS preparations was then estimated by visual inspection of the blots. Complement reagents and assays. The ability of LPS to activate the classical or alternative complement pathways was determined by functional hemolytic assays of Ci, C4, C2, and C3 as previously described (31). Alternative pathway activation was measured by incubating LPS in NHS chelated with 10 mM EGTA [ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid] (Sigma) plus 10 mM MgCl2. The buffer for assays of complement component hemolytic activity was glucose gelatin-Veronal containing MgCl2, CaCl2, and gelatin to final concentrations of 0.5 mM, 0.15 mM, and 0.1% (pH 7.5), respectively, and 75 mM NaCl (21). Functionally purified human (hu) or guinea pig (gp) components, stable cellular intermediates (erythrocytes [,E] optimally sensitized with antibody [Al: EAC19P, EAC4 U, EAC19P4hu), and rabbit anti-E (IgG) and guinea pig serum were prepared as previously described (21, 31) or purchased from Diamedix Corp. (Miami, Fla.). Hemolysis was measured at 412 nm by using a cell concentration of 108/ml. Z values were determined by the equation Z = -ln(1 - Y), where Y is the extent of hemolysis. A plot of the Z values versus complement component input results in a straight line. The titer of a component is defined as the reciprocal of the component dilution at which -ln(1 - Y) is 0.69 (50% lysis). A reagent was developed from NHS which was deficient in Clq but contained C1r2 and Cls2 and was used for the functional hemolytic titration of human Clq (26). Some of the serum complement component titers varied because E from different sheep and different lots of cellular intermediates were used over the course of months during which these experiments were carried out. To facilitate comparison of the results with each new batch of E, a standard reference NHS stored in aliquots at -70°C was tested for each complement component investigated as described previously (31). Hemolytic titers were adjusted to average values on the basis of titers of complement components determined in these laboratories for the past 15 years, and there was good agreement among the adjusted titers. All experiments were repeated at least two times, and all samples within a given experiment were tested in duplicate. Hemolytic titers of individual complement components did not vary more than 5% between replicate experiments. Adsorption of sera with L. pneumophila. Serum samples

INFECT. IMMUN.

used in this study were collected from individuals with no previous history of Legionnaires' disease. In some experiments, NHS was adsorbed with strain Philadelphia-1 to remove anti-Legionella antibodies. Adsorption was accomplished by adding 2 x 109 cells of strain Philadelphia-1 per ml of serum and placing the mixture on ice for 30 min. Bacteria were removed from serum by centrifugation at 10,000 x g. This procedure was repeated three times. After the final treatment, sera were filtered through a 0.22-,um-pore-size filter (Gelman Sciences, Ann Arbor, Mich.) and stored at -80°C. The efficacy of the adsorption procedure was assessed by a colony immunoblot assay. In this assay, single colonies of strain Philadelphia-1 were patched onto nitrocellulose filters (Fisher Scientific Co., Pittsburgh, Pa.) and incubated for 24 to 48 h at 37°C on ACES [N-(2-acetamido)2-aminoethanesulfonic acid]-buffered charcoal yeast extract plates. The filters were incubated in buffer containing 5% nonfat dried milk (Carnation, Los Angeles, Calif.), cut into small pieces, and incubated in serial twofold dilutions of adsorbed or unadsorbed NHS for 2 h at 37°C. After incubation, legionella-containing pieces were washed several times and exposed to horseradish peroxidase-conjugated goat antihuman Ig serum (Cappel) for 1 h at 37°C. Color was developed by using the peroxidase development solution described above. By using this assay, we determined that the titers of anti-Legionella antibodies in adsorbed sera were reduced five- to eightfold compared with those in unadsorbed serum. The titer of anti-Legionella antibodies remaining in adsorbed sera generally ranged from 1:4 to 1:20. The adsorption process resulted in less than a 10% decrease in C3 and C4 titers. Ig-deficient serum (kindly provided by B. Hamilton, University of Miami School of Medicine, Miami, Fla.) was obtained from an AG patient. Functional hemolytic assays indicated that the classical pathway was intact and functional in the AG serum. Additional experiments showed that AG serum contained normal levels of complement components Cl, C2, C3, and C4 and no detectable Ig (data not

