INFECTION AND IMMUNITY, July 1978, p. 310-319 0019-9567/78/0021-0310$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 21, No. 1

Printed in U.S.A.

Strain-Dependent Cytotoxic Effects of Endotoxin for Mouse Peritoneal Macrophages DUANE L. PEAVY," 4 ROBERT E. BAUGHN,2' 5 AND DANIEL M. MUSHER" 3'5 Departments of Microbiology and Immunology,' Dermatology,2 and Medicine,3 Baylor College of Medicine, Houston, Texas, 77030; The Methodist Hospital,4 Houston, Texas, 77030; and the Veterans Administration Hospitals,5 Houston, Texas, 77211 Received for publication 21 December 1977

The cytotoxic effects of bacterial lipopolysaccharides (LPS) on mouse leukocytes have been examined in vivo and in vitro. Intraperitoneal injection of LPS into C57BL/6 mice greatly reduced the recovery of mononuclear cells; LPS was cytotoxic for macrophages, but had a mitogenic effect on lymphocytes. Similar effects of LPS on peritoneal leukocytes were observed in vitro. When monolayers of adherent peritoneal cells were studied in vitro, cytotoxicity was also observed, suggesting that the effect of LPS on macrophages is direct and does not require participation by lymphocytes. Entirely different results were obtained when peritoneal macrophages from LPS-resistant C3H/HeJ mice were studied. LPS failed to activate lymphocytes and was not cytotoxic for macrophages in vitro or in vivo. The effect of LPS on polymorphonuclear leukocytes appeared to be the same in all mouse stains studied. Lipid A was shown to be the most biologically active portion of the LPS molecule. Whereas polysaccharide-deficient endotoxins extracted from rough mutants of Salmonella typhimurium were cytotoxic for macrophages in vitro, polysaccharides that lacked esterified fatty acids did not exhibit this activity. Since LPS may mediate its effects through affminty for mammalian cell membranes, the cellular unresponsiveness of C3H/H3J mice to LPS may reflect an inability of cells from LPS-resistant strains to interact with LPS at the membrane level. When injected into animals, or when gener- varies greatly among inbred strains of mice (36, ated by gram-negative bacteria during naturally 37). One mouse strain, C3H/HeJ, has been or experimentally acquired infections, lipopoly- shown to be particularly refractory to lethal saccharides (LPS) elicit a vast array of biological doses of LPS. Watson and Riblet (40) have events. These include leukocyte mobilization suggested that this susceptibility is determined (34), complement activation (35), kinin genera- by an autosomal dominant gene at one locus. tion (20), intravascular coagulation (4), and in- Subsequent studies using C3H/HeJ mice have creased capillary permeability (47), which fre- shown that LPS fails to act as an adjuvant in quently result in shock and death. Other effects these animals and is only weakly immunogenic include induction of a specific antibody response (32, 41). It has been suggested that the lack of (3), generalized amplification of antibody re- responsiveness to LPS in C3H/HeJ mice is due sponses (17), enhanced reticuloendothelial clear- specifically to the inability of LPS to stimulate ance (B. Benaceraff and M. M. Sebasteyn, Fed. proliferation of bone marrow-derived (B) lymProc. 16:860, 1957), and either increased or de- phocytes (13, 28, 33). However, LPS is known to creased susceptibility to bacterial infection (18), act directly on mononuclear (30) and polymordepending upon the temporal relation between phonuclear (11) phagocytic cells, rendering lyinjection of LPS and bacterial challenge. Despite sosomes unstable and thereby initiating release extensive investigation, a satisfactory explana- of hydrolytic enzymes and pharmacological metion for the capacity of LPS to induce these diators. Chedid et al. (7) have suggested that varied phenomena has never been provided. One macrophages from LPS-resistant C3H/HeJ plausible hypothesis, proposed by Shands et al. mice may be refractory to LPS, since LPS fails (29), is that LPS creates alterations in mamma- to increase nonspecific resistance in these mice lian membranes which trigger responses at the or to activate their peritoneal macrophages to cellular level. inhibit tumor cell growth. Glode et al. (12) have Previous investigations have demonstrated recently presented evidence that macrophages that susceptibility to the lethal effects of LPS from C3H/HeJ mice resist the toxic effect of 310

