Inhibition of Escherichia coli-Induced Meningitis by Carboxyfullerence

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Inhibition of Escherichia coli-Induced Meningitis by Carboxyfullerence

NINA TSAO,

_{PUTHUPARAMPIL P. KANAKAMMA,}

_{TIEN-YAU LUH,}

_{CHEN-KUNG CHOU,}

AND

HUAN-YAO LEI

*

Department of Microbiology & Immunology, College of Medicine, National Cheng Kung University,

Tainan,

_{and Department of Chemistry, National Taiwan University,}

_{and Department}

of Medical Research, Veteran General Hospital,

_{Taipei, Taiwan}

Received 13 January 1999/Returned for modification 26 May 1999/Accepted 10 June 1999

The effect of a water-soluble malonic acid derivative of carboxyfullerence (C60) against Escherichia

coli-induced meningitis was tested. C60 can protect the mice from E. coli-coli-induced death in a dose-dependent

manner. C60 administered intraperitoneally as late as 9 h after E. coli injection was still protective. The

C60-treated mice had less tumor necrosis factor alpha and interleukin-1

␤ production by staining of brain

tissue compared to the levels of production for nontreated mice. The E. coli-induced increases in blood-brain

barrier permeability and inflammatory neutrophilic infiltration were also inhibited. These data suggest that

C60 is a potentially therapeutic agent for bacterial meningitis.

Through the years, bacterial meningitis has remained an

infection with a high mortality rate, particularly in very young

and elderly patients, despite the availability of effective

antibi-otic treatments (21, 30). The pathophysiology of bacterial

men-ingitis involves the invasion and multiplication of bacteria in

the subarachnoidal space of the central nervous system (CNS).

The bacterium itself or its degraded products stimulate the

production and release of proinflammatory mediators such as

cytokines and prostaglandins by leukocytes, endothelial cells,

astrocytes, microglial cells, and other cells in the CNS, and

these subsequently lead to an increase in the permeability of

the blood-brain barrier (BBB). This triggers transendothelial

migration of neutrophils and leakage of plasma proteins that

further damage the brain (2, 20). Proinflammatory cytokines

have been shown to play a critical role in the pathogenesis of

bacterial meningitis. Both tumor necrosis factor alpha

(TNF-␣) and interleukin-1␤ (IL-1␤) were detected in the

ce-rebrospinal fluid of some patients with bacterial meningitis and

in experimental animals (13, 16, 18, 19, 27). In this study, a

model of experimental meningitis induced by direct injection

of Escherichia coli into the brains of B6 mice was set up. As

expected, TNF-␣ and IL-1␤ production was induced, followed

by inflammatory neutrophil infiltration. The vasopermeability

of BBB was also increased.

Buckminsterfullerene (carboxyfullerence [C60]) is

charac-terized as a “radical sponge” due to its avid reactivity with free

radicals (15). A water-soluble malonic acid derivative of C60

has been synthesized and has been found to be an effective

neuroprotective antioxidant both in vitro and in vivo (7, 9, 10,

17). This study tested the effect of C60 on the E. coli-induced

meningitis model, and it was found that C60 could inhibit the

development of E. coli-induced meningitis. Its therapeutic

ap-plication for treatment of bacterial meningitis is discussed.

MATERIALS AND METHODS

Mice.Breeder mice of the strain B6 were purchased from the Jackson Labo-ratory (Bar Harbor, Maine) or Charles River Japan, Inc. (Atsugi, Japan). They were maintained on standard laboratory chow and water ad libitum in the animal facility of the Medical College, National Cheng Kung University, Tainan,

Tai-wan. The animals were raised and cared for by following the guidelines set up by the National Science Council of the Republic of China. Eight- to 12-week-old female mice were used in all experiments.

C60.Two regioisomers of water-soluble carboxylic acid C60 derivatives with C3 or D3 symmetry were synthesized as described previously (7). Both C60 (C3) and C60 (D3) are effective free-radical scavengers. Both compounds are potent inhibitors of neuronal apoptosis in vitro; neuronal apoptosis is associated with increased intracellular free-radical production. In this study, we used C60 (C3) dissolved in phosphate-buffered saline (PBS; 2 mg/ml).

