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c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis
International Journal for Parasitology 41 (2011) 1175–1183
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Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis P.C. Wei a, C.H. Tsai a, P.S. Chiu a, S.C. Lai b,c,⇑ a
Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan Department of Parasitology, Chung Shan Medical University, Taichung 402, Taiwan c Clinical Laboratory, Chung Shan University Hospital, Taichung 402, Taiwan b
a r t i c l e
i n f o
Article history: Received 26 May 2011 Received in revised form 7 July 2011 Accepted 8 July 2011 Available online 6 August 2011 Keywords: Angiostrongylus cantonensis Eosinophilic meningitis Matrix metalloproteinase-12 Elastin
a b s t r a c t The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood–CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Angiostrongyliasis is a parasitic disease caused by the rat nematode lungworm Angiostrongylus cantonensis, which undergoes obligatory intracerebral migration in its hosts (Mackerras and Sandars, 1954). Worms develop to the young adult stage in the meninges, and the inflammatory reaction that is induced by the worms causes severe CNS infection, especially eosinophilic meningitis (Hsu et al., 1990). Diagnosis is based on epidemiological criteria, clinical manifestations, elevated eosinophil count in the CSF and serological tests. Treatment is symptomatic and supportive (Bourée et al., 2010). Matrix metalloproteinases (MMPs) are structurally related extracellular matrix (ECM)-degrading enzymes that are collectively capable of degrading essentially all ECM components (Shapiro, 1998). The integrity of cerebral microvessels is maintained in part by components of the basal lamina (such as collagen IV and elastin). Destruction of basal lamina components by MMPs, especially MMP-9, has been known to occur during subarachnoid haemorrhage (Sehba et al., 2004). MMP-12 is an elastin-degrading proteinase that is associated with vascular injury (Vacek et al., 2010). Elastin is an MMP-12 substrate and has been shown to account for up 4% of the ECM of brain microvessels (Faris et al., 1982; Curci ⇑ Corresponding author at: Department of Parasitology, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung 402, Taiwan. Tel.: +886 4 24730022x1641; fax: +886 4 23823381. E-mail address: [email protected] (S.C. Lai).
et al., 1998). This protein contains two cysteine residues that might be important in the interaction of elastin with other ECM proteins (Rosenbloom, 1984). Arterioles and venules have three layers: an inner endothelium that acts as a membrane; a middle layer of muscle and elastic tissue; and an outer layer of fibrous connective tissue (Bell and Weddell, 1984). Activated T cells can enter the subarachnoid space by extravasation through the cell wall of the meningeal venules, which consists of endothelial cells connected by tight junctions (Goverman, 2009). Elastin is an important factor in the development of vascular wall alterations (Nicoloff et al., 2000). Elastin-derived peptide levels in CSF of patients with ischaemic (Nicoloff et al., 2008) and lacunar (Tzvetanov et al., 2008) stroke are strongly elevated compared with healthy subjects. Therefore, we presume that the raised levels of elastin in CSF might result from vasculopathy. To our knowledge, the presence or absence of MMP-12 and its substrate elastin in mice with angiostrongyliasis meningitis is unclear. The aims of the present study were therefore to investigate whether MMP-12 and elastin are altered in mice with eosinophilic meningitis caused by A. cantonensis.
2. Materials and methods 2.1. Experimental animals Male BALB/c mice (specific-pathogen-free grade and 5 weeks old) were purchased from the National Laboratory Animal Center,
0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.07.002
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Taipei, Taiwan. Mice were maintained in a 12 h alternating light and dark cycle photoperiod, were provided with Purina Laboratory Chow and water ad libitum, and were kept in our laboratory more than 1 week before experimental infection. All procedures that involved animal use and care were approved by the Institutional Animal Care and Use Committee of Chung-Shan Medical University (IACUC Approval No: 431), Taiwan, in accordance with the institutional guidelines for animal experiments.
were sacrificed on day 25 p.i. Brains were rapidly removed and frozen at 80 °C. 2.6. Collection of CSF-like fluid The mice were sacrificed and their brains removed into a 35mm dish. The cranial cavity and cerebral ventricles (lateral, third and fourth ventricles) were rinsed with 1 mL PBS and CSF was thus harvested with PBS; the washing solution being CSF-like fluid.
2.2. Antibodies 2.7. Western blot analysis Rabbit anti-mouse MMP-12 polyclonal antibody was purchased from Abcam (Cambridge, UK), goat anti-mouse tissue inhibitor of metalloproteinase (TIMP)-1 polyclonal antibody was purchased from R&D Systems (Minneapolis, MN, USA), goat anti-mouse elastin polyclonal antibody was purchased from Santa Cruz Biotechnology (CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-goat IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). 2.3. Drugs
Mouse CSF was centrifuged at 12,000g at 4 °C for 10 min and supernatant protein was determined with protein assay kits (BioRad, USA) using BSA as the standard. An equal volume of loading buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2mercaptoethanol and 0.05% bromophenol blue) was added to the samples. The mixture was boiled for 5 min before electrophoresis on SDS–PAGE and electrotransferred to nitrocellulose membranes at a constant current of 190 mA for 90 min. Membranes were saturated with PBS that contained 0.1% Tween 20 (PBS–T) for 30 min at room temperature. Membranes were reacted with primary antibodies (rabbit anti-mouse MMP-12 polyclonal antibody, goat antimouse TIMP-1 polyclonal antibody or goat anti-mouse elastin polyclonal antibody) at a 1:500 dilution at 37 °C for 1 h. Membranes were washed three times with PBS that contained PBS–T, followed by HRP-conjugated secondary antibody (1:5,000 dilution) incubation at 37 °C for 1 h to detect primary bound antibody. Reactive proteins were detected by enhanced chemiluminescence (Amersham, Little Chalfont, Bucks, UK), and the densities of the specific immunoreactive bands were quantified with densitometric scanning analysis.
Albendazole (ZentelÒ, GlaxoSmith Kline, NC, USA) is a benzimidazole derivative. It is widely used in veterinary medicine and has been found to be a safe broad spectrum anthelmintic in animals and humans (Hwang and Chen, 1988; Chen and Lai, 2007). Albendazole acts by binding to b-tubulin in the parasite, inhibiting its polymerisation and impairing glucose uptake, which causes death (Venkatesan, 1998). Doxycycline was purchased from Sigma (St. Louis, MO, USA) and dissolved in PBS, pH 7.4. It is a lipophilic, second-generation tetracycline derivative that has anti-inflammatory actions that are independent of its antimicrobial actions (Yrjänheikki et al., 1998). It crosses the blood–brain, blood–CSF and placental barriers, and thus has excellent brain and body tissue penetration. Doxycycline could prove to be a most attractive pharmacological therapy for cerebral ischaemia due to its ability to protect the brain against pathological apoptotic neuronal cell death and neuroinflammation secondary to brain injury (Jantzie et al., 2005). Doxycycline is a water-soluble, non-specific competitive inhibitor of MMPs. It inhibits MMPs via modulation of plasminogen activators in focal cerebral ischaemia (Burggraf et al., 2007).
The CSF was collected into a centrifuge to spin at 400g for 10 min. The resultant sediments were then gently mixed with 100 lL of Unopette buffer (Vacutainer System; Becton Dickinson, Franklin Lakes, NJ, USA) and 2 lL of acetic acid, and placed in a haemocytometer cell counting chamber (Paul Marienfeld, Lauda-Koenigshofen, Germany) to count eosinophils.
2.4. Larval preparation
2.9. Histology
The infective larvae (L3) of A. cantonensis were originally obtained from wild giant African snails (Achatina fulica) and were propagated for several months in the Wufeng experimental farm (Taichung, Taiwan) by cycling through rats and A. fulica. Tissue larvae were recovered with a modified Parsons and Grieve method (1990). Briefly, snail shells were crushed and the tissues were homogenised, digested in a pepsin–HCl solution (pH 1–2, 500 IU pepsin/g tissue), and incubated with agitation in a 37 °C water bath for 2 h. Host cellular debris was removed from the digest by centrifugation at 1,400g for 10 min. The larvae in the sediment were collected by serial washes in double-distilled water and counted under a microscope. The third stage of larvae of A. cantonensis was confirmed as described by Hou et al. (2004).