shown). Purification of IgM from NHS. Serum was precipitated with 50% (NH4)2SO4 and centrifuged, and the precipitate was dialyzed overnight at 4°C against phosphate-buffered saline (PBS) (pH 7.4). The precipitate was solubilized and applied to a Sephacryl S-300 (Pharmacia LKB) column (1.6 by 100 cm) equilibrated with PBS plus 0.01 M EDTA (pH 7.4). One-milliliter fractions from the IgM-rich first peak were pooled, concentrated by ultrafiltration by using an Amicon PM10 membrane (molecular weight cutoff, 10,000; Amicon, Danvers, Mass.), dialyzed, and passed over a protein G column (0.9 by 10 cm) equilibrated at room temperature with buffer containing 20 mM sodium phosphate and 10 mM EDTA (pH 7.0). The effluent was concentrated by ultrafiltration as described above, dialyzed, and analyzed by immunoelectrophoresis (24) and by functional hemolytic assays for Clq (26) and macromolecular Cl (31). These analyses revealed that neither Clq nor Cl was present in the IgM preparation. However, this preparation did contain small amounts of a contaminant which was subsequently identified as haptoglobin. Coating of sheep E with LPS. EDTA-extracted LPS (4 mg) was incubated in 0.1 NaOH for 18 h at 37°C. Alkali-treated LPS was dialyzed against distilled water for 24 h and then lyophilized. Sheep E (2 x 108) were incubated with 100 jig of NaOH-treated LPS in 300 pAl of gelatin-Veronal buffer (GVB) (pH 7.5) for 1.5 h at 37°C with gentle rotation. E were washed several times with GVB and suspended in 1.0 ml of

VOL. 60, 1992

ACTIVATION OF COMPLEMENT BY LEGIONELLA PNEUMOPHILA LPS

TABLE 1. Effect of AP-LPS on human serum C4' Serum sample

1

2

C4 titer

_ +

35,028 1,331

9

-

35,555 10,141

71.5

_ +

17,940 4,306

7.

+

34,210

99.2 9

-

35,127 23,184

34.0

30,697 1,535

9.

+

3 4

5

6

+

7 8

76.0

95.0

+

10,232

+

27,286 1,091

9.

+

34,926 70

99.8

-

27,490

+

852

85.7

1,463

9

10

96.2

274

+

consumption of C4 (21%) in 50% NHS. There was no apparent difference in the ability of EDTA-extracted LPS or AP-LPS to activate complement (data not shown). To further assess the effect of LPS on complement, individual serum samples obtained from 10 normal healthy adults were treated with AP-LPS and assayed for C4 hemolytic activity. In these experiments, AP-LPS (1 mg/ml) or Veronal-buffered saline (VBS) was incubated in 50% NHS for 2 h at 37°C. After incubation, each mixture was assayed for C4 hemolytic activity and the percent consumption of C4 was calculated. Table 1 shows the variability of C4 consumption by LPS among individual serum samples. This ranged from a high of 99.8% (serum sample 9) to a low of 34% (serum sample 5). Classical pathway activation by LPS. To show that LPSmediated consumption of C4 in NHS resulted from classical pathway activation rather than a selective inactivation of C4, we measured the consumption of Cl, C2, and C3, in addition to C4, in three individual serum samples incubated with AP-LPS. These sera were chosen because of the large differences in the levels of LPS-mediated C4 consumption, which were high for serum sample 9, moderate for serum sample 2, and low for serum sample 5. The levels of Cl, C4, C2, and C3 in LPS-treated sera were measured by functional hemolytic assays as described above. The data presented in Table 2 show that LPS caused consumption of Cl, C4, C2, and C3 in each of the three serum samples tested. Cl, C4, and C2 consumption were highest in serum sample 9, moderate in serum sample 2, and lowest in serum sample 5. For unknown reasons, C2 consumption was higher in serum sample 5 than in serum sample 2. Approximately one-third of the C3 was consumed in serum samples 9 and 2, and only 11% was consumed in serum sample 5. These results clearly indicated that LPS activated the classical complement pathway in NHS. Activation of the alternative pathway by LPS. To determine if activation of the alternative pathway contributed to the consumption of C3 observed in LPS-treated sera, LPS or VBS was incubated in serum samples 9, 2, and 5 treated with 10 mM EGTA and excess (10 mM) Mg2+. EGTA treatment inhibits the classical pathway but leaves the alternative pathway intact and reactive (5). After 2 h of incubation at 37°C, C3 hemolytic titers were measured and the percent consumption of C3 was calculated. Before the initiation of these experiments, we determined by functional hemolytic assays that serum treated with EGTA and Mg2+ was devoid of classical pathway activity (data not shown). The results from these experiments showed that Legionella LPS caused only slight activation of the alternative pathway (Fig. 1). In contrast, incubation of EGTA-treated

