Elevation of plasminogen activators in cerebrospinal fluid of mice with

eosinophilic meningitis caused by Angiostrongylus cantonensis

Roger F. Houa, Wu-Chun Tua, Hsiu-Hsiung Leeb, Ke-Min Chenb,Hui-Lin Choub, Shih-Chan Laib,*

aDepartment of Entomology, National Chung-Hsing University, Taichung 402, Taiwan, ROCbDepartment of Parasitology, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung 402, Taiwan, ROC

Received 23 July 2004; received in revised form 25 August 2004; accepted 27 August 2004

Abstract

A hallmark of parasitic meningitis is the infiltration of eosinophils into the subarachnoid space. Infection with Angiostrongylus

cantonensis in mice induced proteinase activity in parallel with the pathological changes of eosinophilic meningitis. Zymogram analysis

demonstrated that 70 and 55 kDa proteinases from cerebrospinal fluid (CSF) were active against the casein/plasminogen substrate. The

proteinase activities were clearly inhibited by phenylmethanesulphonyl fluoride but not by ethylenediamine tetraacetic acid, 1,10-

phenanthroline or leupeptin. Western blotting confirmed these enzymes to be tissue-type plasminogen activator and urokinase-type

plasminogen activator, respectively. High activities of tissue-type plasminogen activator and urokinase-type plasminogen activator were

detected in the CSF of mice with eosinophilic meningitis, and correlated positively with CSF eosinophil numbers and total protein,

respectively. Immunohistochemistry demonstrated that tissue-type plasminogen activator and urokinase-type plasminogen activator

localised in the endothelial cells of blood vessels, in blood clots and in infiltrated leukocytes. These results suggest that tissue-type

plasminogen activator and urokinase-type plasminogen activator may be play a role in the pathogenesis of eosinophilic meningitis of

angiostrongyliasis.

q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Angiostrongylus cantonensis; Eosinophilic meningitis; Plasminogen activator; Proteinase; Blood–brain barrier

1. Introduction

Mature adults of the zoonotic parasitic nematode

Angiostrongylus cantonensis reside in the pulmonary

arteries of the permissive hosts (rats) (Alicata and Jindrak,

1970). However, in non-permissive hosts (humans and

mice), the immature adults remain in the central nervous

system (CNS) of the host, this infection being the main

cause of eosinophilic meningitis and eosinophilic menin-

goencephalitis (Hsu et al., 1990; Ismail and Arsura, 1993).

In mice infected with A. cantonensis, the cerebrospinal fluid

(CSF) eosinophilia reaches a peak at around 3 weeks and

parallels the pathogenesis of eosinophilic meningitis

0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by

doi:10.1016/j.ijpara.2004.08.010

* Corresponding author. Tel.: C886 4 2473 0022/1641; fax: C886 4 238

23381.

E-mail address: shih@csmu.edu.tw (S.-C. Lai).

(Sugaya and Yoshimura, 1988; Sasaki et al., 1993). The

blood–brain barrier (BBB) serves to protect the CNS from

invasive agents, such as inflammatory cells and bacteria, as

well as from chemical agents. Elevation of CSF total protein

indicates damage to the BBB (Fryden et al., 1978) and such

an elevation has been reported in angiostrongylosis (Yii,

1976; Wan and Weng, 2004).

Plasminogen activators (PAs) are serine proteases that

convert the zymogen, plasminogen, into the active serine

protease, plasmin. There are two types—tissue-type PA

(tPA) and urokinase-type PA (uPA) (Vassalli et al., 1991).

In normal plasma and in tissue, they are inactive and

complexed to plasminogen activator inhibitors, of which

type 1 plasminogen activator inhibitor (PAI-1) is believed to

be the most important (Vassalli et al., 1991; Loskutoff et al.,

1993; Blasi, 1997). It is well known that tPA plays a primary

role in the plasmin generation required for fibrinolysis,

International Journal for Parasitology 34 (2004) 1355–1364

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Elsevier Ltd. All rights reserved.