VOL. 21, 1978

CYTOTOXICITY OF LPS FOR MOUSE PERITONEAL CELLS

LPS in vitro. These authors use these findings to support the view that LPS exerts its effects by acting directly on mononuclear cell membranes. In the studies reported in the present paper, we investigated the morphological and functional effects of LPS on peritoneal leukocytes from LPS-susceptible and LPS-resistant strains of mice in vivo and in vitro. Our results show that the cellular unresponsiveness of C3H/HeJ mice is widespread, involving both B lymphocytes and macrophages. MATERIALS AND METHODS Animals. A, C3H/HeJ, and C57BL/6 mice (6 to 8 weeks old) were purchased from Jackson Laboratories, Bar Harbor, Maine. Mice were housed 15 to a cage and fed acidified, chlorinated water and laboratory chow ad libitum. Endotoxins. LPS extracted from Salmonella typhimurium 7 (STM 7) and Escherichia coli B were generously provided by J. W. Shands, Jr., Department of Medicine, University of Florida College of Medicine, Gainesville. Several mutants of S. typhimurium LT-2, characterized by biosynthetic defects in cell wall synthesis, were also obtained from Shands, including TV 119, chemotype Ra; SL 684, chemotype Rc; and SL 1102, chemotype Re. J. A. Rudbach, Department of Microbiology, University of Montana, Bozeman, supplied LPS from Brucella abortus, E. coli O111:B4, E. coli 0113, Neisseria gonorrheae, S. enteritidis, S. typhi, and Serratia marcescens and native protoplasmic polysaccharides (NPP) from E. coli O11L:B4 and E. coli 0113. LPS from E. coli K-235 was provided by F. C. McIntire, Department of Oral Biology, School of Dentistry, University of Colorado, Denver. All LPS preparations were extracted by the phenol/water technique (43), resuspended in RPMI 1640 (Grand Island Biological Co., Grand Island, N.Y.), sonically disrupted, and stored at 4°C until used; the time from resuspension until use did not exceed 3 weeks. LPS preparations used in this study have been shown to be mitogenic for splenic B lymphocytes from C57BL/6 and A strain mice (21-23); however, they failed to significantly stimulate C3H/HeJ spleen cells when tested over a wide dosage range (38). Hydrolysis of LPS. Lipid A and free polysaccharide were prepared from extracted LPS by acid hydrolysis; LPS was deesterified by treatment with mild alkali (23). Effects of LPS on leukocytes in vivo. Mice received LPS in 0.2 ml of RPMI intraperitoneally at the start of each experiment. Control animals received RPMI without LPS. At the indicated times thereafter, groups of three LPS-treated and three control mice were sacrificed. Peritoneal cells were collected in RPMI containing 10 U of preservative-free heparin (Fisher Scientific Co., Fair Lawn, N.J.) per ml, washed twice in RPMI, and resuspended in complete tissue culture medium (TCM; Eagle minimal essential medium, Microbiological Associates, Bethesda, Md.) supplemented with glutamine, penicillin, streptomycin, and 10% fetal calf serum (24).