% Consumption'

LPS

2771

96.0

96.9

a Serum samples (50%) from 10 individuals were incubated with AP-LPS (1 mg/ml) or buffer for 2 h at 37°C. C4 titers were measured by functional hemolytic assays. " Percent consumption = 1 - (C4 titer of serum + LPS)/(C4 titer of serum - LPS) x 100.

GVB. The presence of bound LPS on treated E was confirmed by hemagglutination by using hyperimmune rabbit sera raised against L. pneumophila Philadelphia-1 (produced in our laboratory). RESULTS Activation of complement by LPS. Initial experiments showed that maximal activation of complement occurred with 1 mg of affinity-purified LPS (AP-LPS) per ml after 2 h of incubation at 37°C in NHS. Incubation of this concentration of AP-LPS in 50% NHS resulted in 98% consumption of C4. From dose-response experiments, we determined that as little as 10 ,ug of AP-LPS per ml was sufficient to cause

TABLE 2. Effect of AP-LPS on human serum Cl, C4, C2 and C3Y Serum titer

Serum sample

LPS

9

+ + +

C1

38,457

1,515 (60.6)" 35,694 18,597 (47.9) 35,470 31,568 (11.0)

C4

C2

34,790 139 (99.3)

3,222 100 (96.9)

C3

10,466

7,159 (31.6) 14,713 4,121 1,648 (60.0) 10,078 (31.5) 7,019 8,373 5 22,686 (34.0) 1,502 (78.6) 7,452 (11.0) Serum samples (50%) from three individuals were incubated with AP-LPS (1 mg/ml) for 2 h at 37°C. Functional hemolytic assays were used to measure complement components in LPS-treated and control sera. ' Values in parentheses indicate percent consumption. Percent consumption 1 (titer of complement component in serum + LPS)/(titer of complement 2

35,541 11,309 (68.1) 34,372

a

=

component in serum

-

LPS)

x

100.

-

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INFECT. IMMUN.

MINTZ ET AL.

20 -

100 -

z

0

80 -

P IL C,) z

0

z

0

10

P IL

60 -

C')

z

0

wo-

C.)

LPS FIG. 1. Activation of the alternative pathway by AP-LPS. The AP-LPS (1 mg/ml) was incubated in 50% NHS treated with 10 mM EGTA plus 10 mM MgCl2 for 2 h at 37°C. Percent C3 consumption represents the decrease in C3 titers in serum incubated with LPS compared with those in serum incubated with buffer. Symbols: E., serum sample 2; El, serum sample 9; O, serum sample 5.