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, and embedded in paraffin at 55 °C for 24 h. Several serial sections were cut at 5-lm thickness for each organ from each mouse. Sections were deparaffinised, stained with H&E (Muto, Japan) and pathological changes were examined under a light microscope.
2.5. Animal infection A total of 120 male BALB/c mice were randomly allocated to six groups (control, D5, D10, D15, D20 and D25) of 20 mice each. Food and water were withheld for 12 h before infection. The mice in five experimental groups (D5, D10, D15, D20 and D25) were infected with 50 A. cantonensis larvae by oral inoculation, and were sacrificed on days 5, 10, 15, 20 or 25 p.i. Control mice received only water and
2.8. Eosinophil counts in the CSF
2.10. Immunohistochemistry Mouse brains were prepared as described in Section 2.10 and the histological methods have been described previously (Chen et al., 2004). Relatively thick (10 lm) serial sections of waxembedded brains were cut, mounted on glass slides, dewaxed and rehydrated for immunohistochemistry. Sections were blocked with 3% BSA at room temperature for 1 h before incubation with a 1:100 dilution of rabbit anti-mouse MMP-12 polyclonal antibody in 1% BSA at 37 °C for 1 h. Sections were washed three times in PBS, incubated with the HRP-conjugated secondary antibodies diluted 1:200 in 1% BSA at 37 °C for 1 h, and washed again in PBS. Sections were incubated for 3 min at room temperature with 3,30 -diaminobenzidine (0.3 mg/mL) in 100 mM Tris (pH 7.5) that
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contained 0.3 lL H2O2/mL. Sections were mounted in 50% glycerol in PBS after final washes and examined under a light microscope. 2.11. Co-immunoprecipitation
were rinsed with 1 mL of PBS and CSF was thus harvested with PBS. The average concentration of Evans blue in the CSF was then measured at 620 nm by a spectrophotometer (U3000; Hitachi, Tokyo, Japan).
To prevent non-specific adsorption, protein A/G agarose beads (Santa Cruz Biotechnology) were washed five times in PBS and incubated with 5% BSA at 4 °C for 30 min. The solution was centrifuged twice at 10,000g for 2 min to remove supernatant before use. MMP-12 antibody was incubated with brain lysates (1 mg) at 4 °C overnight and collected by binding to protein A/G agarose beads. The beads were washed twice in dissociation buffer (0.5 M Tris– HCl, pH 8.0, 120 mM NaCl, 0.5% Triton X-100). Bound proteins were resolved by SDS–PAGE and target protein (elastin) association was determined by blotting.
2.14. Statistical analysis
2.12. Treatment of animals
CSF protein levels of MMP-12 and TIMP-1 were analysed by western blotting. The MMP-12 bands were significantly increased (P < 0.05) on days 10, 15, 20 and 25 p.i. However, the TIMP-1 level was unchanged and remained at basal levels at all time points, even in uninfected mice. The MMP-12/TIMP-1 ratio was significantly elevated (P < 0.05) on days 10, 15, 20 and 25 p.i. (Fig. 1).
A total of 50 mice were randomly divided into five treated groups (10 mice/group), and subdivided into two different time points of administration (five mice/time point). Food and water were withheld for 12 h before infection. The uninfected control mice were administered only distilled water orally on days 5 and 15 p.i. All other groups were infected with 50 larvae, including the infected-untreated control mice, which were treated with only distilled water orally on days 5 and 15 p.i.; the albendazole-treated mice, which were treated with albendazole (10 mg/kg/day) alone for seven consecutive days on days 5 and 15 p.i.; the doxycycline-treated mice, which were treated with doxycycline (30 mg/ kg/day) alone for seven consecutive days on days 5 and 15 p.i.; and albendazole–doxycycline combination therapy mice, which were treated with albendazole (10 mg/kg/day) and doxycycline (30 mg/kg/day) for seven consecutive days on days 5 and 15 p.i. All groups were sacrificed on day 22 p.i. and the CSF was collected for biochemical analysis. 2.13. Measurement of blood–CSF barrier permeability Blood–CSF barrier permeability was evaluated by assessing Evans blue concentration in CSF. Two hours before being sacrificed, mice were administered a tail vein injection of 2% Evans blue (100 mg/kg body weight; Sigma) in saline. The mice were sacrificed and their brains removed into a 35-mm dish. The cranial cavity and cerebral ventricles (lateral, third and fourth ventricles)
(A)
Results among different mouse groups were compared with the non-parametric Kruskal–Wallis test followed by post-testing using Dunn’s multiple comparison of means. All results were presented as mean ± S.D. Statistical significance was established for P < 0.05. 3. Results 3.1. Kinetic studies for MMP-12/TIMP-1 ratio in CSF
3.2. Correlation of MMP-12/TIMP-1 ratio with CSF eosinophils CSF eosinophilia was found only in infected mice. The MMP-12/ TIMP-1 ratio correlated significantly (r = 0.88, P < 0.05) with the CSF eosinophil count (Fig. 2). 3.3. Immunohistochemical distribution of MMP-12 in the cell wall of meningeal vessels and leucocytes of mouse subarachnoid space Immunohistochemical staining of brain sections showed that MMP-12 had a strong positive signal in the vessel walls, polynuclear leucocytes and macrophages of the subarachnoid space. However, there was a weak signal in the vessel wall and leucocytes detected with normal serum (Fig. 3). 3.4. Kinetic studies for protein levels of elastin in CSF The CSF elastin protein levels were analysed with western blotting using goat anti-mouse elastin polyclonal antibody. The 68, 58 and 55 kDa elastin bands were detected in mouse CSF. The 68 kDa elastin band was significantly increased (P < 0.05) on days 10, 15,
(B)
MMP-12
TIMP-1
MMP-12/TIMP-1 ratios
25
*
20
* 15 10
* *
5
Co nt ro l D ay 5 D ay 10 D ay 15 D ay 20 D ay 25
0
Fig. 1. Kinetic studies of the matrix metalloproteinase (MMP)-12/tissue inhibitor of metalloproteinase (TIMP-1) ratio in CSF of Angiostrongylus cantonensis-infected mice. (A) The protein bands of MMP-12 and TIMP-1 from six different time points. (B) Quantitative analysis of MMP-12 and TIMP-1 was performed with a computer-assisted imaging densitometer system. The MMP-12/TIMP-1 ratio was significantly increased (⁄P < 0.05) on days 10, 15, 20 and 25 p.i.
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r = 0.88
MMP-12/TIMP-1 ratios
30
The effects of doxycycline alone or combined with albendazole were investigated in a murine angiostrongyliasis model. Western blotting measured the protein level of MMP-12, which was significantly increased (P < 0.05) in the CSF of infected mice, compared with uninfected controls. For mice treated beginning on day 5 p.i., albendazole alone or albendazole–doxycycline combination therapy significantly reduced (P < 0.05) MMP-12 levels. However, there was no significant inhibition (P > 0.05) of this enzyme when treatment began on day 15 p.i. (Fig. 6).
25 20 15 10 5 0
3.6. Influence of treatment on MMP-12
0
100
200
300
400
3
Eosinophil counts (x10 ) Fig. 2. Correlation of the matrix metalloproteinase (MMP)-12/tissue inhibitor of metalloproteinase (TIMP-1) ratio with CSF eosinophils from Angiostrongylus cantonensis-infected mice. The MMP-12/TIMP-1 ratio correlated significantly (r = 0.88) with CSF eosinophil count, using Spearman’s ranking correlation test.
20 and 25 p.i. The 58 kDa degraded elastin band was significantly increased (P < 0.05) on days 20 and 25 p.i. The 55 kDa degraded elastin band was significantly increased (P < 0.05) on days 10, 15, 20 and 25 p.i. (Fig. 4). 3.5. Interaction of MMP-12 and elastin in brain homogenates Co-immunoprecipitation is a common method used to determine protein–protein interactions. We examined the relationship between MMP-12 and elastin density from A. cantonensis-infected mouse homogenates and found that MMP-12 interacted with elastin (Fig. 5).
