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. 2014 Oct 1;178(2):245–252. doi: 10.1111/cei.12413

Modulation of the kallikrein/kinin system by the angiotensin-converting enzyme inhibitor alleviates experimental autoimmune encephalomyelitis

A Uzawa 1, M Mori 1, J Taniguchi 1, S Kuwabara 1
PMCID: PMC4233374  PMID: 24996009

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Bradykinin is the end-product of the kallikrein/kinin system, which has been recognized as an endogenous target for combating CNS inflammation. Angiotensin-converting enzyme (ACE) inhibitors influence the kallikrein/kinin system and reportedly have immunomodulatory characteristics. The objectives of this study were to determine whether bradykinin is involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and whether bradykinin control by the ACE inhibitor could be a therapeutic target in MS. The ACE inhibitor enalapril (1·0 or 0·2 mg/kg/day) was administered orally to EAE mice and the serum levels of bradykinin and cytokines in EAE mice were analysed. As a result, the administration of enalapril increased serum bradykinin levels, decreased the clinical and pathological severity of EAE and attenuated interleukin-17-positive cell invasion into the CNS. Additionally, bradykinin receptor antagonist administration reduced the favourable effects of enalapril. Our results suggest that bradykinin is involved in the pathomechanism underlying CNS inflammation in EAE, possibly through inhibiting cell migration into CNS. Control of the kallikrein/kinin system using ACE inhibitors could be a potential therapeutic strategy in MS.

Keywords: angiotensin-converting enzyme inhibitor, bradykinin, enalapril, experimental autoimmune encephalomyelitis, multiple sclerosis

Introduction

Multiple sclerosis (MS) is an autoimmune-mediated inflammatory disease of the central nervous system (CNS) that is manifested by demyelination and axonal degeneration [1].

Bradykinin, the end-product of the kallikrein/kinin system (KKS), is a peptide causing vasodilation, thereby decreasing blood pressure. Angiotensin-converting enzyme (ACE) catalyzes the production of angiotensin II and the inactivation of bradykinin. Bradykinin is released during various types of tissue injury, including inflammation [2,3]. Because bradykinin is inactivated by ACE, ACE inhibitors up-regulate bradykinin and bradykinin receptor 1 (B1R) activity. B1R is present at low levels under normal conditions, but can be selectively induced by tissue injury and inflammatory mediators [4]. KKS has recently been recognized as an endogenous target for combating CNS inflammation [3,5]. The activation of B1R has been reported recently to limit encephalitogenic T lymphocyte recruitment into the CNS [3], providing insight into the mechanism of ACE inhibitor immunomodulatory function. An association between bradykinin and CNS inflammation is thus expected. In addition, an association between ACE inhibitors and cytokine production has been reported, with ACE inhibitors decreasing circulating levels of interleukin (IL)-6 [6], increasing levels of IL-10 [7], abolishing angiotensin II-mediated up-regulated expression of proinflammatory chemokines [8] and inhibiting angiotensin II release from human monocytes stimulated with tumour necrosis factor (TNF)-α and granulocyte–macrophage colony-stimulating factor [9].

Studies have reported beneficial effects of ACE inhibitors in the treatment of inflammatory diseases, including experimental autoimmune encephalomyelitis (EAE) [5,10,11]. It has been reported that ACE inhibitors suppress production of interferon (IFN)-γ and IL-12, and may have an immunomodulatory effect by inhibiting IL-12 [12]. However, the mechanism of ACE inhibitor action in decreasing inflammatory responses has not been studied extensively. The objectives of this study were to determine whether bradykinin is involved in the pathogenesis of EAE and whether bradykinin control by the ACE inhibitor enalapril might show therapeutic potential for MS treatment.