serum with Salmonella minnesota LPS (1 mg/ml), by using the conditions described above, resulted in 95% consumption of C3 (data not shown). These results suggested that L. pneumophila LPS primarily activates complement in NHS via the classical pathway. Role of antibodies in the activation of the classical pathway by LPS. The role of antibodies in the activation of the classical pathway by LPS was tested by incubating AP-LPS in NHS or NHS adsorbed with strain Philadelphia-1 and by measuring C4 hemolytic titers. LPS caused similar amounts of C4 consumption in both NHS and adsorbed NHS (Fig. 2). Activation of the classical pathway by LPS in adsorbed NHS could be explained by two possible mechanisms: activation occurred in an antibody-independent fashion possibly mediated by the lipid A portion of Legionella LPS (17) or activation was mediated by antibodies present in NHS that were not removed by adsorption. To distinguish between these possibilities, we tested LPS for its ability to activate the classical pathway in Ig-deficient serum obtained from an AG patient. Incubation of LPS in AG serum did not cause consumption of C4 (Fig. 2). This result suggested that antibodies were responsible for classical pathway activation in LPS-treated sera. Identification of the LPS-reactive antibodies in NHS. To identify the putative "anti-LPS" antibodies in NHS, serum sample 9 was fractionated by ammonium sulfate precipitation or by protein G affinity chromatography. Serum sample 9 was chosen for these experiments because it exhibited the greatest consumption of C4 after LPS treatment (Table 1), which suggested that it contained greater quantities of the antibodies than other serum samples did. The Ig fraction of unadsorbed serum sample 9 was obtained by 50% ammonium sulfate precipitation. The precipitate was washed several times with 55% ammonium sulfate and then solubilized and dialyzed against PBS (pH 7.5). In a separate experiment, IgG was isolated by adding unadsorbed serum sample 9 to a protein G-Sepharose column equilibrated with buffer containing 20 mM phosphate and 10 mM

40 -

20 -

1'.

0.1

LPS (mg/ml)

2.0

FIG. 2. Classical pathway activation by AP-LPS in unadsorbed or adsorbed serum sample 9 or AG serum. Each serum sample (50%) was incubated with the indicated concentration of LPS for 2 h at 37°C. Percent C4 consumption represents the decrease in C4 titers in sera incubated with LPS compared with those in sera incubated with buffer. Symbols: F1, NHS; El, adsorbed NHS; [1, AG serum.

EDTA (pH 7.0). The column was washed several times with equilibration buffer, and the effluent was collected. The IgG-containing fraction was eluted from the column by using 100 mM glycine-HCI buffer (pH 2.7). The column effluent and eluate were individually concentrated by membrane filtration and dialyzed against PBS (pH 7.5). To determine which fraction(s) of serum sample 9 contained the anti-LPS antibodies, portions of the ammonium sulfate fraction, the protein G effluent, or the protein G eluate were incubated with LPS or with VBS in 0.3 mM CaCl2 and 1 mM MgCl2 (VBS2+), added to AG serum, and tested for the ability to activate the classical pathway (Fig. 3). The final concentration in the reaction mixtures of the ammonium sulfate fraction, the protein G effluent, and the protein G eluate was 0.1 mg/ml. The ammonium sulfate precipitate activated the classical pathway and resulted in 95% consumption of C4. The effluent from the protein G column also activated the classical pathway and caused 45% consumption of C4. In contrast, the protein G eluate (which contained the IgG fraction of serum sample 9) caused approximately 5% C4 consumption. This suggested that the LPS-reactive antibodies were not IgG and probably belonged to the IgM class. To test this further, we purified the IgM fraction from serum sample 9 by ammonium sulfate precipitation and then by molecular sieve and protein G affinity chromatography. Purified IgM contained trace amounts of haptoglobin and did not contain any detectable Clq or macromolecular Cl as determined by functional hemolytic assays (24, 31) described in Materials and Methods. To determine whether the IgM fraction from serum sample 9 contained anti-LPS antibodies, different amounts of purified IgM in VBS2+ were incubated with AP-LPS (2 mg/ml) for 1 h at 37°C. After incubation, an equal volume of 10% AG serum was added to each mixture

ACTIVATION OF COMPLEMENT BY LEGIONELLA PNEUMOPHILA LPS

VOL. 60, 1992 100-

80-

z 0

C) 60z

0

C.)