3.7. Influence of treatment on elastin Western blotting measured the elastin protein level, which was significantly increased (P < 0.05) in the CSF of infected mice. For mice in which treatment began on day 5 p.i., albendazole alone or albendazole–doxycycline significantly reduced (P < 0.05) 68, 58 and 55 kDa elastin levels. Albendazole–doxycycline treatment on day 15 p.i. significantly reduced (P < 0.05) 68 and 58 kDa elastin levels (Fig. 7).
3.8. Influence of treatment on blood–CSF barrier permeability The presence of Evans blue dye in the CSF was used to assess blood–CSF barrier breakdown. Levels of Evans blue were significantly increased (P < 0.05) in the CSF of infected mice compared with uninfected controls. Albendazole alone or albendazole–doxycycline combination therapy significantly reduced (P < 0.05) Evans blue levels. However, there was no significant reduction (P > 0.05) of Evans blue when treated with doxycycline only (Fig. 8).
Fig. 3. Immunohistochemical distribution of matrix metalloproteinase (MMP)-12 in the subarachnoid space of Angiostrongylus cantonensis-infected mice. (A) The polynuclear leucocytes (black arrowheads) and macrophages (white arrowheads) could be detected as a weak signal with normal serum. (B) MMP-12 showed a strong positive signal in the polynuclear leucocytes (black arrowheads) and macrophages (white arrowheads) in the subarachnoid space. (C) The vascular wall (arrow) could be detected as a weak signal with normal serum. M, meninge. (D) MMP-12 showed a strong positive signal in the vascular wall (arrow) of the subarachnoid space. M, meninge.
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(A) 68 kDa
IP: elastin IB: MMP-12
58 kDa 55 kDa
(B)
*
60 40
Fig. 5. Interaction of matrix metalloproteinase (MMP)-12 and elastin in brain homogenates. We examined the relationship between MMP-12 and elastin from Angiostrongylus cantonensis-infected mouse brain and uninfected controls. MMP-12 interacted with elastin. H, heavy chain; IP, immunoprecipitation; IB, immunoblotting.
*
*
80
68kDa 58kDa 55kDa
*
*
100
* * *
*
* 4. Discussion
20
25 ay
D
Fig. 4. Kinetic studies for protein levels of elastin in the CSF of Angiostrongylus cantonensis-infected mice. (A) The 68, 58 and 55 kDa elastin bands were analysed by western blotting using specific antibody from the CSF. (B) The relative CSF elastin band densities were significantly elevated (⁄P < 0.05) in A. cantonensis-infected mice compared with uninfected controls.
3.9. Influence of treatment on pathological changes Optical microscopic examination of tissues stained using H&E revealed that eosinophilic meningitis was induced in BALB/c mice by A. cantonensis infection. Large-scale infiltration of the subarachnoid space by leucocytes was evident 22 days after inoculation of the larvae. Following treatment on day 5 p.i., leucocytes were significantly decreased by albendazole–doxycycline combination therapy, moderately reduced after albendazole treatment and slightly reduced after treatment with doxycycline alone. Following treatment on day 15 p.i., leucocytes were mildly reduced by albendazole alone or by albendazole–doxycycline combination therapy, and there was no significant difference after treatment with doxycycline alone. The larvae were reduced by albendazole treatment alone or albendazole–doxycycline combination therapy (Fig. 9).
(B)
MMP-12
Day 15
8 6
*
4
*
2 0 D ox A BZ +D ox
Day 5
Day 5 Day 15
Co nt ro l In fe ct ed
MMP-12
10
A BZ
(A)
Neurological diseases, such as infection, lead to an increase in the matrix-degrading proteinases (Lukes et al., 1999). MMPs are implicated in the pathogenesis of various inflammatory diseases of the CNS (Kieseier et al., 1999). Investigations of serum, CSF and brain tissue of patients with multiple sclerosis have revealed an increase in MMP-12 activity (Kurzepa et al., 2005). MMP-12 is upregulated during experimental autoimmune encephalomyelitis and might play an active role during mononuclear cell infiltration in response to CNS infection (Pagenstecher et al., 1998). Previous studies have implied that angiostrongyliasis meningitis evokes an eosinophilic reaction and MMP-9 is also involved in angiostrongyliasis (Lee et al., 2004). In the present study, MMP-12 was demonstrated in CSF samples obtained from mice with eosinophilic meningitis. A significant correlation was found between the level of MMP-12 and CSF eosinophilia. Also, MMP-12 was localised in the wall of meningeal vessels and infiltrated leucocytes in the subarachnoid space. Therefore, it was reasonable to assume that MMP-12 was also associated with the inflammatory reaction of angiostrongyliasis meningitis. Both MMP-9 and MMP12 were associated with this pathogenic process. However, the roles of MMP-9 and MMP-12 in angiostrongyliasis meningitis were decided according to substrate specificity. Elastin is an important component of cerebral arteries (Lammie, 2000) and might be involved in some pathological processes (Hornebeck et al., 1984; Satta et al., 1998). The raised levels of elastin-derived peptides in CSF could result from penetration from the
Relative density of MMP-12
ay
20
15 D
10
ay D
ay
ay D
D
Co nt
5
0 ro l
Relative intensity of elastin
MMP-12 H
Fig. 6. Influence of treatment on matrix metalloproteinase (MMP)-12. The test groups were: uninfected mice (control); Angiostrongylus cantonensis-infected untreated mice (infected); albendazole-treated mice (ABZ); doxycycline-treated mice (Dox); and albendazole–doxycycline-treated mice (ABZ + Dox). Mice received treatment for seven consecutive days on days 5 and 15 p.i. ⁄P < 0.05 indicates a significant difference in MMP-12 levels in treated mice versus infected-untreated mice.
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(B)
68 kDa
*
(C)
60
**
40
Relative intensity of elastin
68 kDa 58 kDa 55 kDa
**
20 0
(D) Day 15
*
80
Co nt ro l In fe ct ed
58 kDa 55 kDa
68kDa 58kDa 55kDa
100
D O A BZ X +D O X
Relative intensity of elastin
Day 5
A BZ
(A)
100
*
80
68kDa 58kDa 55kDa
60 40 20
* A BZ +D O X
O X D
BZ A
Co nt ro l In fe ct ed
0
Fig. 7. Influence of treatment on elastin. The test groups were: uninfected mice (control); Angiostrongylus cantonensis-infected untreated mice (infected); albendazole-treated mice (ABZ); doxycycline-treated mice (Dox); and albendazole–doxycycline-treated mice (ABZ + Dox). Mice received treatment for seven consecutive days on days 5 (A and B) and 15 (C and D) p.i. ⁄P < 0.05 indicates a significant difference in elastin levels in treated mice versus infected-untreated mice.
Evans blue units
2.0
Day 5 Day 15
1.5 * *
1.0
* *
0.5
X O
X
+D
O A
BZ
D
BZ A
ct fe In
Co
nt
ro
ed
l
0.0
Fig. 8. Influence of treatment on blood–CSF barrier permeability. The test groups were: uninfected mice (control); Angiostrongylus cantonensis-infected untreated mice (infected); albendazole-treated mice (ABZ); doxycycline-treated mice (Dox); and albendazole–doxycycline-treated mice (ABZ + Dox). Mice received treatment for seven consecutive days on days 5 and 15 p.i. ⁄P < 0.05 indicates significant decrease in Evans blue levels in treated mice versus infected-untreated mice.