311

Cell preparation and tissue culture conditions. Preparation of single-cell suspensions from excised spleens has been described previously (25). Monolayers of adherent cells were prepared by adding 0.3 ml of a peritoneal leukocyte suspension (2 x 106 cells per ml) to a 35-mm culture dish containing a 15- by 15mm glass cover slip and incubating cultures for 30 min at 37°C. Nonadherent cells were removed by vigorously rinsing each cover slip in a 50-ml beaker filled with prewarmed (37°C) TCM. Conditions of incubation. Spleen cells, peritoneal leukocytes, and adherent cell monolayers in 1 ml of TCM were incubated in 35-mm culture dishes at 37°C in a humidified atmosphere of 83% N2, 10% C02, and 7% 02 for the designated periods of time. Morphological and functional characterization. Peritoneal cells from LPS-treated and control mice or peritoneal cells harvested from tissue cultures were processed in an identical manner. Phagocytic cells were first labeled by incubating cell suspensions with 0.1 ml of a 1:10 dilution of India ink (Pelikan 17) for 20 min at 37°C. Cells were washed thoroughly in RPMI and pelleted on glass microscope slides (31) using a cytocentrifuge (Shandon Elliot Corp., Sewickley, Pa.). Differential counts of May-GrunwaldGiemsa-stained slide preparations were performed by light microscopy at a magnification of xl,000. In selected experiments, unstained peritoneal cells were examined under phase microscopy. Detection of immunoglobulin-bearing cells. The number of peritoneal cells bearing surface membrane immunoglobulin was quantitatively assessed by direct immunofluorescence (39) using fluorescein isothiocyanate-conjugated goat anti-mouse gamma globulin (Cappell Laboratories, Downington, Pa.). A sample (5 x 106) of peritoneal cells was suspended in 1 ml of phosphate-buffered saline containing 2% bovine serum albumin (PBS-BSA). A 0.25-ml portion of the cell suspension was reacted with an equal volume of a 1:10 dilution of the fluorescein conjugate in polystyrene centrifuge tubes, and incubated for 30 min at 4°C. Unbound conjugate was removed by washing three times with 1-ml portions of PBS-BSA. The final cell pellet was resuspended in 0.1 ml of phosphate-buffered glycerol (pH 7.2), and a small drop was applied to a glass microscope slide using a Pasteur pipette. A cover slip was placed over the specimen, and slides were examined immediately using a Leitz fluorescent microscope equipped with phase optics and Ploem illumination. At least 300 nucleated cells per slide were examined. B lymphocytes were recognized by a uniform rim of fluorescence encompassing 21/2 the cell circumference. Specificity of the reaction was demonstrated by inhibiting fluorescent staining with unconjugated goat anti-mouse gamma globulin (Cappell Laboratories). Measurement of DNA synthesis. DNA synthesis by spleen cells in vitro was measured by the incorporation of [3H]thymidine into acid-precipitable material (21). Data are expressed as the mean counts per minute per culture of three replicate samples ± the standard error of the mean (SEM). Cell quantitation and viability determination. Nucleated cells in spleen and peritoneal cell suspensions were counted by a model ZBI Coulter Counter

312

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INFECT. IMMUN.

(Coulter Electronics, Hialeah, Fla.). The viability of peritoneal leukocyte suspensions was determined by adding 0.2 ml of 0.2% trypan blue to each culture, staining for 30 s (6), and terminating the reaction with 0.8 ml of 4% acetic acid. Peritoneal adherent cells were treated in an identical manner except that monolayers were rinsed and transferred to new culture dishes containing 1 ml of TCM before staining. Statistical analysis. Means were compared using Student's t test (46). Values of P < 0.05 were considered significant.

RESULTS At 24 h after intraperitoneal injection of 10 )ug of LPS into A and C57BL/6 mice, the number of mononuclear leukocytes recovered from the peritoneal cavity decreased substantially; macrophages, immunoglobulin-bearing lymphocytes, and non-immunoglobulin-bearing lymphocytes all were reduced (Table 1). Morpho-

logical examination by light and phase-contrast microscopy showed that macrophages from LPS-treated C57BL/6 mice had ruffled cytoplasmic membranes, increased granulation, and extensive vacuolation (Fig. la); pyknotic nuclei were sometimes observed. Although the number of lymphocytes was sharply reduced, an increased percentage of large lymphocytes was seen; these cells had prominent nucleoli, clear perinuclear areas, and increased cytoplasmic basophilia. In contrast to these results, intraperitoneal injection of 10 jig of LPS into C3H/HeJ mice increased the numbers of macrophages and lymphocytes recovered from the peritoneal cavity (Table 1) without causing morphological alterations in macrophages or lymphocytes (Fig. lb). In all three mouse strains, i.p. injection of LPS induced a similar degree of polymorphonuclear cell inflammatory response.