Materials and methods

EAE induction in mice

Female wild-type C57BL/6 mice (10–12 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The mice were housed with a maximum of four animals per cage in pathogen-free facilities at Chiba University and had free access to water and powdered rodent chow. EAE was induced by immunization with myelin oligodendrocyte glycoprotein (MOG) using Hooke kits (EK-0115; Hooke Laboratories, Lawrence, MA, USA). At two sites, C57BL/6 mice were injected subcutaneously with a total of 200 μg of MOG peptide 35–55 in complete Freund's adjuvant containing 400 μg of killed Mycobacterium tuberculosis H37Ra (day 1). The mice were also injected intraperitoneally with 500 ng pertussis toxin on days 1 and 2. EAE was scored on the following scale: 0, no clinical signs; 1, partial paralysis of tail; 2, flaccid tail; 3, limp tail and partial weakness of hind legs; 4, limp tail and complete weakness of hind legs; 5, limp tail, complete weakness of hind legs and partial weakness of front legs; and 6, complete hind and front leg paralysis. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Chiba University.

Treatment of EAE mice with the ACE inhibitor enalapril

The effect of the ACE inhibitor enalapril [(S,S,S)-Enalapril Maleate; Wako Pure Chemical Industries, Ltd, Osaka, Japan] on EAE development was evaluated. Enalapril was mixed with powdered chow, and the dosage (1·0 mg/kg/day or 0·2 mg/kg/day per mouse) was calculated from daily food intake (each mouse eats about 3 g/day of powdered chow). Mice were administered either 1·0 mg/kg/day of enalapril [EAE + enalapril (1·0) group; n = 12; two of 12 mice were used for only pathological analyses] or 0·2 mg/kg/day of enalapril [EAE + enalapril (0·2) group; n = 5] from 14 days before immunization with MOG to 30 days after immunization with MOG. For comparison, EAE mice not treated with enalapril were evaluated (EAE group; n = 12; two of 12 mice were used for only pathological analyses). The protocol for this study is summarized in Fig. 1.

Fig. 1.

Fig. 1

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Study protocol. The day of immunization with myelin oligodendrocyte glycoprotein (MOG) is defined as day 1. Enalapril was administered from day −14 to day 30. Blood (100 μl) was collected from the orbital sinus of mice on days −14, 0, 18, and 30 [n = 10, experimental autoimmune encephalomyelitis (EAE) group; n = 10, EAE + enalapril (1·0) group]. Autopsy was performed on day 18 [n = 2, EAE group; n = 2, EAE + enalapril (1·0) group].

B1R antagonist R715 administration in EAE mice treated with enalapril

The effect of the B1R antagonist R715 on EAE + enalapril (1·0) was evaluated separately from the above experiment. The amount of 20 μg (1 mg/kg/day) of R715 (Tocris Bioscience, Bristol, UK) dissolved in 100 μl of sterile phosphate-buffered saline (PBS) [EAE + enalapril (1·0) + R715 group; n = 5] or PBS alone [EAE + enalapril (1·0) + PBS group; n = 5] was injected intraperitoneally on days 11–20 after immunization with MOG. EAE clinical scores in each group were checked on days 1–30.

Measurement of serum bradykinin

Serum bradykinin levels in the EAE mice (n = 10) and EAE + enalapril (1·0) mice (n = 10) on days −14, 0, 18 and 30 were determined using a bradykinin enzyme immunoassay (EIA) kit (EK-009-01; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA), according to the manufacturer's instructions. In brief, an EIA plate coated with secondary antibody was incubated for 2 h with 50 μl of mouse serum diluted 500-fold with assay buffer. After four washes, 100 μl of a streptavidin–horseradish peroxidase solution was added to each well and the plate incubated for 1 h. The plate was washed four times, and 100 μl of substrate solution [3,3′,5,5′-tetramethylbenzidine (TMB)] was added to each well, followed by another 1 h of incubation. Finally, 100 μl of 2N HCl was added. The optical density was measured at 450 nm. Bradykinin levels were calculated by reference to a standard curve. The bradykinin detection range was 5–50 000 ng/ml.