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–13641356

including clot or thrombus lysis. It also promotes BBB

disruption and is involved in the pathophysiology of

bacterial meningitis (Busch et al., 1997). uPA is primarily

involved in cell surface proteolysis and, thus, is important in

extracellular matrix (ECM) degradation and cell invasion

(Blasi et al., 1987). Additionally, the uPA system has the

capacity to promote leukocyte recruitment and BBB

breakdown, and thus may play an important pathophysio-

logical role in bacterial meningitis (Winkler et al., 2002).

The induction of PAs in bacterial meningitis is well

known. However, the relationship between PAs and

parasitic meningitis is still unknown. The current study,

therefore, set out to measure the activity of tPA and uPA in

the CSF in A. cantonensis-infected mice, and to investigate

the correlation between eosinophilic meningitis and PAs in

angiostrongylosis.

2. Materials and methods

2.1. Experimental animals

Five-week-old male mice, BALB/c strain, were pur-

chased from the National Laboratory Animal Center,

Taipei, Taiwan. Mice were maintained at a 12 h light/dark

cycle photoperiod, provided with Purina Laboratory Chow

and water ad libitum, and kept in our laboratory for more

than 1 week before the experimental infection.

2.2. Larval preparation

L3 (infective) larvae of A. cantonensis were obtained

from naturally infected giant African snails, Achatina fulica,

collected from fields in Pingtung County, southern Taiwan.

The larvae within tissues were recovered using the method

of Parsons and Grieve (1990) with slight modifications.

Briefly, the shells were crushed, the tissues were homogen-

ised and digested in a pepsin–HCl solution (pH 1–2,

500 I.U. pepsin/g tissue), and incubated with agitation at

37 8C in a waterbath for 2 h. Host cellular debris was

removed from the digest by centrifugation at 1400 g for

10 min. The larvae in the sediment were observed under the

microscope. The morphological criteria for identification of

the L3 of A. cantonensis 425–524 mm in length and from 23

to 34 mm in width. The posterior end of the tail always

terminates in a fine point were provided by Ash (1970). To

confirm that the larvae found were A. cantonensis, 60 L3

were fed to five rats and then examined their brains (two

rats) 2–3 weeks later for evidence of infection. The other

rats were killed 5–6 weeks later they were found to harbour

the adults in their pulmonary arteries. The morphology of

the adult worms was consistent with that described for

A. cantonensis. The males measured 14–15 mm in length,

the tail with copulatory bursa and long spicules; females

24–26 mm in length, with characteristic barber-pole appear-

ance (Lindo et al., 2002).

2.3. Animal infection

A total of 90 male mice were randomly allocated to six

groups (D0, D5, D10, D15, D20, and D25) of 15 mice each.

They were prohibited food and water for 12 h before

infection. The mice of experimental groups (D5, D10, D15,

D20, and D25) were infected with 60 A. cantonensis larvae

by oral inoculation on day 0 and the groups sacrificed on

days 5, 10, 15, 20, and 25 p.i., respectively. The control

mice (D0) received only water and sacrificed on day 25 p.i.

The mice were sacrified by cervical dislocation, and the

brains and CSF samples were rapidly collected and frozen at

K70 8C before use.