affected cerebral vessels to the CSF, passive diffusion from the serum or abnormal synthesis of elastin-derived peptide precursors (Robert et al., 1984; Sajanti and Majamaa, 1999). These responses might lead to further elastinolysis and production of more elastin-derived peptides, thereby triggering a vicious circle that amplifies vessel wall degradation. Thus, elastin-derived peptides are not
only the by-products and markers of vasculopathy but also participate in the pathogenesis of endothelial failure (Faury et al., 1997), which is responsible for blood–CSF barrier breakdown that allows extravasation of blood components in lacunar infarcts (Wardlaw et al., 2003). In this study, we used western blotting to measure elastin in mouse CSF. Major bands at 68, 58 and 55 kDa were detected by SDS–PAGE. The 68 kDa protein was tropoelastin and others were degradation products that resulted from cleavage by proteinase. These degraded proteins correlated with eosinophil count and pathological changes (data not shown). This implies that degradation of elastin plays an important role in vascular leakage in mice with angiostrongyliasis meningitis. MMPs may be essential for the breakdown of the vessel wall in the meninges and the choroids plexus (Raffetto and Khalil, 2008). Within the MMP family, MMP-12 is able to degrade ECM components such as elastin and various basement membrane components (Vaalamo et al., 1999). Furthermore, 89 cleavage sites in tropoelastin have been identified for MMP-12 (Heinz et al., 2010). In this study, we demonstrated by co-immunoprecipitation that MMP-12 interacted with elastin in mice with angiostrongyliasis meningoencephalitis or meningitis. It is therefore a candidate molecule for the causation of angiostrongyliasis, which is characterised by damage to the elastin. Elastin represents a crucial component of vessel wall structure and a matrix substrate for MMP-12. Degradation and loosening of ECM by MMP-12 could facilitate vascular remodelling. These results suggest that MMP-12 plays an important role in elastin degradation during eosinophilic meningitis or meningoencephalitis. We propose that upregulated MMP-12 and increased elastin degradation in meningitis or
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Fig. 9. Influence of treatment on subarachnoid space infection in Angiostrongylus cantonensis-infected mice. (A) The uninfected controls had normal meninges (arrow) and almost no leucocytes in the subarachnoid space. (B) Mice infected with A. cantonensis showed larva (arrows) and leucocyte (arrowheads) accumulation. Treatment with albendazole beginning on day 5 p.i. (C) significantly decreased the numbers of larvae and leucocytes (arrowhead), but treatment beginning on day 15 p.i. (D) slightly decreased leucocytes (arrowhead). Treatment with doxycycline beginning on day 5 p.i. (E) did not kill the larvae (arrow) and only slightly decreased the leucocytes (arrowhead), and on day 15 p.i. (F) did not kill the larvae (arrows) and permitted significant leucocyte (arrowhead) accumulation. Albendazole–doxycycline combination therapy beginning on day 5 p.i. (G) significantly decreased leucocyte numbers, and beginning on day 15 p.i. (H) slightly decreased leucocyte numbers (arrowheads). Bar = 160 lm.
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Fig. 10. Possible mechanisms of matrix metalloproteinase (MMP)-12 contribution to meningitis. It is proposed that leucocytes cross the blood–CSF barrier and present in the subarachnoid space during Angiostrongylus cantonensis infection. Activated leucocytes can enter the subarachnoid space by extravasation through the wall of the meningeal vessels, which consists of endothelial cells and elastic tissue. This parasitic infection leads to the induction of MMP-12 from infiltrating leucocytes and causes elastin degradation in the meningeal vessels. These processes are associated with the inflammatory response in the subarachnoid space caused by A. cantonensis. BBB, blood–brain barrier; BCB, blood–CSF barrier.
meningoencephalitis could participate in disruption of the blood– CSF barrier and blood–brain barrier, which results in leucocyte recruitment and tissue damage. In the CNS, MMPs have been shown to degrade components of the basal lamina, which leads to disruption of the blood–CSF barrier and contributes to the neuroinflammatory responses in many neurological diseases. Inhibition of MMPs has been shown to prevent progression of these diseases (Mandal et al., 2003). In this study, we use a non-selective MMP inhibitor, doxycycline, to explore the potential for pharmacological inhibition of elastinolysis and inflammation. The results showed that the MMP-12 and elastin were significantly decreased when mice were treated with albendazole alone or albendazole–doxycycline in combination on day 5 p.i. However, there was no significant difference in MMP12 and elastin in infected-untreated or doxycycline-treated mice. To explain this phenomenon, one possibility is that doxycycline cannot kill the nematode. Thus, in the present study, we used simultaneous administration of doxycycline and albendazole to inhibit meningitis and kill the nematode. Treatment on day 15 p.i. with albendazole alone or albendazole–doxycycline in combination significantly reduced elastin levels and Evans blue. However, there was no significant inhibition of MMP-12. A possible explanation is that, in addition to MMP-12, elastin can be degraded by other proteinases. These results suggest that albendazole–doxycycline combination therapy is able to reverse the inflammatory events of angiostrongyliasis. The present study shows that MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. MMP-12 production was correlated with elastin degradation, eosinophil count, blood–CSF barrier permeability and pathological changes in the subarachnoid space. Therefore, we propose the following mechanisms (Fig. 10) to explain the contribution of MMP12 to meningitis. Leucocytes are thought to cross the blood–CSF barrier and present in the subarachnoid space during A. cantonensis infection. Activated leucocytes can enter the subarachnoid space
by extravasation through the wall of meningeal vessels, which consist of endothelial cells and elastic tissue. This parasite infection leads to the induction of MMP-12 from infiltrating leucocytes and causes elastin degradation in the meningeal vessels. These processes are associated with the inflammatory response in the subarachnoid space that is caused by A. cantonensis. In conclusion, our results suggest that MMP-12-induced elastin degradation could highlight blood–CSF barrier dysfunction and vascular damage seen in angiostrongyliasis, and could be used for the pharmacological evaluation of anti-inflammatory mechanism.
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P.C. Wei et al. / International Journal for Parasitology 41 (2011) 1175–1183 Hornebeck, W., Brechemier, D., Jacob, M.P., Frances, C., Robert, L., 1984. On the multiplicity of cellular elastases and their inefficient control by natural inhibitors. Adv. Exp. Med. Biol. 167, 111–119. Hou, R.F., Tu, W.C., Lee, H.H., Chen, K.M., Chou, H.L., Lai, S.C., 2004. Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis. Int. J. Parasitol. 34, 1355– 1364. Hsu, W.Y., Chen, J.Y., Chien, C.T., Chi, C.S., Han, N.T., 1990. Eosinophilic meningitis caused by Angiostrongylus cantonensis. Pediatr. Infect. Dis. J. 9, 443–445. Hwang, K.P., Chen, E.R., 1988. Larvicidal effect of albendazole against Angiostrongylus cantonensis in mice. Am. J. Trop. Med. Hyg. 39, 191–195. Jantzie, L.L., Cheung, P.Y., Todd, K.G., 2005. Doxycycline reduces cleaved caspase-3 and microglial activation in an animal model of neonatal hypoxia–ischemia. J. Cereb. Blood Flow Metab. 25, 314–324. Kieseier, B.C., Paul, R., Koedel, U., Seifert, T., Clements, J.M., Gearing, A.J., Pfister, H.W., Hartung, H.P., 1999. Differential expression of matrix metalloproteinases in bacterial meningitis. Brain 122, 1579–1587. Kurzepa, J., Bartosik-Psujek, H., Suchozebrska-Jesionek, D., Rejdak, K., StryjeckaZimmer, M., Stelmasiak, Z., 2005. Role of matrix metalloproteinases in the pathogenesis of multiple sclerosis. Neurol. Neurochir. Pol. 39, 63–67. Lammie, G.A., 2000. Pathology of small vessel stroke. Br. Med. Bull. 56, 296–306. Lee, H.H., Chou, H.L., Chen, K.M., Lai, S.C., 2004. Association of matrix metalloproteinase-9 in eosinophilic meningitis of BALB/c mice caused by Angiostrongylus cantonensis. Parasitol. Res. 94, 321–328. Lukes, A., Mun-Bryce, S., Lukes, M., Rosenberg, G.A., 1999. Extracellular matrix degradation by metalloproteinases and central nervous system diseases. Mol. Neurobiol. 19, 267–284. Mackerras, M.J., Sandars, D.F., 1954. Life history of the rat lung-worm and its migration through the brain of its host. Nature 173, 956–957. Mandal, M., Mandal, A., Das, S., Chakraborti, T., Sajal, C., 2003. Clinical implications of matrix metalloproteinases. Mol. Cell. Biochem. 252, 305–329. Nicoloff, G., Baydanoff, S., Stanimirova, N., Petrova, C., Christova, P., 2000. An association of anti-elastin IgA antibodies with development of retinopathy in diabetic children. Gen. Pharmacol. 35, 83–87. Nicoloff, G., Tzvetanov, P., Christova, P., Baydanoff, S., 2008. Detection of elastin derived peptides in cerebrospinal fluid of patients with first ever ischaemic stroke. Neuropeptides 42, 277–282. Pagenstecher, A., Stalder, A.K., Kincaid, C.L., Shapiro, S.D., Campbell, I.L., 1998. Differential expression of matrix metalloproteinase and tissue inhibitor of