Measurements of serum cytokines

To examine the possible mechanisms by which enalapril could attenuate EAE, we checked the serum levels of IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α in the EAE mice (n = 10) and EAE + enalapril (1·0) mice (n = 10). Measurements were performed using mouse serum samples obtained from mice on days −14, 0 and 18 after EAE induction using a multiplexed fluorescent magnetic bead-based immunoassay (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. In brief, serum samples were centrifuged and supernatants were collected and analysed simultaneously for the above-mentioned cytokines. All serum samples were diluted fourfold with specific Bio-Plex sample diluents. Anti-cytokine-conjugated beads (50 μl) were added to wells of a 96-well filter plate. Next, 50 μl of either sample or cytokine standard was added to the wells and incubated for 60 min. After three washes, detection antibody (25 μl) was added to each well and incubated for 30 min. The plates were washed three times, and 50 μl of streptavidin–phycoerythrin was added to each well, followed by another 10 min of incubation. Finally, 125 μl of assay buffer was added and analysed using a Bio-Plex array reader (Bio-Rad Laboratories). Cytokine levels were determined by reference to the standard curve for each cytokine.

Histopathological analysis

Mouse spinal cords were removed on day 18 after EAE induction (at the peak of disease severity). Two mice that had median severity scores in the EAE + enalapril (1·0) and EAE groups were killed. Pathological examinations were conducted using formalin-fixed sections of spinal cords. Spinal cord tissue was processed as follows: after initial fixation in formalin, the spinal cord tissue was sectioned at 10 μm in the axial plane from the cervical to lumbar spinal cord and stained with haematoxylin and eosin and Luxol fast blue. Spinal cord sections were stained immunohistochemically using the avidin–biotin complex method with a rabbit monoclonal antibody against mouse IL-17 (Bioss Inc., Boston, MA, USA). After section deparaffinization with xylene and gradual dehydration, endogenous peroxidase activity was blocked with 0·5% H2O2 for 15 min. Tissue sections were incubated with 10% normal goat serum (G9023; Sigma-Aldrich, Tokyo, Japan) in PBS and diluted primary antibody (rabbit monoclonal antibody against mouse IL-17, 1:1000) at 4°C overnight. The sections were washed in PBS containing 0·05% Tween-20 (PBST), followed by incubation with biotinylated goat anti-rabbit immunoglobulin (Ig)G (BA-1000; Vector Laboratories, Burlingame, CA, USA; diluted 1:1000), which acted as the secondary antibody, at 4°C overnight. The sections were then washed in PBST and incubated with Vectastain ABC reagent (PK-6100; Vector Laboratories; diluted 1:1000) for 2 h, and then washed in PBST. Finally, staining was visualized using 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and 0·03% hydrogen peroxide in Tris-buffered saline for 10 min. Stained sections were then examined under a light microscope.

For histological evaluation, we examined 10 transverse sections from the cervical to lumbar spinal cord for each mouse and scored the inflammation (inflammatory index) as follows: 0, no inflammation; 1, cellular infiltration only in the perivascular areas and meninges; 2, mild cellular infiltration in the parenchyma; 3, moderate cellular infiltration in the parenchyma; and 4, severe cellular infiltration in the parenchyma.

Statistical analyses

All data were analysed according to the intention-to-treat principle. For baseline variables, groups were compared using the Wilcoxon signed-rank test for paired continuous measures and the Mann–Whitney U-test for unpaired continuous measures. Spearman's rank correlation coefficient was used to evaluate statistical dependence between two variables. All comparisons were planned, statistical tests were two-sided and P-values of <0·05 were considered statistically significant.

Results

Enalapril decreases the severity of EAE

Compared with the EAE group (n = 10), the EAE + enalapril (1·0) group (n = 10) showed significantly decreased severity of EAE on days 14–28 (P < 0·05) and cumulative EAE scores (P = 0·0016). The cumulative scores were defined as total EAE scores of days 1–30 per mouse in each group. Similarly, the EAE + enalapril (0·2) group (n = 5) demonstrated a significant reduction in the severity of EAE on days 15–19 (P < 0·05) compared with the EAE group (n = 10). Although not significant, the maximum EAE clinical scores were lower in the EAE mice treated with enalapril than in the untreated mice (Fig. 2).

Fig. 2.