Induction of bacterial meningitis.E. coli ATCC 10536 was cultured in Luria-Bertani (LB) broth (1% NaCl, 1% tryptone, 0.5% yeast extract) for 12 h and was subcultured in fresh medium for another 3 h. The concentration of E. coli was determined with a spectrophotometer (Beckman Instrument, Somerset, N.J.), with an optical density at 600 nm of 1 equal to 108_{CFU/ml (32). For the}

induction of meningitis, groups of three to four mice were given intracerebral injections directly into the temporal area of a 20-␮l volume of 5 ⫻ 105_{E. coli cells}

diluted in saline. The 100% lethal dose (LD100) by intracerebral injection in B6

mice is 5⫻ 105_{E. coli cells. The animals were observed every 12 h for a total of}

6 days. In the C60 inhibition experiments, the mice were given an intraperitoneal (i.p.) injection of C60 (40 mg/kg of body weight) three times every 24 h either before or after intracerebral injection of E. coli. The survival curve was pre-sented. In some experiments, the brains were aseptically removed and were homogenized with 3% gelatin (Difco Laboratories, Detroit, Mich.) in PBS. The samples were serial diluted, poured in agar plates, and incubated at 37°C over-night. The number of CFU of E. coli was quantitated and was expressed as the mean⫾ standard deviation per mouse. E. coli was also cultured with various concentrations of C60 in LB broth. The growth curve of E. coli was determined in LB broth to evaluate the direct antimicrobial activity of C60.

Immunohistochemistry.Groups of three to four mice were killed by perfusion via cardiac puncture with PBS. The brains were removed and embedded in OCT compound (Miles Inc., Elkhart, Ind.) and were then frozen in liquid nitrogen. Four-micrometer cryosections were made and were fixed with ice-cold acetone for 3 min. They were then stained with a primary rat anti-TNF-␣ monoclonal antibody (MAb; MAb MP6-XT3; PharMingen, San Diego, Calif.) or a hamster anti-IL-1␤ MAb (Genzyme, Cambridge, Mass.). Secondary antibodies were per-oxidase-conjugated sheep anti-rat immunoglobulin G (IgG), goat anti-hamster IgG, or swine anti-goat IgG (Boehringer Mannheim GmbH, Mannheim, Ger-many). A peroxidase stain with a reddish brown color was developed with an aminoethyl carbazole substrate kit (ZYMED Laboratories, San Francisco, Cal-if.) (8).

Detection of increased vasopermeability of BBB by M4 tracer with

␤-galac-tosidase activity.An E. coli mutant (mutant M4) that constitutively expresses ␤-galactosidase was used as the tracer to detect alterations in the vasoperme-ability of the BBB. M4 was selected from E. coli K-12 that grew in an M63 culture plate [0.3% KH2PO4, 0.7% K2HPO4, 0.2% (NH4)2SO4, 0.1 mM FeSO4]

con-taining 0.2% lactose, 0.002% vitamin B1, 1 mM MgSO4, 0.001%

isoleucine-leucine-valine, and 0.002% 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal). The M4 mutant constitutively expresses␤-galactosidase and has a characteristic blue colony on medium containing X-Gal without induction. Pre-liminary studies have demonstrated that M4 is avirulent (LD50,⬎2 ⫻ 109cells)

to mice and that M4 was rapidly removed from the circulation. It can be used as an inert tracer within 10 min after injection. To each mouse into which E. coli was injected, 2⫻ 108_{cells of the M4 tracer in 0.1 ml were given intravenously 2 min}

before the mice were killed. The brains were removed, cyrosectioned, and fixed in 0.2% glutaldehyde (Merck GmbH, Parmstadt, Germany). The M4 in the

* Corresponding author. Mailing address: Department of

Microbi-ology & ImmunMicrobi-ology, College of Medicine, National Cheng-Kung

University, Tainan, Taiwan, Republic of China. Phone: 886-6-2353535,

ext. 5643. Fax: 886-6-2082705. E-mail: hylei@mail.ncku.edu.tw.