2.4. Casein/plasminogen zymography

The CSF was centrifugated at 12,000 g for 10 min to

remove debris. The protein contents of supernatants were

loaded on 7.5% (mass/volume) SDS-polyacrylamide gels

that had been co-polymerised with 0.1% casein (Sigma,

USA) for plasmin activities, and plasminogen (13 mg/ml,

American Diagnostica) for PAs activities. Stacking gels

were 4% (mass/volume) polyacrylamide and did not contain

casein and plasminogen substrate. Electrophoresis was

performed in running buffer (25 mM Tris, 250 mM glycine,

1% SDS) at room temperature at 120 V for 1 h. The gel was

washed two times at room temperature for 30 min each in

2.5% Triton X-100, and then washed two times with double

distilled H2O for 10 min each. The gel was incubated in

reaction buffer (50 mM Tris–HCl, pH 8.0, containing

10 mM CaCl2, 0.01% NaN3) at 37 8C for 18 h. The gel

was stained with 0.25% Coomassie Brilliant Blue R-250

(Sigma, USA) for 1 h and destained in 15% methanol/7.5%

acetic acid. PAs activities were detected as unstained bands

on a blue background. Quantitative analysis of these

caseinolytic enzymes were performed with a computer-

assisted imaging densitometer system, UN-SCAN-ITe gel

Version 5.1 (Silk Scientific, USA).

2.5. Inhibition of proteinases on casein zymography

To explore the effects of various potential inhibitors on

the caseinolytic activities in the CSF samples, the samples

were run on SDS-polyacrylamide gels as described above.

Following electrophoresis, gels were soaked in 2.5% Triton-

X-100 to replace SDS, washed twice with water, then

incubated at 37 8C for 18 h in activation buffer (50 mM Tris,

pH 8.0, 10 mM CaCl2). For inhibitor studies, 10 mM

ethylenediamine tetraacetic acid (EDTA; Sigma, USA),

20 mM leupeptin (Sigma, USA), or 2 mM phenylmethane-

sulphonyl fluoride (PMSF; Sigma, USA), or 5 mM 1,10-

phenanthroline (Sigma, USA), was added to the Triton and

activation buffers. Zymography gels were stained with

Coomassie Brilliant Blue and destained in 15% metha-

nol/7.5% acetic acid. Proteins with casein activity were

revealed as clear bands on a blue background.

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–1364 1357

2.6. Western blot analysis

The CSF was centrifugated at 12,000 g for 10 min to

remove debris. The protein contents of supernatants were

determined with protein assay kits (Bio-Rad, USA) using

bovine serum albumin (BSA) as the standard. An equal

volume of loading buffer (62.5 mM Tris–HCl, pH 6.8, 10%

glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromo-

phenol blue) was added to the samples, which contained

30 mg of brain tissue protein. The mixture was boiled for

5 min prior to electrophoresis on SDS-polyacrylamide gel

and electrotransferred to nitrocellulose membrane at a

constant current of 190 mA for 90 min. Afterwards, the

membrane was saturated with phosphate buffered saline

(PBS) containing 0.1% Tween 20 for 30 min at room

temperature. The membrane was allowed to react with

rabbit anti-mouse tPA and uPA polyclonal antibodies

(American Diagnostica, USA) diluted 1:100 at 37 8C for

1 h. Then, the membrane was washed three times with PBS

containing 0.1% Tween 20 (PBS-T), followed by incubation

with horseradish peroxidase (HRP)-conjugated goat anti-

rabbit IgG (Jackson ImmunoResearch Laboratories, USA)

diluted 1:5000 at 37 8C for 1 h to detect the bound primary

antibody. The reactive protein was detected by enhanced

chemiluminescence (Amersham, UK). To confirm equival-

ent protein loading, membranes were stripped by incubation

in 62.5 mM of Tris–HCl (pH 6.8), 2% SDS, and 100 mM 2-

mercaptoethanol at 55 8C, subsequently washed with PBS-

T, and reprobed with anti-b-actin antibody (dilution 1:500;

Sigma, USA).