Fig. 2

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Effects of administering enalapril on experimental autoimmune encephalomyelitis (EAE) clinical scores. (a) EAE clinical score was higher in the EAE group compared to the EAE + enalapril (1·0) group [*P < 0·05, days 14–28, n = 10/EAE group, n = 10/EAE + enalapril (1·0) group, mean ± standard error of the mean (s.e.m.) are displayed, Mann–Whitney U-test] and EAE + enalapril (0·2) group [*P < 0·05, days 15–19, n = 10/EAE group, n = 5/EAE+enalapril (0·2) group, mean ± s.e.m. are displayed, Mann–Whitney U-test]. (b) The mean ± s.e.m. of the cumulative EAE scores and maximum EAE scores of each group (from days 0 to 30). The cumulative EAE scores were higher in the EAE group compared to the EAE + enalapril (1·0) group [*P = 0·016, n = 10/EAE group, n = 10/EAE+enalapril (1·0) group, Mann–Whitney U-test].

R715 reduced the enalapril-induced effects

EAE + enalapril (1·0) + R715 group (n = 5) showed higher EAE clinical scores compared with those of the EAE + enalapril (1·0) + PBS group (n = 5) (Fig. 3). The differences were significant at days 20–23 (P < 0·05). The cumulative and maximum EAE clinical scores tended to be higher in the EAE + enalapril (1·0) mice treated with R715 than in the untreated mice.

Fig. 3.

Fig. 3

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Effects of R715 on clinical scores of experimental autoimmune encephalomyelitis (EAE) treated with enalapril. (a) EAE clinical score was higher in EAE + enalapril (1·0) + R715 compared to EAE + enalapril (1·0) + phosphate-buffered saline (PBS) group [*P < 0·05, days 20–23, n = 5 in each group, mean ± standard error of mean (s.e.m.) are displayed, Mann–Whitney U-test] (b) The mean ± s.e.m. of the cumulative EAE scores and maximum EAE scores of each group (from days 0 to 30).

Bradykinin levels are elevated by enalapril and correlate with EAE severity

In the EAE + enalapril (1·0) group (n = 10), bradykinin levels were significantly higher on day 0 than on day −14 (P = 0·012) and significantly lower on day 18 than on day 0 (P = 0·007). In the EAE group (n = 10), bradykinin levels were significantly lower on day 18 than on day 0 (P = 0·028) (Fig. 4a). The ratio of bradykinin on day 0 to day −14 (day 0/day −14) showed a negative correlation with the cumulative EAE score (P = 0·039, r = −0·467) (Fig. 4b).

Fig. 4.

Fig. 4

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Bradykinin levels in experimental autoimmune encephalomyelitis (EAE) mice. (a) Bradykinin levels in EAE + enalapril (1·0) mice increased on day 0 compared with day −14 (P = 0·012) and decreased on day 18 compared with day 0 (P = 0·007). In EAE mice, bradykinin levels on day 18 were decreased compared with those on day 0 (P = 0·028). (b) The bradykinin ratio correlated negatively with the EAE cumulative score (r = −0·467, P = 0·039, n = 10/EAE group and n = 10/EAE + enalapril (1·0) group, Spearman's rank correlation coefficient).

Enalapril has no effect on serum cytokine levels in EAE mice

Serum IL-6 and IL-17 levels in untreated EAE mice (n = 10) were significantly higher on day 18 than on day 0. Other cytokines did not change between days 0 and 18. These cytokine changes were the same in EAE mice (n = 10) treated with enalapril (1·0 mg/kg/day). Treatment with enalapril did not affect serum cytokine levels (Fig. 5).

Fig. 5.

Fig. 5

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Serum cytokine levels in enalapril-treated and untreated experimental autoimmune encephalomyelitis (EAE) mice. Serum cytokine levels were determined in EAE + enalapril (1·0 mg/kg/day) mice (•) and EAE mice (♦). Serum interleukin (IL)-6 and IL-17 levels in the EAE + enalapril (1·0) and EAE groups were increased on day 18 compared with those on day 0 [*P < 0·05, n = 10/EAE group, n = 10/EAE + enalapril (1·0) group, Wilcoxon's signed-rank test]. Dashed lines indicate the mean level of each cytokine.