2273

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tissues was detected by X-Gal staining (1 mg of X-Gal per ml in 20 mM potas-sium ferricyanide, 20 mM potaspotas-sium ferrocyanide, and 2 mM magnepotas-sium chlo-ride) at 37°C for 2 h.

RESULTS

Inhibition of experimental E. coli-induced meningitis by

C60.

Intracerebral injection of E. coli in B6 mice induced

TNF-␣ and IL-1␤ production in the brain and recruited

neu-trophil infiltration into the brain at 6 to 9 h postinjection. The

authors were interested in the effect of C60 on the regulation

of brain inflammatory responses. Without treatment the mice

will die within 36 h of intracerebral injection of 5

⫻ 10

_{E. coli}

cells (the LD

for mice). However, pretreatment of each

mouse with 40 mg of C60 per kg i.p. protects the mouse from

E. coli-induced death. The inhibition is dose-dependent; 20 mg

of C60 per kg protected 40% of the mice, while 30 mg/kg

protected 75% of the mice (Fig. 1). This inhibitory effect was

better than that of dexamethasone (6 mg/kg), which protected

only 20% of the mice. The inhibitory effect of C60 was further

studied. As shown in Fig. 2B, the increased vasopermeability of

the BBB detected with the M4 tracer was manifested at 24 h in

E. coli-treated mice. However, in the C60-pretreated mice,

these increases in BBB permeability were inhibited (Fig. 2C).

Furthermore, the TNF-␣ and IL-1␤ staining intensities on

arterioles or infiltrating neutrophils were lower in C60-treated

mice than in nontreated mice (Fig. 2G and K versus 2F and J).

This is consistent with the observation that less neutrophil

infiltration occurs in C60-treated mice. Apparently, the C60

treatment decreases the level of cytokine production in the

brain, which consequently inhibits the increase in BBB

perme-ability and protects the mice from E. coli-induced death.

Therapeutic effect of C60 on E. coli-induced meningitis in B6

mice.

The therapeutic effect of C60 on E. coli-induced

menin-gitis was examined next. As shown in Fig. 3A, the mice died

within 36 h after the injection of 5

⫻ 10

_{E. coli cells. In}

contrast, intraperitoneal administration of 40 mg of C60 per kg

as late as 6 h after E. coli injection protected 80% of the mice

from E. coli-induced death. There was still a 50% survival rate

if C60 was given at 9 h postinfection. Its protective effect was

better than that of dexamethasone, which had only a

preven-tive effect (Fig. 3B). The cytokine expression, increase in BBB

permeability, and neutrophil infiltration were also inhibited in

the groups treated with C60 postinfection (Fig. 2D, H, and L).

Immunomodulatory effect of C60 in the brain.

The

inhibi-tion of E. coli-induced meningitis by C60 is not due to its direct

antimicrobial activity. C60 did not inhibit the growth of E. coli

in an in vitro LB broth culture (Fig. 4A). Furthermore, the

growth of E. coli in the brain after intracerebral injection was

determined after C60 treatment. As shown in Fig. 4B, the

number of E. coli cells in the brain was not lower in

C60-treated mice than in nonC60-treated mice 12 h after intracerebral

injection of E. coli. The E. coli cells were cleared from the

brain after 24 h in C60-treated mice, while they replicated

significantly in nontreated mice, suggesting that C60 might

have enhanced the natural antibacterial defenses in the brain.

This was supported by the observation that TNF-␣ expression

was found in brain endothelial cells or ependymal cells from

naive mice treated with C60 alone (Fig. 5). On the basis of the

information presented above, we conclude that C60 may be a

therapeutic agent for bacterial meningitis.