2.7. Reverse transcriptase polymerase chain reaction

(RT-PCR) analysis

The CSF was centrifugated at 12,000 g for 10 min and

the liquid removed. Total RNA was isolated from the cell

pellets using Trizol reagent (Invitrogen, USA), according to

the manufacturer’s instructions. One microgram of total

RNA was used for first strand cDNA synthesis in 20 ml of

reaction volume using 50 units of Superscripte II reverse

transcriptase (Invitrogen, USA). PCR was performed under

standard conditions using Taq DNA polymerase (Invitro-

gen, USA) and primers. Forward (5 0–3 0) and reverse (5 0–3 0)

primers, respectively, were 5 0-GACATCAAGAAGGT-

GGTGAAGC-3 0 and 5 0-TGTCATTGAGAGCAATGC-

CAGC-3 0 for glyceraldehyde-3-phosphate dehydrogenase

(GAPDH), GGGAGGTTCAGAAGAGGAGCCCGG-3 0

and 5 0-GCGTTTCCCTACAAATCCATCAGGG-3 0 for

tPA (de Vries et al., 1995), 5 0-TGCCCAAGGAAATTC-

TGCCCAAGGAAATTCCAGGG-3 0 and 5 0-GCCAAT-

CTGCACATAGCACC-3 0 for uPA (de Vries et al., 1995),

5 0-CACAAGTCTGATGGCAGCAC-3 0 and 5 0-CAGG-

CATGCCCAACTTCTC-3 0 for PAI-1 (Yamamoto and

Loskutoff, 1996). PCR cycling conditions for GAPDH,

tPA, uPA and PAI-1 were denaturation at 94 8C for 45 s,

annealing at 55 8C for 1 min, primer extension at 72 8C for

2 min, and then holding at 4 8C; this was repeated for 30

cycles for tPA, uPA and PAI-1; 25 cycles for GAPDH. Ten

microlitre of the amplified product were then subjected to

electrophoresis in 1% agarose gels containing 20 mg/ml

ethidium bromide in Tris borate-EDTA buffer. Gels

were visualised on a UV transilluminator (Taiwan), and

digital images were taken using DGIS-5 Digital Gel

Image System (Taiwan). Quantitative analysis was per-

formed with a computer-assisted imaging densitometer

system, UN-SCAN-ITe gel Version 5.1 (Silk Scientific,

USA).

2.8. Cell counts in the CSF

The mice were sacrificed and their brains removed into a

35 mm dish. The cranial cavity and cerebral ventricles

(lateral, third and fourth ventricles) were rinsed with 1 ml

PBS each. The washing solution was collected into a

centrifuge to spin at 400g for 10 min. The resultant

sediments were then resuspended with 30 ml PBS from

each mouse for enumerating a total number of leukocytes on

hemacytometer. The differential cell count was assessed

with Wright–Giemsa staining (Sigma, Taufkirchen,

Germany) in 3 ml/smear. The percentages of eosinophils

were determined in 200 leukocytes/smear.

2.9. The measurement of CSF total protein

The CSF was centrifuged at 12,000 g at 4 8C for 10 min,

and the protein contents of the supernatants were deter-

mined with protein assay kits (Bio-Rad, USA) using BSA as

the standard. Protein concentration was determined by

absorbencies at 595 nm using a HITACHI U1100 spectro-

photometer (Japan).

2.10. Histology

The mouse brains were fixed separately in 10% neutral

buffered formalin for 24 h. The fixed specimens were

dehydrated in a graded ethanol series (50, 75, and 100%)

and xylene, then embedded in paraffin at 55 8C for 24 h.

Several serial sections were cut at a 5 mm thickness for each

organ from each mouse. Sections were deparaffinised,

stained with H&E using standard techniques and examined

under a light microscope.

2.11. Scanning electron microscopy

The mouse brains were fixed in 2.5% glutaraldehyde

(Electron Microscopy Science, USA) in 0.15 M PBS buffer,

pH 7.4, for 3 h at 4 8C, and post-fixed in 1% osmium

tetroxide (Electron Microscopy Science, USA) in the same

buffer for 1 h at 4 8C. The fixed specimens were dehydrated

in a graded ethanol series (30–100%) and dried in LADD

28000 critical point dryer (USA). The dried specimens were

mounted on stubs, coated with 20 nm gold in JBS E5150

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–13641358

sputter coater (UK), and photographed with a TOPCON

ABT-150S scanning electron microscope (Japan).