TABLE 1. Effects of LPS on peritoneal cell populations PMLb

Mononuclear leukocytes Strain

Treatment

Neutro

Baso

Eosino

Control LPS

1.28 0.21

Ig' lymphd 0.93 0.27

Ig- lymph'

A

0.61 0.16

0.13 1.91

0.19 0.05

0.01 0.05

C57BL/6

Control LPS

2.14 0.49

1.36 0.74

1.02 0.45

0.10 2.18

0.19 0.25

0.05 0.04

C3H/HeJ

Control LPS

1.73 3.62

MW

0.87 0.65 0.04 0.29 0.04 1.32 1.35 4.00 0.26 0.26 a Results are expressed as the number of cells in each category X 106 This value was obtained by multiplying the percent differential by the total number of cells recovered from each mouse and averaging differential results for three mice to obtain the mean. b Neutro, neutrophils; Baso, basophils or mast cells; Eosino, eosinophils. M+, macrophages; all mononuclear leukocytes containing five or more phagocytized carbon particles. d Lymphocytes and blast cells staining positively for surface membrane immunoglobulin (Ig). Lymphoblasts not staining for immunoglobulin (Ig).

FIG. 1. Morphological alterations in mononuclear leukocytes induced by LPS. (a) Photomicrograph of an extensively vacuolated macrophage obtained from the peritoneum of C57BL/6 mice 24 h after intraperitoneal injection of 10 ,ug of LPS; (b) a peritoneal macrophage from similarly treated C3H/HeJ mice. May-Grinwald-Giemsa stain. Magnification x2,220.

313

CYTOTOXICITY OF LPS FOR MOUSE PERITONEAL CELLS

VOL. 21, 1978

These experiments detected differences in re- phages from C57BL/6 mice decreased, suggestsponsiveness to LPS of mononuclear leukocytes ing a cytolytic effect (Table 2). Corresponding in LPS-susceptible and LPS-resistant mouse increases in large lymphocytes and blast cells strains. Since studies in vivo do not allow dis- were noted. In vitro exposure to LPS induced tinction between cell damage and loss of acces- the same morphological alterations in C57BL/6 sibility of cells due to either adherence to peri- mononuclear leukocytes that had been observed toneum or migration out of the peritoneal cavity, in vivo; macrophages were vacuolated and pykin vitro experiments were designed to determine notic, while small lymphocytes had transformed the direct effects of LPS on macrophages and into blast cells. Viability studies of these cells lymphocytes. Normal peritoneal leukocytes (2 that remained, using trypan blue exclusion, x 106) from C57BL/6 and C3H/HeJ mice were showed that, 24 h after addition of 50 [Lg of LPS, incubated in 1 ml of TCM with or without 50 22% of C57BL/6 leukocytes were no longer via,ig of LPS. At 24 and 48 h later, cells were ble (Fig. 2). Extending the time of incubation to harvested and counted, and differential counts 48 h resulted in increased cytotoxicity, with were performed. The total number of cells re- fewer than half of the leukocytes remaining vicovered from cultures after 48 h decreased in able as compared to 87% of controls. the case of both strains. In the presence of LPS, As predicted from the results of studies in the percentage and absolute number of macro- vivo, LPS had no observable effect in vitro on TABLE 2. Effects of LPS on mouse peritoneal cells in vitro Differential classification of recovered cells Strain

Time (h)

Treatment

24

Control LPS Control LPS

C57BL/6

48

Nucleated cells per

1.43 1.57 1.16 1.09

± 0.04 ± 0.14 ± 0.05 ± 0.06

MO

SL

LL

Blasts

PML

59 42 58 37

20 14 16 10

15 26 16 27

3 12 5 18

3 6 5 8

12 2 1.49 ± 0.12 62 15 9 Control 1.72 ± 0.10 57 20 14 3 6 LPS 22 15 5 3 48 Control 0.97 ± 0.08 55 2 9 0.92 ± 0.04 55 19 15 LPS aMean counts of three replicate cultures x 106 ± SEM. b Cells were classified according to morphological criteria described in Table 1. Results are expressed as percentages based on counts of -200 cells. MO, macrophages; SL, small lymphocytes; LL, large lymphocytes.

C3H/HeJ

24

100 C)

I

-J -J

80 _

w -J

60 _

w C.)

z w V

w 0.