DISCUSSION

The pathogenesis of bacterial meningitis is determined by

several factors including bacterial load, production of

proin-flammatory cytokines such as TNF-␣ and IL-1␤, increased

permeability of BBB, and infiltration of inflammatory

neutro-phils. The pathophysiologic sequelae of meningitis that result

from the interaction between the bacteria and the host

consti-tutes a complex cascade. A single intervention is insufficient to

halt the disease process, especially from the therapeutic point

of view. Studies with adjunctive therapies, including

anticyto-kine drugs, antiinflammatory modulators, and

glucocorticoste-roids, have shown promising results (21, 30). In this study, it

was found that a water-soluble malonic acid derivative of C60

(C3) could interfere with the inflammatory response in

exper-imental E. coli-induced meningitis. C60 not only suppressed

cytokine production as well as increased permeability of the

BBB, but it also inhibited neutrophil infiltration into the brain.

C60 can also modulate the natural antibacterial defense in the

brain. Furthermore, C60 is still effective 9 h after E. coli

injec-tion, indicating that it can interfere with neutrophil activation.

Fullerenes have attracted much attention since their

discov-ery and large-scale synthesis. Fullerenes have an unique cage

structure that allows them to interact with biomolecules and to

have avid reactivity with free radicals. These properties of

fullerenes have generated great interest in their use in

biomed-ical research (14, 15). It is necessary to convert hydrophobic

C60 into water-soluble derivatives before using it as

free-rad-ical scavenger or an antioxidant in medfree-rad-ical or therapeutic

ap-plications. Several strategies have been used to enhance its

water solubility and were reported to have protective effects in

various systems (4–6, 23, 25, 28). A newly synthesized

trima-lonic acid derivative of C60, C

[(COOH)

]

, is one of the

compounds that not only protected cultured cortical neurons

from excitotoxic injury in vitro but that also delayed the

neu-ronal deterioration in a transgenic model of familial

amyotro-phic lateral sclerosis (7, 9, 10, 17). In this study, the authors

have demonstrated for the first time that C60 has a protective

effect against bacterial meningitis. The C3 regioisomer of C60

can be used as late as 9 h postinjection. The dosage used in this

study (40 mg/kg of body weight three times by i.p. injection

every 24 h) was far below the toxic dose. The LD

s by i.p.

injection in rats and mice are approximately 600 and 1,000

mg/kg of body weight, respectively (3, 29).

The CNSs of mammals are considered to be

immunologi-cally privileged sites because of a lack of lymphatic drainage

and separation from the blood compartment by the BBB. The

BBB, by virtue of its selective permeability, plays an important

role in mediating the migration of inflammatory cells into the

brain microenvironment (1, 12, 20). Inflammatory cytokines

such as TNF-␣, IL-1␤, and IL-6 are known to be produced in

the cerebrospinal fluid during bacterial meningitis. Their

mu-tual stimulation might be responsible for the alteration of the

FIG. 1. C60 pretreatment inhibited E. coli-induced death in B6 mice. Groups of 10 B6 mice were inoculated intracerebrally with 5⫻ 105_{E. coli per mouse.} Various doses of C60 were administrated i.p. before E. coli injection. Dexameth-asone (6 mg/kg of body weight given i.p.) was used as a control treatment. The reagents were given again at 24 and 48 h. The mice were monitored for death every 12 h for 6 days.

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FIG. 2. C60 treatment inhibited the brain inflammation induced by E. coli in B6 mice. Groups of three B6 mice were inoculated intracerebrally with 5⫻ 105_E.

coli cells per mouse, and the mice were killed at 24 h postinjection. (C, G, and K) C60 (40 mg/kg per mouse) was administered i.p. before E. coli injection. (D, H, and L) C60 (40 mg/kg per mouse) was administrated i.p. 6 h after E. coli injection. The M4 tracer (2⫻ 108_{cells in 0.1 ml) was injected intravenously 2 min before the mice}

were killed. Four-micrometer cryosections of frozen brain tissues were stained with X-Gal (A to D) or anti-TNF␣ (E to H) and anti-IL-1␤ (I to L), as described in Materials and Methods. (A, E, and I) Mock control; (B, F, and J) E. coli; (C, G, and K); E. coli and C60 pretreatment; (D, H, and L) E. coli and C60 posttreatment. 3, M4 deposition; ➔, arteriole; ➜, infiltrating neutrophil. Magnifications,⫻400.