2.12. Transmission electron microscopy

The mouse brains were fixed in 2.5% glutaraldehyde

(Electron Microscopy Science, USA) in 0.15 M PBS buffer,

pH 7.4, for 3 h at 4 8C, and post-fixed in 1% osmium

tetroxide (Electron Microscopy Science, USA) in the same

buffer for 1 h at 4 8C. The fixed specimens were dehydrated

in a graded ethanol series (30–100%), and embedded in LR

White resin (Spi Supplies, USA) for 24 h at 54 8C. Ultrathin

sections were cut with an ultramicrotome (Reichert Ultracut

S, Austria) and were doubly stained in 2% uranyl acetate

(Merck, Germany) for 30 min and 1% lead citrate (Merck,

Germany) for 12 min. The sections were examined and

photographed using a 100 kV electron microscope (JOEL

1200 EX II, Japan).

2.13. Immunohistochemistry

The mouse brains were fixed separately in 10% neutral

buffered formalin for 24 h. The fixed specimens were

dehydrated in a graded ethanol series (50, 75, and 100%)

and xylene, then embedded in paraffin at 55 8C for 24 h. Ten

micrograms of paraffin-embedded sections were prepared

and mounted on glass slides. Serial sections were depar-

affinised with xylene and a graded series of ethanol.

Sections were treated with 3% H2O2 in methanol for

10 min to inativate endogenous peroxidase, and washed

three times with PBS, pH 7.4 for 5 min. Sections were

blocked non-specific reactions with 3% BSA at room

temperature for 1 h, incubated with primary antibodies

(rabbit anti-mouse tPA and uPA polyclonal antibodies;

American Diagnostica, USA) diluted 1:50 in 1% BSA at

37 8C for 1 h, and washed three times in PBS for 5 min each.

Sections were incubated with HRP-conjugated goat anti-

rabbit IgG (Jackson ImmunoResearch Laboratories, USA)

diluted 1:100 in 1% BSA at 37 8C for 1 h, and washed three

times in PBS for 5 min each. Sections were incubated in

DAB (3,3 0-diaminobenzidine; 0.3 mg/ml in 100 mM Tris

pH 7.5 containing 0.3 ml H2O2/ml) at room temperature for

3 min, and washed three times in PBS for 5 min each.

Mounted slides with 50% glycerol in PBS were examined

under a light microscope.

Fig. 1. Time-course studies for caseinolytic activity from CSF. (a) The

molecular mass 70 kDa bands were detected at all time points, and the

intensity increased gradually from days 5 to 25 p.i. The 55 kDa bands were

detected on day 10 p.i. and reached a high intensity from days 15 to 25 p.i.

but were undetectable in the uninfected control. (b) Quantitative analysis of

the proteolytic enzyme was performed with a computer-assisted imaging

densitometer system. The relative intensity of the bands in Angiostrongylus

cantonensis-infected mice showed significant increase (*P!0.05) com-

pared with uninfected control.

2.14. Statistical analysis

Results in the different groups of mice were compared

using the non-parametric Kruskal–Wallis test followed by

post-testing using Dunn’s multiple comparison of means.

Correlations between CSF laboratory parameters and PAs

were quantified using the Spearman’s ranking correlation

test. The best fitting regression curve was drawn using

Microsoftw Excel 2000 analysis software. All results were

presented as meanGstandard deviation (SD). P values of

!0.05 were considered statistically significant.

3. Results

3.1. Time-course studies for caseinolytic activity from CSF

Bands corresponding to 70 kDa were detected at all time

points tested, including in uninfected mice, and the intensity

increased gradually from days 5 to 25 p.i. 55 kDa bands

were detected on day 10 p.i. and reached a high intensity

from days 15 to 25 p.i. An increased activity of PAs was

observed in mice with meningitis (Fig. 1a). The relative

activity of PAs in A. cantonensis-infected mice showed a

significant increase (P!0.05) compared with uninfected

controls (Fig. 1b).