40 _

h-- Control LPS

20 1 -

C57BL/6

C3H/HeJ

0 12

24

36

48

1

I

12

24

36

48

HOURS AFTER CULTURE INITIATION FIG. 2. Strain-dependent cytotoxic effects of LPS for mouse peritoneal leukocytes. Normal peritoneal leukocytes were incubated with (A) or without (A) 50 jig of LPS from S. typhimurium 7. Results are expressed as the mean percentage of viable cells in triplicate cultures + SEM.

314

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INFECT. IMMUN.

the numbers, proportions, or morphology of peritoneal leukocytes from C3H/HeJ mice. Cell viabilities in LPS-treated C3H/HeJ peritoneal cells and control cultures were not significantly different at any time tested. To study the direct cytotoxic effect of LPS on mouse macrophages, adherent cell monolayers were prepared by overlaying sterile glass cover slips with peritoneal leukocytes from C57BL/6 or C3H/HeJ mice. After incubation for 30 min at 370C, cover slips were washed to remove nonadherent cells and incubated at 370C in the presence of varying concentrations of LPS. Differential counts at the start of the incubation period showed that about 95% of adherent cells were macrophages, the remainder consisting of lymphocytes and granulocytes. LPS produced cytotoxic effects on C57BL/6 adherent cells; only 56% of these cells survived incubation with 50 ,g of LPS, compared to 95% of adherent cells incubated in the absence of LPS (Fig. 3). No reduction in cell viability was seen in cultures of

adherent C3H/HeJ peritoneal leukocytes. Cytotoxicity for mouse peritoneal macrophages depended upon the preparation of LPS and its concentration. The dose-response relation for LPS extracted from several gram-negative organisms is shown in Fig. 4. Considerable variation was noted in the potency of different LPS preparations. For example, 50 ,ug of LPS from N. gonorrheae reduced cell viability to 14%, whereas LPS from E. coli O111:B4, S. enteritidis, and Serratia marcescens produced a similar degree of cytotoxicity at one-hundreth the concentration. Furthermore, LPS derived

from B. abortus, E. coli B, E. coli 0113, E. coli K235, and S. typhi were not cytotoxic for C57BL/6 macrophages at any concentration tested (data not shown). None of these LPS preparations decreased the viability of C3H/HeJ macrophages. To determine the LPS moiety (19) responsible for cytotoxicity, lipid A, free polysaccharide, or deesterified LPS was added to cultures of mouse 100

-J 6' 80 w 4

60

U 40

c. 20

C57BL/6 C3H/KJ o---o Unfractionated- *- Adherent

0

-

5 50 0.5 0.05 MICROGRAMS LPS PER CULTURE

FIG. 3. Cytotoxicity of LPS forglass-adherent peritoneal leukocytes. Glass-adherent subpopulations were incubated for 48 h with the indicated doses of LPS from S. typhimurium 7. Data are also shown for additional controls, which are studied using unfractionated peritoneal leukocytes. Results of five separate experiments are expressed as the mean of duplicate cultures ± SEM.

100 Un -J -J w

w

-J

OD 4

80F 60F

--

* z w

oE.coli

* N. gonorrheae

S. enteritidis

40~

*' Ser. marcescons

u

w

a.

201-

C57 BL/6 0

.05

C3H /HeJ .5

5

50

.05

.5

5

50

MICROGRAMS LPS PER CULTURE

FIG. 4. Cytotoxic effects of LPS derived from different gram-negative bacteria. LPS extracted from the designated bacterial species was added to cultures containing adherent cell monolayers. Cell viabilities, determined after 48 h by trypan blue exclusion, are represented as the mean of triplicate cultures ± SEM.

CYTOTOXICITY OF LPS FOR MOUSE PERITONEAL CELLS

VOL. 21, 1978

peritoneal macrophages; untreated LPS from which these components had been derived (S. typhimurium 7) was used as a positive control. Lipid A was cytotoxic for C57BL/6 macrophages over a wide range of doses and killed more cells at each dose than the untreated LPS from which it had been derived (Fig. 5). In contrast, soluble polysaccharide was not cytotoxic. Removal of esterified fatty acids from LPS by treatment with NaOH reduced but did not abolish cytotoxic activity. Macrophages from C3H/HeJ mice were resistant to LPS or lipid A at concentrations ranging from 0.05 to 50 jug/ml. LPS extracted from rough mutants of S. typhimurium possess an intact lipid moiety but