Enalapril attenuates CNS inflammation, demyelination and infiltration of IL-17-positive cells into the CNS in EAE mice

EAE mice treated with (n = 2) and without (n = 2) enalapril (1·0 mg/kg/day) were killed on day 18 after EAE induction (at the peak of disease severity). The median clinical score of the untreated EAE mice was 4, whereas that of the enalapril-treated EAE mice was 1. Enalapril treatment decreased cell infiltration and demyelination in the spinal cord of EAE mice. In addition, the infiltration of IL-17-positive cells into the spinal cord was decreased in the EAE + enalapril (1·0) group. Enalapril treatment also significantly decreased the inflammatory index [P < 0·001, n = 20 transverse sections/EAE group, 2·7 ± 0·2 (mean ± standard error); n = 20 transverse sections/EAE + enalapril (1·0) group, 0·6 ± 0·2, Mann–Whitney U-test] (Fig. 6).

Fig. 6.

Fig. 6

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Pathological findings of enalapril-treated experimental autoimmune encephalomyelitis (EAE) and untreated EAE mice. Hematoxylin and eosin (H&E), Luxol fast blue (LFB) and interleukin (IL)-17 staining of the lumbar spinal cord of EAE mice and EAE + enalapril (1·0 mg/kg/day) mice. The spinal cords of EAE + enalapril mice showed diminished infiltration of cells and decreased demyelination compared with those of EAE mice. IL-17 staining was confirmed the expression of IL-17 in untreated but not enalapril-treated EAE mice. Inflammatory index of enalapril-treated EAE mice was significantly lower than that of untreated EAE mice [**P < 0·001, n = 20 transverse sections/EAE group, n = 20 transverse sections/EAE + enalapril (1·0) group, Mann–Whitney U-test]. Yellow bars = 100 μm.

Discussion

In this study, we found significant increases in serum bradykinin levels, reductions in EAE clinical scores and decreased demyelination and inflammation of the CNS in enalapril-treated EAE mice. These findings suggest that KKS, including bradykinin, is involved in EAE pathogenesis.

ACE may be critically involved in promoting autoimmune inflammatory diseases, mediating inflammation and T cell stimulation and influencing the permeability of the blood–brain barrier [3,5,10,11]. In fact, an ACE gene polymorphism is associated with the susceptibility to MS [13], and increased ACE levels have been reported in MS patients [14,15]. ACE inhibitors not only block angiotensin II production but also affect bradykinin pathways. Both elevated bradykinin levels and reduced angiotensin II levels play an important role in EAE amelioration [3,5,11,16]. These pathways may modulate autoimmunity in the CNS, because activation of B1R has been shown to limit encephalitogenic Th17 cell recruitment to the CNS [3]. Blocking ACE induces potent regulatory T cells and modulates Th1- and Th17-mediated autoimmunity in EAE [11]. The present study results show that the ACE inhibitor enalapril suppresses certain immune functions and decreases EAE scores in mice. We presume that the mechanism of enalapril action involves an increase in bradykinin levels, resulting in B1R activation and subsequent inhibition of IL-17-positive cell migration into the CNS; thereby, the ACE inhibitor enalapril attenuated the severity of EAE (Fig. 7). Bradykinin was consumed and decreased by binding B1R at the peak of disease severity. Therefore, bradykinin elevation at day 0 (before mediating CNS inflammation) is important. In fact, the bradykinin ratios (day 0/–14) were correlated negatively with the EAE cumulative score, and IL-17 staining of the spinal cord was reduced in enalapril-treated EAE mice, although serum IL-17 levels were elevated. From these findings, we assume that bradykinin is the major mediator for favourable results for EAE via inhibiting Th17 lymphocyte recruitment into the CNS. Thus, there is a potential interaction between bradykinin and the inflammatory processes in the CNS of EAE mice.

Fig. 7.