3.2. Identification of the proteinases

Casein/plasminogen zymography on day 20 p.i. showed

70 and 55 kDa proteinases present in mice infected with

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–1364 1359

A. cantonensis, whereas uninfected mice showed only a low

activity of tPA, while uPA was undetectable. The activity of

proteinases was significantly inhibited by PMSF, but not by

EDTA, 1,10-phenanthroline or leupeptin (Fig. 2a and b).

Western blot analysis with polyclonal antibodies of tPA

Fig. 2. Identification of the proteinases. (a) Casein zymography presented

70 and 55 kDa proteinases bands in Angiostrongylus cantonensis-infected

mice on day 20 p.i. whereas the uninfected control were low intensity (at

70 kDa) or undetectable (at 55 kDa). Inhibition of proteinases with

phenylmethanesulphonyl fluoride, EDTA, 1,10-phenanthroline and leu-

peptin on casein zymography. (b) Quantitative analysis of the 70 and

55 kDa bands were performed with a computer-assisted imaging densi-

tometer system. The proteinase was clearly inhibited (*P!0.05) by

phenylmethanesulphonyl fluoride, but not affected by EDTA, 1,10-

phenanthroline and leupeptin. (c) Western blot analysis from uninfected

control and mice infected with A. cantonensis on day 20 p.i. The molecular

weight of 70 and 55 kDa proteinase bands were detected with polyclonal

antiserum against tissue-type plasminogen activator and urokinase-type

plasminogen activator in CSF, respectively. b-actin was used as a loading

control.

and uPA confirmed that the 70 and 55 kDa proteinases were

tPA and uPA, respectively (Fig. 2c).

3.3. The mRNA expression of tPA, uPA, and PAI-1

in the CSF

Total RNA isolated from CSF cells was assayed for RT-

PCR analysis by using tPA, uPA, PAI-1 and GAPDH-

specific primers. The mRNA of tPA was expressed at all

time points and showed upregulation from days 10 to 25 p.i.

The uPA mRNA was detected on days 10 to 25 p.i. but not

detected in uninfected controls. Similarly, PAI-1 mRNA

was found to be higher on days 15, 20, and 25 p.i. (Fig. 3a).

The tPA/PAI-1 and uPA/PAI-1 ratios showed a statistically

Fig. 3. The mRNA levels of tPA, uPA, and PAI-1 in the CSF. (a) The

mRNA expression of tPA was upregulated at all time points, and showing a

high expression from days 10 to 25 p.i. The uPA mRNA was detected from

days 10 to 25 p.i. and undetectable in uninfected control. PAI-1 mRNA was

showed a higher expression from days 15 to 25 p.i. glyceraldehyde-3-

phosphate dehydrogenase mRNA was used as a loading control.

(b) Densitometric scanning quantification of six mice expressed as the

ratio of the signal intensity of PAs to that of PAI-1 at each time point. Ratios

of tPA/PAI-1 and uPA/PAI-1 showed statistically significant elevation

(*P!0.05) on days 10, 15, 20, and 25 p.i.

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–13641360

significant elevation (P!0.05) on days 10, 15, 20, and 25

p.i. (Fig. 3b).

3.4. Correlation of CSF eosinophilia with tPA and uPA

Only infected mice showed CSF pleocytosis. The

leukocytes were identified as eosinophils cells by

Wright–Giemsa staining. The time-course studies showed

a mild eosinophilia on day 10 p.i. and a plateau response

from days 15 to 25 p.i. Using Spearman’s ranking

correlation test, the CSF eosinophilia showed a significant

correlation (P!0.05) with the activity of tPA (rZ0.93)

(Fig. 4a), and uPA (rZ0.91) (Fig. 4b). The relation was

best fitted using a regression curve, and the elative

intensity of PAs reached a plateau at 15–37% eosinophils

in CSF.