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matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states. Am. J. Pathol. 152, 729–741. Parsons, J.C., Grieve, R.B., 1990. Effect of egg dosage and host genotype on liver trapping in murine larval toxocariasis. J. Parasitol. 76, 53–58. Raffetto, J.D., Khalil, R.A., 2008. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease. Biochem. Pharmacol. 75, 346–359. Robert, L., Jacob, M.P., Frances, C., Godeau, G., Hornebeck, W., 1984. Interaction between elastin and elastase and its role in the aging of the arterial wall, skin and other connective tissues. Mech. Ageing Dev. 28, 155–166. Rosenbloom, J., 1984. Elastin: relation of protein and gene structure to disease. Lab. Invest. 51, 605–623. Sajanti, J., Majamaa, K., 1999. Detection of meningeal fibrosis after subarachnoid haemorrhage by assaying procollagen propeptides in cerebrospinal fluid. J. Neurol. Neurosurg. Psychiatry 67, 185–188. Satta, J., Laurila, A., Pääkkö, P., Haukipuro, K., Sormunen, R., Parkkila, S., Juvonen, T., 1998. Chronic inflammation and elastin degradation in abdominal aortic aneurysm disease: an immunohistochemical and electron microscopic study. Eur. J. Vasc. Endovasc. Surg. 15, 313–319. Sehba, F.A., Mostafa, G., Knopman, J., Friedrich Jr., V., Bederson, J.B., 2004. Acute alterations in microvascular basal lamina after subarachnoid hemorrhage. J. Neurosurg. 101, 633–640. Shapiro, S.D., 1998. Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr. Opin. Cell Biol. 10, 602–608. Tzvetanov, P., Nicoloff, G., Rousseff, R., Christova, P., 2008. Increased levels of elastin-derived peptides in cerebrospinal fluid of patients with lacunar stroke. Clin. Neurol. Neurosurg. 110, 239–244. Vaalamo, M., Kariniemi, A.L., Shapiro, S.D., Saarialho-Kere, U., 1999. Enhanced expression of human metalloelastase (MMP-12) in cutaneous granulomas and macrophage migration. J. Invest. Dermatol. 112, 499–505. Vacek, T.P., Gillespie, W., Tyagi, N., Vacek, J.C., Tyagi, S.C., 2010. Hydrogen sulfide protects against vascular remodeling from endothelial damage. Amino Acids 39, 1161–1169. Venkatesan, P., 1998. Albendazole. J. Antimicrob. Chemother. 41, 145–147. Wardlaw, J.M., Sandercock, P.A., Denis, M.S., Starr, J., 2003. Is breakdown of the blood–brain barrier responsible for lacunar stroke, leukoaraiosis, and dementia? Stroke 34, 806–812. Yrjänheikki, J., Keinänen, R., Pellikka, M., Hökfelt, T., Koistinaho, J., 1998. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc. Natl. Acad. Sci. USA 95, 15769–15774.
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis P.C. Wei a, C.H. Tsai a, P.S. Chiu a, S.C. Lai b,c,⇑ a
Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan Department of Parasitology, Chung Shan Medical University, Taichung 402, Taiwan c Clinical Laboratory, Chung Shan University Hospital, Taichung 402, Taiwan b
a r t i c l e
i n f o
Article history: Received 26 May 2011 Received in revised form 7 July 2011 Accepted 8 July 2011 Available online 6 August 2011 Keywords: Angiostrongylus cantonensis Eosinophilic meningitis Matrix metalloproteinase-12 Elastin
a b s t r a c t The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood–CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Angiostrongyliasis is a parasitic disease caused by the rat nematode lungworm Angiostrongylus cantonensis, which undergoes obligatory intracerebral migration in its hosts (Mackerras and Sandars, 1954). Worms develop to the young adult stage in the meninges, and the inflammatory reaction that is induced by the worms causes severe CNS infection, especially eosinophilic meningitis (Hsu et al., 1990). Diagnosis is based on epidemiological criteria, clinical manifestations, elevated eosinophil count in the CSF and serological tests. Treatment is symptomatic and supportive (Bourée et al., 2010). Matrix metalloproteinases (MMPs) are structurally related extracellular matrix (ECM)-degrading enzymes that are collectively capable of degrading essentially all ECM components (Shapiro, 1998). The integrity of cerebral microvessels is maintained in part by components of the basal lamina (such as collagen IV and elastin). Destruction of basal lamina components by MMPs, especially MMP-9, has been known to occur during subarachnoid haemorrhage (Sehba et al., 2004). MMP-12 is an elastin-degrading proteinase that is associated with vascular injury (Vacek et al., 2010). Elastin is an MMP-12 substrate and has been shown to account for up 4% of the ECM of brain microvessels (Faris et al., 1982; Curci ⇑ Corresponding author at: Department of Parasitology, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung 402, Taiwan. Tel.: +886 4 24730022x1641; fax: +886 4 23823381. E-mail address: [email protected] (S.C. Lai).
et al., 1998). This protein contains two cysteine residues that might be important in the interaction of elastin with other ECM proteins (Rosenbloom, 1984). Arterioles and venules have three layers: an inner endothelium that acts as a membrane; a middle layer of muscle and elastic tissue; and an outer layer of fibrous connective tissue (Bell and Weddell, 1984). Activated T cells can enter the subarachnoid space by extravasation through the cell wall of the meningeal venules, which consists of endothelial cells connected by tight junctions (Goverman, 2009). Elastin is an important factor in the development of vascular wall alterations (Nicoloff et al., 2000). Elastin-derived peptide levels in CSF of patients with ischaemic (Nicoloff et al., 2008) and lacunar (Tzvetanov et al., 2008) stroke are strongly elevated compared with healthy subjects. Therefore, we presume that the raised levels of elastin in CSF might result from vasculopathy. To our knowledge, the presence or absence of MMP-12 and its substrate elastin in mice with angiostrongyliasis meningitis is unclear. The aims of the present study were therefore to investigate whether MMP-12 and elastin are altered in mice with eosinophilic meningitis caused by A. cantonensis.
2. Materials and methods 2.1. Experimental animals Male BALB/c mice (specific-pathogen-free grade and 5 weeks old) were purchased from the National Laboratory Animal Center,
0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.07.002
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Taipei, Taiwan. Mice were maintained in a 12 h alternating light and dark cycle photoperiod, were provided with Purina Laboratory Chow and water ad libitum, and were kept in our laboratory more than 1 week before experimental infection. All procedures that involved animal use and care were approved by the Institutional Animal Care and Use Committee of Chung-Shan Medical University (IACUC Approval No: 431), Taiwan, in accordance with the institutional guidelines for animal experiments.
were sacrificed on day 25 p.i. Brains were rapidly removed and frozen at 80 °C. 2.6. Collection of CSF-like fluid The mice were sacrificed and their brains removed into a 35mm dish. The cranial cavity and cerebral ventricles (lateral, third and fourth ventricles) were rinsed with 1 mL PBS and CSF was thus harvested with PBS; the washing solution being CSF-like fluid.
2.2. Antibodies 2.7. Western blot analysis Rabbit anti-mouse MMP-12 polyclonal antibody was purchased from Abcam (Cambridge, UK), goat anti-mouse tissue inhibitor of metalloproteinase (TIMP)-1 polyclonal antibody was purchased from R&D Systems (Minneapolis, MN, USA), goat anti-mouse elastin polyclonal antibody was purchased from Santa Cruz Biotechnology (CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-goat IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). 2.3. Drugs
Mouse CSF was centrifuged at 12,000g at 4 °C for 10 min and supernatant protein was determined with protein assay kits (BioRad, USA) using BSA as the standard. An equal volume of loading buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2mercaptoethanol and 0.05% bromophenol blue) was added to the samples. The mixture was boiled for 5 min before electrophoresis on SDS–PAGE and electrotransferred to nitrocellulose membranes at a constant current of 190 mA for 90 min. Membranes were saturated with PBS that contained 0.1% Tween 20 (PBS–T) for 30 min at room temperature. Membranes were reacted with primary antibodies (rabbit anti-mouse MMP-12 polyclonal antibody, goat antimouse TIMP-1 polyclonal antibody or goat anti-mouse elastin polyclonal antibody) at a 1:500 dilution at 37 °C for 1 h. Membranes were washed three times with PBS that contained PBS–T, followed by HRP-conjugated secondary antibody (1:5,000 dilution) incubation at 37 °C for 1 h to detect primary bound antibody. Reactive proteins were detected by enhanced chemiluminescence (Amersham, Little Chalfont, Bucks, UK), and the densities of the specific immunoreactive bands were quantified with densitometric scanning analysis.