40-

20-

LPS (1 mg/ml) FIG. 3. The effect of various fractions of NHS on classical pathway activation by LPS. AP-LPS (1 mg/ml) was ir icubated with 0.1 mg of solubilized and dialyzed (NH4)2SO4 precipit. ate, protein G effluent, or protein G eluate per ml for 1 h at 37°C. Aft4 er incubation, each of the mixtures was then incubated in 5% AG serum for an additional 1 h at 37°C, the C4 hemolytic titers were af rieasured, and the percent C4 consumption was calculated. LPS pl us VBS incubated in 5% AG serum served as a control in these experiments. control; El, (NH4)2SO4; E3, protein G effluent; 0, Symbols: protein G eluate. U,

5%) and incutated for 1 h were measurced as previously described. Incubation of LPS with increas ing amounts of purified IgM (0.1 to 1.3 mg/ml) resulted in a (concomitant increase in C4 consumption. Maximum consumiption of C4 (40%) was observed after incubation of LPS wit]h 0.51 mg of purified IgM per ml. Incubation of LPS with IgN4 at concentrations greater than 0.51 mg/ml resulted in a m )dest reduction (ca. 10%) in C4 consumption. The reduiction in C4 consumption may have resulted from solubilizal tion of LPSIgM complexes caused by the presence of excess LPSspecific IgM antibodies. Incubation of purifiecd IgM (0.51 mg/ml) with increasing amounts of AP-LPS (0.1 to 1.0 mg/ml) also resulted in a concomitant increase in C4 consumption. Maximum consumption of C4 (47%) Mvas obtained after incubation of IgM with AP-LPS at a conc-entration of 0.5 mg/ml. Incubation of purified IgM with LP'S at concentrations greater than 0.5 mg/ml caused a slight re duction (ca. 5%) in C4 consumption. This may have beeni caused by solubilization of LPS-IgM complexes by excess Eantigen, i.e., LPS. Finally, adsorption of the purified IgM fi raction with insolubilized goat anti-human IgM antibody ( 1) caused a fourfold reduction in the ability of this prepara tion to activate the classical pathway (data not shown). IgM antibodies recognize the carbohydrate porrtion of LPS. Our inability to completely remove the antiboclies reactive with LPS from NHS after repeated adsorptionss with strain Philadelphia-1 suggested that the antibodies maty have recognized antigenic sites on Legionella LPS th at were not

(final concentration of AG

serum,

at 37°C, and hemolytic C4 titers

2773

normally exposed in its native configuration. To test this idea, we coated sheep E with NaOH-treated LPS (E-LPS). By using this method, LPS partitions into the E membrane via lipid A, leaving the hydrophilic carbohydrate portion exposed to the medium as it is in the bacterial membrane (35). Uncoated E or E-LPS (108) were incubated in EDTAtreated NHS, AG serum (possible antibody sources), or buffer for 1 h at 37°C. After incubation, E or E-LPS were washed several times with GVB, restandardized to 108/ml, and used as indicator cells to determine the whole complement titer in AG serum as described by Mayer (18). Results from our previous experiments indicated that anti-LPS IgM antibodies were present in NHS (Table 1) but absent in AG serum (Fig. 3). Therefore, we reasoned that E-LPS preincubated in NHS but not AG serum or buffer should bind the IgM antibodies. The IgM-coated E-LPS should then lyse via complement fixation during the second incubation in AG serum because this serum contains normal levels of complement components. The results from these experiments showed that E-LPS preincubated in NHS activated the classical complement pathway in AG serum (titer in AG serum, 160). In contrast, E-LPS preincubated in AG serum or buffer failed to promote classical pathway activation (no lysis occurred). In no instance did uncoated E preincubated in NHS, AG serum, or buffer activate complement (data not shown). These results confirmed that LPS-induced activation of the classical pathway was dependent upon antibody and suggested that the antibodies that mediated activation recognized antigenic sites on Legionella LPS that were exposed in their native configurations. DISCUSSION It is well established that LPS produced by enteric bacteria such as Salmonella spp. and Escherichia coli activate complement (16, 32). The polysaccharide portion of LPS is thought to be responsible for alternative pathway activation (16), whereas the lipid A portion can activate the classical pathway independent of antibody (17, 32). The incubation of enterobacterial LPS in NHS primarily results in activation of the alternative pathway because the sites on lipid A that interact with classical pathway components are normally not exposed (32). The results of our study showed that L. pneumophila LPS can activate both the classical and alternative complement pathways after incubation in NHS (Table 2 and Fig. 1). However, in contrast to enterobacterial LPS activation, complement activation proceeded primarily through the classical pathway. The LPSs produced by several other bacterial species, including Brucella abortus (6), Yersinia enterocolita (33), and Pseudomonas aeruginosa (4, 23), also do not appreciably activate the alternative pathway. Each of these LPSs and L. pneumophila LPS have structural motifs similar to that of enterobacterial LPS, i.e., an O-polysaccharide, a core oligosaccharide, and lipid A. However, the sugar content and nature of the linkages between the sugars in the polysaccharide portions of each of these LPSs are different than those observed in enterobacterial LPS (4, 6, 17, 19, 23, 28). It is likely that these differences account for the reduced ability of these LPSs to activate the alternative pathway. Activation of the classical pathway by L. pneumophila LPS was dependent upon antibodies of the IgM class. These antibodies caused variable consumption of C4 in the serum samples tested (Table 1) but were clearly absent in Igdeficient serum (Fig. 2). Attempts to completely remove these antibodies from NHS by repeated adsorptions with