315

lack varying portions of 0 and R polysaccharides (2). As shown in Table 3, 50,ug of LPS lacking 0 side chains (TV 119), LPS lacking 0 side chains and part of the R core (SL 684), or LPS completely deficient in 0 and R polysaccharides (SL 1102) was mitogenic for C57BL/6 spleen cells; similar results were obtained using 5 and 200 jig of LPS per ml. Each LPS was also cytotoxic for C57BL/6 macrophages; in fact, these preparations had had greater activity than complete LPS (STM 7). In contrast to these results, SL 684, SL 1102, and STM 7 failed to elicit a mitogenic or cytotoxic response when added to cultures of spleen or peritoneal adherent cells from C3H/HeJ mice. LPS derived from TV 119 in-

U w

_

o-TSTM 7

60

*

40

_ ^-~Lipid A A NoOH-treoted STM 7

\

_

Polysocchoride

A

z

C3H/HeJ

C57BL/6

5 50 5 .5 50 .05 MICROGRAMS LPS PER CULTURE FIG. 5. Characterization of the LPS moiety responsible for macrophage cytotoxicity. Soluble polysaccharide, lipid A, or complete LPS from S. typhimurium 7 was added to cultures containing adherent cell monolayers. Experimental details and organization of this figure are as described for Fig. 4. 0

.05

.5

TABLE 3. Cytotoxicity of LPS extracted from smooth and rough strains of S. typhimurium" C3H/HeJ

C57BL/6 Strain

None STM 7 TV 119

LPS preparation

Heptose backbone, core and side chains Heptose backbone and

[ 'H]thymidine in-

Macrophage

corporation 1,697 ± 143 11,606 ± 284

viability' 97 ± 2 69 ± 4

26,678 ± 3,116

35 ± 6

1,779 ± 30 1,827 ± 116

Macrophage viability" 96 ± 1 94 ± 2

4,287 ± 299

88 ± 1

[t H]thymidine incorporationh

core

95 ± 2 33 ± 6 3,036 ± 127 Glucose and heptose back24,413 ± 5,427 bone 97 ± 2 41 ± 2 2,901 ± 416 SL 1102 KDOd and lipid A 18,182 ± 586 x a A 50-,sg sample of LPS from each strain was added to cultures containing 2 10'l spleen cells or monolayers of peritoneal adherent cells in 1 ml of TCM. Proliferative responses by spleen cells were measured by the incorporation of [3H]thymidine during the final 24 h of a 48-h culture period; cytotoxicity for adherent cells was determined after 48 h by trypan blue exclusion. All data represent mean values obtained from three replicate cultures. ' Mean counts per minute per 106 cells ± SEM. 'Mean percentage of viable cells ± SEM. d KDO, 2-Keto-3-deoxyoctulosonic acid.

SL 684

316

INFECT. IMMUN.

PEAVY, BAUGHN, AND MUSHER

creased proliferation by C3H/HeJ spleen cells twofold and decreased viability of macrophages 8%. NPP is similar to LPS in polysaccharide composition and possesses serological reactivity, but lacks esterified fatty acids (2). NPP or LPS from E. coli O 1:B4 was added to cultures containing C57BL/6 macrophages or spleen cells. LPS increased proliferative responses by splenic lymphocytes approximately 10-fold and decreased viability of macrophages 41% (Table 4). NPP from E. coli O111:B4 was neither mitogenic nor cytotoxic, showing that haptenic polysaccharides that lack the lipid moiety are devoid of activity in vitro. DISCUSSION The results of this investigation demonstrate that LPS derived from gram-negative bacteria is cytotoxic for mouse peritoneal macrophages. When LPS was administered to mice intraperitoneally or added to cultures of peritoneal leukocytes, the number of recoverable macrophages decreased. Those macrophages that remained had altered membrane permeability and a loss of cellular integrity as shown by the presence of extensively vacuolated cytoplasms, pyknotic nuclei, and the failure to exclude trypan blue. LPS was also cytotoxic in vitro for isolated preparations of adherent peritoneal cells, thus suggesting that cell damage was independent of the presence of lymphocytes and resulted from the direct interaction of LPS with macrophage membranes. These observations confirm the findings of previous investigators, which provide morphological evidence that LPS is toxic for cells of the monocyte-macrophage series. Heilman (15) and Heilman and Bernton (16) originally noted that LPS inhibited the migration of cells from rabbit spleen and liver organ cultures; the migrating cells resembled macrophages that appeared ruffled and extensively vacuolated after incubation with LPS. Shands et al. (30) reported similar morphological alterations in mouse macro-