Albendazole (ZentelÒ, GlaxoSmith Kline, NC, USA) is a benzimidazole derivative. It is widely used in veterinary medicine and has been found to be a safe broad spectrum anthelmintic in animals and humans (Hwang and Chen, 1988; Chen and Lai, 2007). Albendazole acts by binding to b-tubulin in the parasite, inhibiting its polymerisation and impairing glucose uptake, which causes death (Venkatesan, 1998). Doxycycline was purchased from Sigma (St. Louis, MO, USA) and dissolved in PBS, pH 7.4. It is a lipophilic, second-generation tetracycline derivative that has anti-inflammatory actions that are independent of its antimicrobial actions (Yrjänheikki et al., 1998). It crosses the blood–brain, blood–CSF and placental barriers, and thus has excellent brain and body tissue penetration. Doxycycline could prove to be a most attractive pharmacological therapy for cerebral ischaemia due to its ability to protect the brain against pathological apoptotic neuronal cell death and neuroinflammation secondary to brain injury (Jantzie et al., 2005). Doxycycline is a water-soluble, non-specific competitive inhibitor of MMPs. It inhibits MMPs via modulation of plasminogen activators in focal cerebral ischaemia (Burggraf et al., 2007).
The CSF was collected into a centrifuge to spin at 400g for 10 min. The resultant sediments were then gently mixed with 100 lL of Unopette buffer (Vacutainer System; Becton Dickinson, Franklin Lakes, NJ, USA) and 2 lL of acetic acid, and placed in a haemocytometer cell counting chamber (Paul Marienfeld, Lauda-Koenigshofen, Germany) to count eosinophils.
2.4. Larval preparation
2.9. Histology
The infective larvae (L3) of A. cantonensis were originally obtained from wild giant African snails (Achatina fulica) and were propagated for several months in the Wufeng experimental farm (Taichung, Taiwan) by cycling through rats and A. fulica. Tissue larvae were recovered with a modified Parsons and Grieve method (1990). Briefly, snail shells were crushed and the tissues were homogenised, digested in a pepsin–HCl solution (pH 1–2, 500 IU pepsin/g tissue), and incubated with agitation in a 37 °C water bath for 2 h. Host cellular debris was removed from the digest by centrifugation at 1,400g for 10 min. The larvae in the sediment were collected by serial washes in double-distilled water and counted under a microscope. The third stage of larvae of A. cantonensis was confirmed as described by Hou et al. (2004).
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, and embedded in paraffin at 55 °C for 24 h. Several serial sections were cut at 5-lm thickness for each organ from each mouse. Sections were deparaffinised, stained with H&E (Muto, Japan) and pathological changes were examined under a light microscope.
2.5. Animal infection A total of 120 male BALB/c mice were randomly allocated to six groups (control, D5, D10, D15, D20 and D25) of 20 mice each. Food and water were withheld for 12 h before infection. The mice in five experimental groups (D5, D10, D15, D20 and D25) were infected with 50 A. cantonensis larvae by oral inoculation, and were sacrificed on days 5, 10, 15, 20 or 25 p.i. Control mice received only water and
2.8. Eosinophil counts in the CSF
2.10. Immunohistochemistry Mouse brains were prepared as described in Section 2.10 and the histological methods have been described previously (Chen et al., 2004). Relatively thick (10 lm) serial sections of waxembedded brains were cut, mounted on glass slides, dewaxed and rehydrated for immunohistochemistry. Sections were blocked with 3% BSA at room temperature for 1 h before incubation with a 1:100 dilution of rabbit anti-mouse MMP-12 polyclonal antibody in 1% BSA at 37 °C for 1 h. Sections were washed three times in PBS, incubated with the HRP-conjugated secondary antibodies diluted 1:200 in 1% BSA at 37 °C for 1 h, and washed again in PBS. Sections were incubated for 3 min at room temperature with 3,30 -diaminobenzidine (0.3 mg/mL) in 100 mM Tris (pH 7.5) that
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contained 0.3 lL H2O2/mL. Sections were mounted in 50% glycerol in PBS after final washes and examined under a light microscope. 2.11. Co-immunoprecipitation
were rinsed with 1 mL of PBS and CSF was thus harvested with PBS. The average concentration of Evans blue in the CSF was then measured at 620 nm by a spectrophotometer (U3000; Hitachi, Tokyo, Japan).
To prevent non-specific adsorption, protein A/G agarose beads (Santa Cruz Biotechnology) were washed five times in PBS and incubated with 5% BSA at 4 °C for 30 min. The solution was centrifuged twice at 10,000g for 2 min to remove supernatant before use. MMP-12 antibody was incubated with brain lysates (1 mg) at 4 °C overnight and collected by binding to protein A/G agarose beads. The beads were washed twice in dissociation buffer (0.5 M Tris– HCl, pH 8.0, 120 mM NaCl, 0.5% Triton X-100). Bound proteins were resolved by SDS–PAGE and target protein (elastin) association was determined by blotting.
2.14. Statistical analysis
2.12. Treatment of animals
CSF protein levels of MMP-12 and TIMP-1 were analysed by western blotting. The MMP-12 bands were significantly increased (P < 0.05) on days 10, 15, 20 and 25 p.i. However, the TIMP-1 level was unchanged and remained at basal levels at all time points, even in uninfected mice. The MMP-12/TIMP-1 ratio was significantly elevated (P < 0.05) on days 10, 15, 20 and 25 p.i. (Fig. 1).
A total of 50 mice were randomly divided into five treated groups (10 mice/group), and subdivided into two different time points of administration (five mice/time point). Food and water were withheld for 12 h before infection. The uninfected control mice were administered only distilled water orally on days 5 and 15 p.i. All other groups were infected with 50 larvae, including the infected-untreated control mice, which were treated with only distilled water orally on days 5 and 15 p.i.; the albendazole-treated mice, which were treated with albendazole (10 mg/kg/day) alone for seven consecutive days on days 5 and 15 p.i.; the doxycycline-treated mice, which were treated with doxycycline (30 mg/ kg/day) alone for seven consecutive days on days 5 and 15 p.i.; and albendazole–doxycycline combination therapy mice, which were treated with albendazole (10 mg/kg/day) and doxycycline (30 mg/kg/day) for seven consecutive days on days 5 and 15 p.i. All groups were sacrificed on day 22 p.i. and the CSF was collected for biochemical analysis. 2.13. Measurement of blood–CSF barrier permeability Blood–CSF barrier permeability was evaluated by assessing Evans blue concentration in CSF. Two hours before being sacrificed, mice were administered a tail vein injection of 2% Evans blue (100 mg/kg body weight; Sigma) in saline. The mice were sacrificed and their brains removed into a 35-mm dish. The cranial cavity and cerebral ventricles (lateral, third and fourth ventricles)
(A)
Results among different mouse groups were compared with the non-parametric Kruskal–Wallis test followed by post-testing using Dunn’s multiple comparison of means. All results were presented as mean ± S.D. Statistical significance was established for P < 0.05. 3. Results 3.1. Kinetic studies for MMP-12/TIMP-1 ratio in CSF
3.2. Correlation of MMP-12/TIMP-1 ratio with CSF eosinophils CSF eosinophilia was found only in infected mice. The MMP-12/ TIMP-1 ratio correlated significantly (r = 0.88, P < 0.05) with the CSF eosinophil count (Fig. 2). 3.3. Immunohistochemical distribution of MMP-12 in the cell wall of meningeal vessels and leucocytes of mouse subarachnoid space Immunohistochemical staining of brain sections showed that MMP-12 had a strong positive signal in the vessel walls, polynuclear leucocytes and macrophages of the subarachnoid space. However, there was a weak signal in the vessel wall and leucocytes detected with normal serum (Fig. 3). 3.4. Kinetic studies for protein levels of elastin in CSF The CSF elastin protein levels were analysed with western blotting using goat anti-mouse elastin polyclonal antibody. The 68, 58 and 55 kDa elastin bands were detected in mouse CSF. The 68 kDa elastin band was significantly increased (P < 0.05) on days 10, 15,
(B)
MMP-12
TIMP-1
MMP-12/TIMP-1 ratios
25
*
20
* 15 10
* *
5
Co nt ro l D ay 5 D ay 10 D ay 15 D ay 20 D ay 25
0
Fig. 1. Kinetic studies of the matrix metalloproteinase (MMP)-12/tissue inhibitor of metalloproteinase (TIMP-1) ratio in CSF of Angiostrongylus cantonensis-infected mice. (A) The protein bands of MMP-12 and TIMP-1 from six different time points. (B) Quantitative analysis of MMP-12 and TIMP-1 was performed with a computer-assisted imaging densitometer system. The MMP-12/TIMP-1 ratio was significantly increased (⁄P < 0.05) on days 10, 15, 20 and 25 p.i.