Fig. 4. Correlation of CSF eosinophil with tPA and uPA. The percentages of

CSF eosinophil significant correlated (*P!0.05) with the intensity of tPA

(a), and uPA (b) using the Spearman’s ranking correlation test.

3.5. Correlation of CSF total protein with tPA and uPA

The appearance of plasma proteins in CSF is a hallmark

of numerous CNS disorders with presumed or overt BBB

disruption. In this experimental eosinophilic meningitis of

angiostrongyliasis, CSF total protein significantly correlated

(P!0.05) with the activity of tPA (rZ0.81) (Fig. 5a), and

uPA (rZ0.82) (Fig. 5b) by Spearman’s ranking correlation

test. The relation was best fitted using a regression curve,

and the elative intensity of PAs reached a plateau at the total

protein concentration of 0.75–1.7 mg/ml.

3.6. Histopathological observations in

the subarachnoid space

In brain sections stained with haematoxylin and

eosin, uninfected mice had no inflammatory cells in

Fig. 5. Correlation of CSF total protein with tPA and uPA. The CSF total

protein significant correlated (*P!0.05) with the intensity of tPA (a), and

uPA (b) using the Spearman’s ranking correlation test.

Fig. 6. Histopathological observations in the subarachnoid space. (a) H&E stain showing severe inflammatory reaction (arrowheads) and hemorrhage (arrow).

C, cortex. (b) Enlargement of the portion shown in rectangle of (a). Inflammatory reaction consisting of polymorphonuclear (arrowhead) and mononuclear

(arrow) leukocytes. H, hemorrhage. (c) SEM showing the inflammatory cells (arrowheads) accumulate on the brain surface. (d) Red blood cells (arrowheads)

and inflammatory cells (arrows) aggregated on the brain surface by SEM. (e) Ultrastructural observations showing red blood cells (R) and lymphocytes (L)

aggregated in the subarachnoid space. E, endothelial cell of meninge. (f) TEM showing eosinophils with a bi-lobed nucleus (N) containing condensed

chromatin and the cytoplasm packed with many large, membrane-enclosed, dense crystalloid-containing ovoid granules (arrowheads).

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–1364 1361

the subarachnoid space and the meninges were normal (data

not shown). A gradual increase in pathological effects after

infection culminated in a severe infiltration of leukocytes,

edema and hemorrhage from days 15 to 25 p.i. Inflamma-

tory reaction consisting of polymorphonuclear and mono-

nuclear leukocytes were observed in the brain tissue on day

20 p.i. (Fig. 6a and b). Scanning electron micrographs

showed red blood cells and inflammatory cells accumulated

on the brain surface (Fig. 6c and d). Ultrastructural

observations showed red blood cells and eosinophils

aggregated on the subarachnoid space. The eosinophils

showed many crystalloid-containing secretory granules in

the cytoplasm (Fig. 6e and f).

3.7. Distribution of tPA and uPA in the subarachnoid space

Positive signals for tPA (Fig. 7a and b) and for uPA

(Fig. 7c and d) were localised in the endothelial cells of

blood vessels, in blood clot and in infiltrated polymorpho-

nuclear and mononuclear cells. No positive signal was

detected in these structures in uninfected mice (Fig. 7e).

4. Discussion

The activities of serine proteinases increased is associ-

ation with the inflammatory disease (Tarlton et al., 2000)

Fig. 7. Immunohistochemical distribution of tPA and uPA in the subarachnoid space. (a) tPA localised in endothelial cells (arrowhead) of blood vessel, in blood

clot (C) and in infiltrated leukocytes (arrows), and presented brown colour. (b) Enlargement of the portion shown in rectangle of (a). Polymorphonuclear

(arrowheads) and mononuclear (arrow) cells presented positive signal for tPA. (c) uPA localised brown colour in blood clot (C) and infiltrated leukocytes

(arrowheads). (d) The endothelial cells (arrowhead) and in infiltrated leukocytes (arrow) contained a positive signal for uPA. (e) No positive signal (brown

colour) could be detected with normal serum in the blood clot (C) nor in infiltrating leukocytes (arrowheads).