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BBB permeability (11, 22, 24, 26, 27, 31). The increase in BBB

permeability also precedes leukocyte migration. It was found

that neutralization of the proinflammatory cytokines inhibited

the increase in BBB permeability, as well as neutrophil

infil-tration (unpublished observation). The hydrophobic nature of

C60 and its ability to intercalate into biological membranes

were found to allow it to penetrate the BBB when it is

admin-istered intravenously (33). This unique property of C60 would

help to suppress local cytokine production and inhibit the BBB

opening, as well as brain inflammation.

The action mechanism of C60 is intriguing. C60 is a potent

free-radical scavenger. C60 at the range of 5 to 500

␮g/ml

tested in the present study did not inhibit the in vitro growth of

E. coli. Although the E. coli cells in the C60-treated mouse

brain were almost cleared at 24 h after injection, direct

inhi-FIG. 3. Therapeutic effect of C60 on E. coli-induced death in B6 mice. Groups of six B6 mice were inoculated intracerebrally with 5⫻ 105_{E. coli cells} per mouse. (A) C60 (40 mg/kg per mouse) was administered i.p. at various times after E. coli injection. (B) Dexamethasone (5 mg/kg of body weight given i.p.) was used as a control treatment. The reagents were given again at 24 and 48 h. The mice were monitored for death every 12 h for 6 days.

FIG. 4. Effect of C60 on the in vitro and in vivo growth of E. coli. (A) In vitro,

E. coli (4⫻ 106_{/ml cells) was cultured with various concentrations of C60 in LB} broth, and the growth curve was determined with a spectrophotometer. (B) In vivo, groups of three mice were given intracerebral injections of 5⫻ 105_{E. coli} cells. C60 (40 mg/kg per mouse) was administered i.p. before E. coli injection. At various times postinjection, the brains were aseptically removed and homoge-nized, and the numbers of CFU of E. coli were quantitated in an agar plate and are expressed as the mean⫾ standard deviation per mouse.

FIG. 5. C60 treatment induced TNF-␣ expression in the brain. Groups of three B6 mice were inoculated i.p. with C60 (40 mg/kg per mouse) and were killed at 24 h postinjection. Four-micrometer cryosections of frozen brain tissues were stained with anti-TNF-␣ as described in Materials and Methods. (A) Ar-teriole (magnification,⫻100); (B) ependymal cells (magnification, ⫻100); (C) ependymal cells (magnification,⫻400). ➔, arteriole; 7, ependymal cells.

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bition of E. coli growth at early times (12 h before) was not

found (Fig. 4B). When C60 was coinjected with E. coli

intra-cerebrally, C60 directly inhibited the local inflammation

(un-published observation). Since the BBB was open during the

development of meningitis (Fig. 2), it is possible that C60

would enter the brain via the bloodstream circulation after i.p.

administration. C60 was also found to intercalate into the brain

lipid membrane (7). This suggests that C60 may suppress

cy-tokine production, inhibit opening of the BBB, and interfere

with the neutrophil-mediated inflammatory reaction in the

brain. Furthermore, i.p. injection of C60 into naive mice

in-duced increased expression of TNF-␣ in brain arterioles and

ependymal cells (Fig. 5). It seems that C60 can also modulate

the natural antibacterial defense to clear the bacteria from the

brain. Since the proinflammatory cytokines induced in

menin-gitis interact in a complex cascade, no single intervention is

effective. Dexamethasone is used clinically, and it primarily

inhibits cytokine production. However, it can only delay or

partially inhibit the experimental E. coli-induced death (Fig.

3). C60 is more effective than dexamethasone, probably

be-cause of its multiple effects.

ACKNOWLEDGMENT

This study was supported by grant DOH88-HR-717 from the

Na-tional Health Research Institute of the Department of Health of the

Republic of China.

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