Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis

International Journal for Parasitology 34 (2004) 1355–1364 www.parasitology-online.com

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,* a

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

b

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, 1,10phenanthroline 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 meningoencephalitis (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 * Corresponding author. Tel.: C886 4 2473 0022/1641; fax: C886 4 238 23381. E-mail address: [email protected] (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,

0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.08.010

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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 pathophysiological 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 purchased 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 homogenised 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 appearance (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 computerassisted 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% TritonX-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 phenylmethanesulphonyl fluoride (PMSF; Sigma, USA), or 5 mM 1,10phenanthroline (Sigma, USA), was added to the Triton and activation buffers. Zymography gels were stained with Coomassie Brilliant Blue and destained in 15% methanol/7.5% acetic acid. Proteins with casein activity were revealed as clear bands on a blue background.

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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% bromophenol 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 antirabbit 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 equivalent protein loading, membranes were stripped by incubation in 62.5 mM of Tris–HCl (pH 6.8), 2% SDS, and 100 mM 2mercaptoethanol at 55 8C, subsequently washed with PBST, 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 (Invitrogen, USA) and primers. Forward (5 0 –3 0 ) and reverse (5 0 –3 0 ) primers, respectively, were 5 0 -GACATCAAGAAGGTGGTGAAGC-3 0 and 5 0 -TGTCATTGAGAGCAATGCCAGC-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 -TGCCCAAGGAAATTCTGCCCAAGGAAATTCCAGGG-3 0 and 5 0 -GCCAATCTGCACATAGCACC-3 0 for uPA (de Vries et al., 1995), 5 0 -CACAAGTCTGATGGCAGCAC-3 0 and 5 0 -CAGGCATGCCCAACTTCTC-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

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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 performed 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 determined 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 spectrophotometer (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

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sputter coater (UK), and photographed with a TOPCON ABT-150S scanning electron microscope (Japan).

presented as meanGstandard deviation (SD). P values of !0.05 were considered statistically significant.

2.12. Transmission electron microscopy

3. Results

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

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

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 deparaffinised 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 antirabbit 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. 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

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) compared with uninfected control.

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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

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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 RTPCR analysis by using tPA, uPA, PAI-1 and GAPDHspecific 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. 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 leupeptin on casein zymography. (b) Quantitative analysis of the 70 and 55 kDa bands were performed with a computer-assisted imaging densitometer system. The proteinase was clearly inhibited (*P!0.05) by phenylmethanesulphonyl fluoride, but not affected by EDTA, 1,10phenanthroline 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.

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-3phosphate 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.

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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.

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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).

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. Inflammatory reaction consisting of polymorphonuclear and mononuclear 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 polymorphonuclear 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 association with the inflammatory disease (Tarlton et al., 2000)

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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).

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

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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,

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for technical assistance in electron microscopy. This study was supported by a research grant NO. NSC 92-2314-B040-027 from the National Science Council, ROC.

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