phages when LPS was injected intraperitoneally or was added to cultures of peritoneal leukocytes. Although Bianco and Edelson (Fed. Proc. 36:1263, 1977) suggested that the effects of LPS or macrophages were mediated through B lymphocytes, Wiener and co-workers (44, 45) observed two direct effects of LPS on mouse macrophages; low concentrations of LPS (0.01 fig/ml) induced the rapid accumulation of intracellular hydrolases, whereas high concentrations (10 to 100 tig/ml) resulted in their extracellular release. Pinosome formation (5) and phagocytic uptake (Benacerraf and Sebasteyn, Fed. Proc. 16:860, 1957) of LPS by macrophages may lead to synthesis of lysosomal enzymes. Excessive stimulation by LPS labilizes newly created lysosomes (42). The instability of lysosomal and plasma membranes may result in destruction of the macrophage by autolytic processes. Sultzer (36, 37) demonstrated that depletion of mononuclear cells following intraperitoneal administration of LPS to mice was strain dependent; LPS failed to elicit this response in C3H/HeJ mice. Our investigations showed vacuolation of macrophages and blast transformation of lymphocytes; decreases among macrophages and immunoglobulin-positive and -negative lymphocytes were also observed. None of these changes was observed after injection of LPS into C3H/HeJ mice. In vitro investigations have previously demonstrated that macrophages from C3H/HeJ mice are resistant to the cytotoxic effects of LPS (12) and that lymphocytes from these animals are unresponsive to mitogenic effects of LPS (28). Our studies show that mononuclear leukocytes from LPS-sensitive and LPSresistant mouse strains respond to LPS in vitro in a fashion identical to that reported previously in vitro. LPS attracted polymorphonuclear leukocytes (PML) into the peritoneal cavity of both LPSsusceptible and LPS-resistant strains of mice, a fact demonstrated previously by Sultzer (36, 37). Whether this accumulation represents a direct cellular interaction of LPS on PML or results

TABLE 4. Cytotoxicity of NPP and LPSG Strain

Prepn

C57BL/6 Macrophage ['H]thymidine incor-

C3H/HeJ

[3H]thymidine incor-

Macrophage

viability' porationb porationb viability' 96 ± 1 ± 71 ± 1 98 128 1,086 None 1,914 + 98 ± 3 987 ± 43 57 ± 4 LPS E. coli O111:B4 18,252 + 488 99 ± 1 953 ± 35 95 ± 1 NPP E. colOl111:B4 1,725 + 216 " A 50-tig sample of LPS or 50 jig of NPP, extracted from the homologous strain, was added to cultures containing spleen cells or adherent cell monolayers. Mitogenic responses by spleen cells and cytotoxicity for adherent cells were determined as described in Table 3. Results are expressed as the mean of triplicate cultures.

Mean counts per minute per 10' cells ± SEM. Mean percentage of viable cells ± SEM.