2774

MINTZ ET AL.

strain Philadelphia-1 were unsuccessful. These data suggested that the anti-LPS antibodies described here may be natural antibodies. We (24) and others (32) have noted that natural antibodies have low affinities and are difficult to remove by adsorption. Vukajlovich (32) reported that all NHS contains natural anti-rabbit E antibodies of the IgG and IgM classes that cross-react with antigenic determinants found on LPS polysaccharide. This conclusion was based on the observation that adsorption of NHS with rabbit E reduced the ability of LPS from Serratia marcescens, E. coli, and Y. enterocolitica to activate the classical complement pathway. From the results of these and additional experiments, the author suggested that the antibodies may recognize galactose, glucose, or glucosamine residues. In another study, Schultz and Arnold (24) identified natural antibodies in NHS of the IgM class with specificity for N-acetyl-D-glucosamine. Although the specificity of the IgM antibodies identified in our study is unknown, it is likely that the antibodies recognized antigenic sites in the polysaccharide portion of L. pneumophila LPS since E-containing LPS inserted into the E membrane via lipid A activated the classical pathway (see Results). It is of interest that recent chemical analyses of the LPS produced by L. pneumophila Philadelphia-1 revealed the presence of large amounts of glucosamine and trace amounts of glucose (10, 28). Therefore, it is possible that the IgM antibodies identified in our study may be similar to the antibodies described by Schultz and Arnold. Additional experiments are in progress to more clearly define the specificity of the LPS-reactive antibodies described in this investigation. Verbrugh et al. (30) determined that incubation of viable whole cells of L. pneumophila Philadelphia-1 in NHS resulted in activation of the classical pathway. It is of interest that they were unable to detect any activation of the alternative pathway during incubation of legionellae in NHS. Although we did not study complement activation by using whole bacterial cells, we clearly showed that solubilized LPS or LPS inserted into membranes of sheep E activated the classical complement pathway in NHS. More recently, we confirmed the findings of Verbrugh et al. by showing that incubation of viable cells of strain Philadelphia-1 in NHS resulted in activation of the classical pathway but no detectable activation of the alternative pathway (25). Our results, along with the findings of Verbrugh et al., demonstrate that activation of complement in NHS by L. pneumophila proceeds via the classical pathway. Since LPS is a major constituent of the L. pneumophila outer membrane (19, 28), it is likely that LPS mediates classical pathway activation during incubation of legionellae in NHS. In contrast with our results and those of Verbrugh et al., Bellinger-Kawahara and Horwitz (2) recently reported that activation of complement by L. pneumophila Philadelphia-1 occurred solely via the alternative pathway during incubation in NHS. Differences in the assay systems used to measure complement activation in these studies may partially account for the seemingly disparate results. We, as well as Verbrugh et al., used functional hemolytic assays to measure residual complement activity in NHS, whereas Bellinger-Kawahara and Horwitz measured complement activation by quantitating the amount of C3 bound to legionellae during incubation in NHS by a whole-cell ELISA. Future experiments using serum depleted of alternative pathway factor B or D should enable us to conclusively determine whether the alternative pathway has any significant role in activation of complement during incubation of legionellae in NHS.