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r = 0.88
MMP-12/TIMP-1 ratios
30
The effects of doxycycline alone or combined with albendazole were investigated in a murine angiostrongyliasis model. Western blotting measured the protein level of MMP-12, which was significantly increased (P < 0.05) in the CSF of infected mice, compared with uninfected controls. For mice treated beginning on day 5 p.i., albendazole alone or albendazole–doxycycline combination therapy significantly reduced (P < 0.05) MMP-12 levels. However, there was no significant inhibition (P > 0.05) of this enzyme when treatment began on day 15 p.i. (Fig. 6).
25 20 15 10 5 0
3.6. Influence of treatment on MMP-12
0
100
200
300
400
3
Eosinophil counts (x10 ) Fig. 2. Correlation of the matrix metalloproteinase (MMP)-12/tissue inhibitor of metalloproteinase (TIMP-1) ratio with CSF eosinophils from Angiostrongylus cantonensis-infected mice. The MMP-12/TIMP-1 ratio correlated significantly (r = 0.88) with CSF eosinophil count, using Spearman’s ranking correlation test.
20 and 25 p.i. The 58 kDa degraded elastin band was significantly increased (P < 0.05) on days 20 and 25 p.i. The 55 kDa degraded elastin band was significantly increased (P < 0.05) on days 10, 15, 20 and 25 p.i. (Fig. 4). 3.5. Interaction of MMP-12 and elastin in brain homogenates Co-immunoprecipitation is a common method used to determine protein–protein interactions. We examined the relationship between MMP-12 and elastin density from A. cantonensis-infected mouse homogenates and found that MMP-12 interacted with elastin (Fig. 5).
3.7. Influence of treatment on elastin Western blotting measured the elastin protein level, which was significantly increased (P < 0.05) in the CSF of infected mice. For mice in which treatment began on day 5 p.i., albendazole alone or albendazole–doxycycline significantly reduced (P < 0.05) 68, 58 and 55 kDa elastin levels. Albendazole–doxycycline treatment on day 15 p.i. significantly reduced (P < 0.05) 68 and 58 kDa elastin levels (Fig. 7).
3.8. Influence of treatment on blood–CSF barrier permeability The presence of Evans blue dye in the CSF was used to assess blood–CSF barrier breakdown. Levels of Evans blue were significantly increased (P < 0.05) in the CSF of infected mice compared with uninfected controls. Albendazole alone or albendazole–doxycycline combination therapy significantly reduced (P < 0.05) Evans blue levels. However, there was no significant reduction (P > 0.05) of Evans blue when treated with doxycycline only (Fig. 8).
Fig. 3. Immunohistochemical distribution of matrix metalloproteinase (MMP)-12 in the subarachnoid space of Angiostrongylus cantonensis-infected mice. (A) The polynuclear leucocytes (black arrowheads) and macrophages (white arrowheads) could be detected as a weak signal with normal serum. (B) MMP-12 showed a strong positive signal in the polynuclear leucocytes (black arrowheads) and macrophages (white arrowheads) in the subarachnoid space. (C) The vascular wall (arrow) could be detected as a weak signal with normal serum. M, meninge. (D) MMP-12 showed a strong positive signal in the vascular wall (arrow) of the subarachnoid space. M, meninge.
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(A) 68 kDa
IP: elastin IB: MMP-12
58 kDa 55 kDa
(B)
*
60 40
Fig. 5. Interaction of matrix metalloproteinase (MMP)-12 and elastin in brain homogenates. We examined the relationship between MMP-12 and elastin from Angiostrongylus cantonensis-infected mouse brain and uninfected controls. MMP-12 interacted with elastin. H, heavy chain; IP, immunoprecipitation; IB, immunoblotting.
*
*
80
68kDa 58kDa 55kDa
*
*
100
* * *
*
* 4. Discussion
20
25 ay
D
Fig. 4. Kinetic studies for protein levels of elastin in the CSF of Angiostrongylus cantonensis-infected mice. (A) The 68, 58 and 55 kDa elastin bands were analysed by western blotting using specific antibody from the CSF. (B) The relative CSF elastin band densities were significantly elevated (⁄P < 0.05) in A. cantonensis-infected mice compared with uninfected controls.
3.9. Influence of treatment on pathological changes Optical microscopic examination of tissues stained using H&E revealed that eosinophilic meningitis was induced in BALB/c mice by A. cantonensis infection. Large-scale infiltration of the subarachnoid space by leucocytes was evident 22 days after inoculation of the larvae. Following treatment on day 5 p.i., leucocytes were significantly decreased by albendazole–doxycycline combination therapy, moderately reduced after albendazole treatment and slightly reduced after treatment with doxycycline alone. Following treatment on day 15 p.i., leucocytes were mildly reduced by albendazole alone or by albendazole–doxycycline combination therapy, and there was no significant difference after treatment with doxycycline alone. The larvae were reduced by albendazole treatment alone or albendazole–doxycycline combination therapy (Fig. 9).
(B)
MMP-12
Day 15
8 6
*
4
*
2 0 D ox A BZ +D ox
Day 5
Day 5 Day 15
Co nt ro l In fe ct ed
MMP-12
10
A BZ
(A)
Neurological diseases, such as infection, lead to an increase in the matrix-degrading proteinases (Lukes et al., 1999). MMPs are implicated in the pathogenesis of various inflammatory diseases of the CNS (Kieseier et al., 1999). Investigations of serum, CSF and brain tissue of patients with multiple sclerosis have revealed an increase in MMP-12 activity (Kurzepa et al., 2005). MMP-12 is upregulated during experimental autoimmune encephalomyelitis and might play an active role during mononuclear cell infiltration in response to CNS infection (Pagenstecher et al., 1998). Previous studies have implied that angiostrongyliasis meningitis evokes an eosinophilic reaction and MMP-9 is also involved in angiostrongyliasis (Lee et al., 2004). In the present study, MMP-12 was demonstrated in CSF samples obtained from mice with eosinophilic meningitis. A significant correlation was found between the level of MMP-12 and CSF eosinophilia. Also, MMP-12 was localised in the wall of meningeal vessels and infiltrated leucocytes in the subarachnoid space. Therefore, it was reasonable to assume that MMP-12 was also associated with the inflammatory reaction of angiostrongyliasis meningitis. Both MMP-9 and MMP12 were associated with this pathogenic process. However, the roles of MMP-9 and MMP-12 in angiostrongyliasis meningitis were decided according to substrate specificity. Elastin is an important component of cerebral arteries (Lammie, 2000) and might be involved in some pathological processes (Hornebeck et al., 1984; Satta et al., 1998). The raised levels of elastin-derived peptides in CSF could result from penetration from the
Relative density of MMP-12
ay
20
15 D
10
ay D
ay
ay D
D
Co nt
5
0 ro l
Relative intensity of elastin
MMP-12 H
Fig. 6. Influence of treatment on matrix metalloproteinase (MMP)-12. The test groups were: uninfected mice (control); Angiostrongylus cantonensis-infected untreated mice (infected); albendazole-treated mice (ABZ); doxycycline-treated mice (Dox); and albendazole–doxycycline-treated mice (ABZ + Dox). Mice received treatment for seven consecutive days on days 5 and 15 p.i. ⁄P < 0.05 indicates a significant difference in MMP-12 levels in treated mice versus infected-untreated mice.