VOL. 21, 1978

CYTOTOXICITY OF LPS FOR MOUSE PERITONEAL CELLS

from the generation of physiologically active molecules (for example, endogenous vasoactive substances) through the interaction of LPS with other cell lines or with soluble constituents is unknown. We are inclined to favor the latter explanation. Although LPS binds to PML in vitro (9) and is internalized by endocytosis, causing major effects on cellular metabolism (11), it is difficult to see how effects on a cell membrane would cause cells to accumulate. In constrast, several biologically potent mediators generated by the complement, kinin, and blood coagulation systems increase vascular permeability and chemotactically attract PML. Previous studies have shown that these pathways may be activated by LPS (4, 20, 35). Snyderman et al. have suggested that the LPS-induced intraperitoneal accumulation of PML is due to C5 activation, since the inflammatory response was greatly diminished in C5deficient mice (35). However, similar studies in our laboratory using C5-deficient A, B1O.D2/oSn, and DBA/2 mice have not confirmed these findings (unpublished data). In any case, the presence of PML in the peritoneums of C3H/HeJ mice following administration of LPS suggests that at least one of these mechanisms is operative in what is an otherwise LPS-refractory strain. LPS are macromolecular complexes composed of lipid A covalently linked to a heteropolysaccharide moiety which, depending on the species, may comprise 10 to 60% of its total content. Results from this investigation provided direct evidence that lipid A was responsible for macrophage cytotoxicity. LPS extracted from Salmonella mutants that lacked 0 and R polysaccharides and purified lipid A had activity similar to that of LPS from smooth strains of Salmonella. In contrast, NPP and soluble polysaccharides that were freed of lipid by acid and alkaline hydrolysis did not possess this activity. These results support the hypothesis that LPS mediates its effect through the affinity of lipid A for mammalian cell membranes. Although all LPS preparations used in this study possess biological activity in vivo and are mitogenic for B lymphocytes in vitro, substantial variation in their cytotoxicity for macrophages was observed. Since lipid A from all the Enterobacteriaceae is structurally closely similar (1, 27), these results suggest that additional physical and chemical properties conferred by polysaccharides may contribute to the cytotoxicity of LPS. In aqueous solution, LPS exists as heterogeneous aggregates formed by hydrophobic interaction of individual subunits. The molecular weight of these aggregates, estimated by velocity sedimentation, varies from 1 x 106 to 25

317

x 106 (14, 26), a weight within the range of phagocytized macromolecules. If cytotoxicity is dependent upon membrane binding and internalization, the activity of LPS could be influenced substantially by its polysaccharide content. Due to their hydrophilic nature, polysaccharides solubilize LPS, thereby reducing the particulate nature of LPS and decreasing its uptake. Previous investigations have reported that administration of LPS results in nonspecific resistance to a variety of experimental infections (10). Increased resistance has been attributed to several host defense mechanisms (10). Cellular responses appear to play an essential role; macrophages and PML display augmented phagocytic activity after interaction with LPS. Several humoral substances with antimicrobial activity such as immunoglobulins, bactericidal factors, interferons, lysozymes, and activated complement components are increased in serum upon injection of LPS. Due to the variety of LPSinduced effects and to the complexity of factors influencing infection, it is difficult to determine precisely which mechanism(s) is involved in resistance to a particular microorganism (8). Identification of strain-dependent differences in the response of host leukocytes to LPS should aid in these investigations. For example, results reported here demonstrate that macrophages and B lymphocytes from C3H/HeJ mice are refractory to LPS in vivo; however, the chemotactic response by PML appears to be intact. Whether or not PML are activated functionally by LPS has yet to be determined. Nevertheless, comparative studies of LPS-induced resistance in these inbred strains should provide a powerful tool for investigating host mechanisms contributing to resistance against microbial pathogens. ACKNOWLEDGMENTS The authors are grateful to Roberta Cooper for her excellent technical assistance and to Elaine Bowers and Mona Thomas for preparation of the manuscript. This work was supported by Public Health Service grants BRSG-77 P4 and AI-12618 from the National Institute of Allergy and Infectious Diseases, by grants IM-138 and IN-27Q from the American Cancer Society, and by research funds from the Veterans Administration Hospital, Houston, Texas.

LITERATURE CIMD 1. Adams, G. A., and P. P. Singh. 1970. Structural features of lipid A preparations isolated from Escherichia coli and Shigella flexneri. Biochim. Biophys. Acta 202:553-555. 2. Anacker, R. L., R. A. Finkelstein, W. T. Haskins, M. Landy, K. C. Milner, E. Ribi, and P. W. Stashak. 1964. Origin and properties of naturally occurring hapten from Escherichia coli. J. Bacteriol. 88:1705-1720. 3. Andersson, B., and H. Blomgren. 1971. Evidence for thymus-independent humoral antibody production in mice against polyvinylpyrrolidone and E. coli lipopolysaccharide. Cell. Immunol. 2:411-424.

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