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Recently, it was reported that L. pneumophila MOMP can activate complement (2). By using liposomes that contained purified MOMP or isolated MOMP in Western blot experiments, Bellinger-Kawahara and Horwitz (2) demonstrated that MOMP bound activated C3 during incubation in NHS. Moreover, activation of complement by MOMP was reported to occur via the alternative pathway. It has been shown that LPS preparations may contain small amounts of outer membrane proteins as contaminants (13). We have determined by ELISA that our LPS preparations contained trace amounts of MOMP (0.4%, wt/wt). Therefore, the possibility existed that the small amount of contaminating MOMP found in our LPS preparations was responsible for complement activation after incubation of LPS in NHS. However, there are several lines of experimental evidence that argue against this possibility. First, as previously mentioned, incubation of LPS in NHS primarily results in activation of the classical pathway and slight activation of the alternative pathway. If MOMP selectively activates the alternative pathway, classical pathway activation in NHS by LPS cannot be ascribed to contamination of our LPS preparations with MOMP. Second, we have recently determined by using purified MOMP in functional hemolytic assays that the trace amount of MOMP contained in our LPS samples (ca. 4 jig of MOMP per mg of LPS) is insufficient to activate either the classical or alternative complement pathway in NHS (25). Taken together, these findings clearly demonstrate that LPS, not MOMP, activates complement after incubation of LPS in NHS. Although Bellinger-Kawahara and Horwitz showed that isolated MOMP or MOMP-containing liposomes bind C3 during incubation in NHS, they were unable to directly detect MOMP in the C3-acceptor complex formed on the surface of opsonized legionellae (2). Consequently, it is not possible to conclude that MOMP is the only complement activator on the L. pneumophila cell surface. It is possible that surface ligands other than or in addition to MOMP may serve as complement activators on L. pneumophila. Our data clearly show that L. pneumophila LPS is a potent complement activator. Moreover, complement-mediated lysis of E-LPS in AG serum after incubation in NHS provides evidence that L. pneumophila LPS inserted into a biological membrane via lipid A can bind activated C3 (see Results). Currently, experiments are under way to determine if activated C3 can bind to LPS molecules found on the L. pneumophila cell surface. On the basis of all available evidence, it is possible that both LPS and MOMP act as C3 acceptors on the L. pneumophila cell surface. In support of this idea, E. coli OllB4 LPS and outer membrane proteins have been reported to bind C3 during incubation of this strain in NHS (13). The amount of fluid-phase L. pneumophila LPS required to activate complement in NHS is in agreement with the findings of several groups working with LPSs from other bacteria (6, 13, 32). Although we used LPS at concentrations of 1 mg/ml in most of the experiments described in this report, as little as 10 ,ug of LPS per ml was sufficient to activate the classical complement pathway. It is not possible to determine how much cell-associated or free LPS would be present at the initial site of pulmonary infection with L. pneumophila. However, the work of Huebner et al. (11) showed that as few as 20 CFU of aerosolized L. pneumophila were sufficient to cause disease in the guinea pig model of legionellosis. Therefore, it is likely that small amounts of L. pneumophila LPS present in lung lesions may be sufficient to activate complement and promote the phagocytosis

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of legionellae by resident and recruited mononuclear phagocytes. A role for C3 and corresponding phagocyte complement receptors CR1 and CR3 has been suggested for the uptake of L. pneumophila by human mononuclear phagocytes (9). The identification of LPS as a complement activator suggests that this molecule may participate in the uptake process. In support of our data showing classical pathway activation by LPS, activated C4 (C4b) also has an affinity for the C3b receptor CR1 (22). Therefore, activated C4 as well as C3 found on the surface of opsonized organisms may promote the uptake of legionellae by mononuclear phagocytes. Future experiments using L. pneumophila LPS mutants that produce structurally altered LPS should provide greater insight into the roles of LPS and complement in the uptake of L. pneumophila by alveolar macrophages and monocytes. ACKNOWLEDGMENTS We thank Churchill McKinney for help in preparing the figures presented in this article. This work was supported in part by Public Health Service grant Al 26532 from the National Institutes of Health awarded to W.

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