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(B)
68 kDa
*
(C)
60
**
40
Relative intensity of elastin
68 kDa 58 kDa 55 kDa
**
20 0
(D) Day 15
*
80
Co nt ro l In fe ct ed
58 kDa 55 kDa
68kDa 58kDa 55kDa
100
D O A BZ X +D O X
Relative intensity of elastin
Day 5
A BZ
(A)
100
*
80
68kDa 58kDa 55kDa
60 40 20
* A BZ +D O X
O X D
BZ A
Co nt ro l In fe ct ed
0
Fig. 7. Influence of treatment on elastin. The test groups were: uninfected mice (control); Angiostrongylus cantonensis-infected untreated mice (infected); albendazole-treated mice (ABZ); doxycycline-treated mice (Dox); and albendazole–doxycycline-treated mice (ABZ + Dox). Mice received treatment for seven consecutive days on days 5 (A and B) and 15 (C and D) p.i. ⁄P < 0.05 indicates a significant difference in elastin levels in treated mice versus infected-untreated mice.
Evans blue units
2.0
Day 5 Day 15
1.5 * *
1.0
* *
0.5
X O
X
+D
O A
BZ
D
BZ A
ct fe In
Co
nt
ro
ed
l
0.0
Fig. 8. Influence of treatment on blood–CSF barrier permeability. The test groups were: uninfected mice (control); Angiostrongylus cantonensis-infected untreated mice (infected); albendazole-treated mice (ABZ); doxycycline-treated mice (Dox); and albendazole–doxycycline-treated mice (ABZ + Dox). Mice received treatment for seven consecutive days on days 5 and 15 p.i. ⁄P < 0.05 indicates significant decrease in Evans blue levels in treated mice versus infected-untreated mice.
affected cerebral vessels to the CSF, passive diffusion from the serum or abnormal synthesis of elastin-derived peptide precursors (Robert et al., 1984; Sajanti and Majamaa, 1999). These responses might lead to further elastinolysis and production of more elastin-derived peptides, thereby triggering a vicious circle that amplifies vessel wall degradation. Thus, elastin-derived peptides are not
only the by-products and markers of vasculopathy but also participate in the pathogenesis of endothelial failure (Faury et al., 1997), which is responsible for blood–CSF barrier breakdown that allows extravasation of blood components in lacunar infarcts (Wardlaw et al., 2003). In this study, we used western blotting to measure elastin in mouse CSF. Major bands at 68, 58 and 55 kDa were detected by SDS–PAGE. The 68 kDa protein was tropoelastin and others were degradation products that resulted from cleavage by proteinase. These degraded proteins correlated with eosinophil count and pathological changes (data not shown). This implies that degradation of elastin plays an important role in vascular leakage in mice with angiostrongyliasis meningitis. MMPs may be essential for the breakdown of the vessel wall in the meninges and the choroids plexus (Raffetto and Khalil, 2008). Within the MMP family, MMP-12 is able to degrade ECM components such as elastin and various basement membrane components (Vaalamo et al., 1999). Furthermore, 89 cleavage sites in tropoelastin have been identified for MMP-12 (Heinz et al., 2010). In this study, we demonstrated by co-immunoprecipitation that MMP-12 interacted with elastin in mice with angiostrongyliasis meningoencephalitis or meningitis. It is therefore a candidate molecule for the causation of angiostrongyliasis, which is characterised by damage to the elastin. Elastin represents a crucial component of vessel wall structure and a matrix substrate for MMP-12. Degradation and loosening of ECM by MMP-12 could facilitate vascular remodelling. These results suggest that MMP-12 plays an important role in elastin degradation during eosinophilic meningitis or meningoencephalitis. We propose that upregulated MMP-12 and increased elastin degradation in meningitis or
P.C. Wei et al. / International Journal for Parasitology 41 (2011) 1175–1183
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Fig. 9. Influence of treatment on subarachnoid space infection in Angiostrongylus cantonensis-infected mice. (A) The uninfected controls had normal meninges (arrow) and almost no leucocytes in the subarachnoid space. (B) Mice infected with A. cantonensis showed larva (arrows) and leucocyte (arrowheads) accumulation. Treatment with albendazole beginning on day 5 p.i. (C) significantly decreased the numbers of larvae and leucocytes (arrowhead), but treatment beginning on day 15 p.i. (D) slightly decreased leucocytes (arrowhead). Treatment with doxycycline beginning on day 5 p.i. (E) did not kill the larvae (arrow) and only slightly decreased the leucocytes (arrowhead), and on day 15 p.i. (F) did not kill the larvae (arrows) and permitted significant leucocyte (arrowhead) accumulation. Albendazole–doxycycline combination therapy beginning on day 5 p.i. (G) significantly decreased leucocyte numbers, and beginning on day 15 p.i. (H) slightly decreased leucocyte numbers (arrowheads). Bar = 160 lm.
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Fig. 10. Possible mechanisms of matrix metalloproteinase (MMP)-12 contribution to meningitis. It is proposed that leucocytes cross the blood–CSF barrier and present in the subarachnoid space during Angiostrongylus cantonensis infection. Activated leucocytes can enter the subarachnoid space by extravasation through the wall of the meningeal vessels, which consists of endothelial cells and elastic tissue. This parasitic infection leads to the induction of MMP-12 from infiltrating leucocytes and causes elastin degradation in the meningeal vessels. These processes are associated with the inflammatory response in the subarachnoid space caused by A. cantonensis. BBB, blood–brain barrier; BCB, blood–CSF barrier.
meningoencephalitis could participate in disruption of the blood– CSF barrier and blood–brain barrier, which results in leucocyte recruitment and tissue damage. In the CNS, MMPs have been shown to degrade components of the basal lamina, which leads to disruption of the blood–CSF barrier and contributes to the neuroinflammatory responses in many neurological diseases. Inhibition of MMPs has been shown to prevent progression of these diseases (Mandal et al., 2003). In this study, we use a non-selective MMP inhibitor, doxycycline, to explore the potential for pharmacological inhibition of elastinolysis and inflammation. The results showed that the MMP-12 and elastin were significantly decreased when mice were treated with albendazole alone or albendazole–doxycycline in combination on day 5 p.i. However, there was no significant difference in MMP12 and elastin in infected-untreated or doxycycline-treated mice. To explain this phenomenon, one possibility is that doxycycline cannot kill the nematode. Thus, in the present study, we used simultaneous administration of doxycycline and albendazole to inhibit meningitis and kill the nematode. Treatment on day 15 p.i. with albendazole alone or albendazole–doxycycline in combination significantly reduced elastin levels and Evans blue. However, there was no significant inhibition of MMP-12. A possible explanation is that, in addition to MMP-12, elastin can be degraded by other proteinases. These results suggest that albendazole–doxycycline combination therapy is able to reverse the inflammatory events of angiostrongyliasis. The present study shows that MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. MMP-12 production was correlated with elastin degradation, eosinophil count, blood–CSF barrier permeability and pathological changes in the subarachnoid space. Therefore, we propose the following mechanisms (Fig. 10) to explain the contribution of MMP12 to meningitis. Leucocytes are thought to cross the blood–CSF barrier and present in the subarachnoid space during A. cantonensis infection. Activated leucocytes can enter the subarachnoid space
by extravasation through the wall of meningeal vessels, which consist of endothelial cells and elastic tissue. This parasite infection leads to the induction of MMP-12 from infiltrating leucocytes and causes elastin degradation in the meningeal vessels. These processes are associated with the inflammatory response in the subarachnoid space that is caused by A. cantonensis. In conclusion, our results suggest that MMP-12-induced elastin degradation could highlight blood–CSF barrier dysfunction and vascular damage seen in angiostrongyliasis, and could be used for the pharmacological evaluation of anti-inflammatory mechanism.
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