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–13641362

and meningitis (Winkler et al., 2002). Meningitis may be

caused by viruses or bacteria and less often by other

pathogens, such as rickettsia, fungi, and parasites (Zhang

and Tuomanen, 1999; Casadevall and Pirofski, 2000).

Angiostrongylosis of the meninges is a chronic meningitis

characterised by the aggregation of eosinophils in

the subarachnoid space (Reid and Wallis, 1984). The

distinct expression profiles found in the present study

indicate a role for uPA and tPA in the pathogenesis of

parasitic meningitis. The role of exaggerated extracellular

R.F. Hou et al. / International Journal for Parasitology 34 (2004) 1355–1364 1363

proteolysis in the CNS caused by PAs was further

strengthened by the high correlation between PAs and

eosinophil counts in the CSF.

Proteolysis becomes pathological when an imbalance

between proteinases and their inhibitors occurs. PAI-1,

which binds to and inactivates both tPA and uPA, is the

primary regulator of plasminogen activation in vivo

(Vassalli et al., 1991). Fibrinolysis and coagulation in

patients with infectious disease and sepsis showed that the

uPA level is markedly increased, but concomitant marked

PAI-1 upregulation (Philippe et al., 1991; Robbie et al.,

2000). Similarly, the present study showed that an

imbalance between tPA and PAI-1 and between uPA and

PAI-1 may be associated with eosinophilic pleocytosis in

the subarachnoid space in angiostrongylosis. Additionally,

the mRNA expression of the PAs coincided with proteolytic

activity, suggesting that increased PAs activity may be

transcriptionally regulated.

The breakdown of BBB is regarded as an important

pathophysiological event in bacterial meningitis. It causes

extravasation of different neurotoxic factors and results in

brain edema with consequent increased intracranial pressure

(Leib and Tauber, 1999). A possible role for uPA in BBB

breakdown was also found in a mouse model of brain

trauma, which showed that uPA deficiency resulted in

decreased extravasation of proteins into the CNS (Kataoka

et al., 2000). In the present study, the time of increase in PAs

corresponds with the time of CSF eosinophilia and total

protein. Increased activities of PAs might threaten the

integrity of BBB and may thereby lead to BBB damage, and

increased influx of inflammatory cells into subarachnoid

space. Therefore, it is plausible to assume that increased PA

activity may promote eosinophilic meningitis by disruption

of the BBB.

tPA, uPA, and PAI-1 have been implicated in fibrin

formation or removal and each are regulated during

inflammatory/thrombotic events. tPA is synthesised by

endothelial cells in normal blood vessels (Kristensen

et al., 1984), and functions in physiological thrombolysis

in vivo (Collen and Lijnen, 1991). Studies in uPA knockout

mice indicated that uPA is also involved in fibrinolysis

(Carmeliet et al., 1994). Additionally, elevations in PAI-1

activity have been demonstrated in a number of clinical

conditions associated with a predisposition to thrombosis

(Tabernero et al., 1989). The present study showed that tPA

and uPA localise in the endothelial cells of blood vessels

and blood clot. These data suggest that the imbalance

between PAs and PAI-1 in angiostrongyliasis may facilitate

cellular infiltration into the subarachnoid space and

thrombolysis.

Acknowledgements

We wish to thank Y.S. Lin and P.C. Chao, the

Instrumentation Center, National Chung Hsing University,

for technical assistance in electron microscopy. This study

was supported by a research grant NO. NSC 92-2314-B-

040-027 from the National Science Council, ROC.

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