Fig. 7

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Assumed mechanism of ACE inhibitor action in experimental autoimmune encephalomyelitis (EAE). Angiotensin-converting enzyme (ACE) inhibitor causes an increase of bradykinin levels, resulting in B1R activation and subsequent inhibition of interleukin (IL)-17-positive cell migration into the central nervous system (CNS). Thus, ACE inhibitor could attenuate the severity of EAE.

ACE inhibitors have been reported to ameliorate EAE severity [5,10,11]. Constantinescu et al. reported the effects of the ACE inhibitor captopril on EAE in Lewis rats. Animals treated with captopril (30 mg/kg/day) from the time of immunization to day 21 had significantly lower mean and cumulative clinical scores compared with untreated animals [10]. They proposed a role for captopril in modulating the immune response that is independent of its anti-hypertensive properties. Treatment with enalapril (10 mg/kg/day) 3 days before immunization in MOG-EAE mice and treatment with lisinopril (1 or 10 mg/kg/day) 2 days before immunization and day 15 in proteolipid-protein-induced EAE mice resulted in a significant amelioration of EAE severity [5,11]. The authors proposed that the mechanism underlying these effects involves the blockade of the renin–angiotensin system, especially blockade of the angiotensin II type 1 receptor, decreased numbers of CD11b or CD11c antigen-presenting cells and impaired expression of chemokines. However, the doses of ACE inhibitors used in these studies were much higher than the clinical doses used in humans. Importantly, we show here that enalapril dosages as low as 0·2–1·0 mg/kg/day are effective in preventing EAE severity in mice, and KKS is also involved in the EAE pathogenesis. Administration of B1R antagonist R715 to EAE mice treated with enalapril reduced enalapril-induced favourable effects, which indicates not only that the renin–angiotensin system, but also KKS is involved in the pathomechanism of EAE attenuation by enalapril administration. We consider that bradykinin is the major mediator for favourable results for EAE via inhibiting Th17 lymphocyte recruitment into the CNS, as reported previously [3].

IL-17 and IL-6 played predominant roles in EAE pathogenesis [17–20]. Our serum cytokine analysis in EAE mice also revealed significant up-regulation of IL-17 and IL-6. However, enalapril did not affect serum cytokine levels. Interestingly, our immunohistochemical studies revealed the presence of IL-17-positive cells in the spinal cord of EAE mice, whereas these immunoreactive cells were reduced in ACE inhibitor-treated EAE mice. Specifically, serum IL-17 levels were increased, but the number of IL-17-positive cells that entered the CNS was decreased in enalapril-treated EAE mice. These results provide direct evidence that this ACE inhibitor modulates blood–brain barrier function in EAE mice and are consistent with the findings of a previous study [3]. Although we analysed IL-17 expression of the CNS in this study, analyses of other cytokine expressions will be required.

ACE inhibitors are inexpensive, safe and used worldwide for cardiovascular indications, with good tolerability. Targeting KKS, ACE inhibitors could be attractive therapeutic agents for application to human MS. Recently, Doerner et al. have reported the clinical analysis that failed to demonstrate a benefit of concomitant administration of ACE inhibitors compared to only IFN-β-1b therapy [21]. There may be a difference in response to ACE inhibitors between human and mouse. Prospective and larger studies in MS patients with ACE inhibitors are highly warranted.

In conclusion, we present evidence implicating a pivotal role for KKS in the pathogenesis of CNS inflammation in EAE. Our results show that ACE inhibitor administration ameliorated IL-17-positive cell invasion to the CNS, elevating serum bradykinin levels and significantly decreasing the severity of EAE in mice. ACE inhibitors may be efficacious in controlling autoimmune responses through their ability to permeate the blood–brain barrier. KKS control is considered to be a critical factor affecting the pathogenesis of EAE and MS.

Acknowledgments

This work was supported partly by research grants from the Ministry of Education, Science, and Technology (A.U.).

Author contributions

All authors were involved in drafting the article or revising it critically for important intellectual content and have read and approved the final version of the manuscript.

Disclosure

The authors declare that there are no conflicts of interest.

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