The non-thiol angiotensin-converting enzyme inhibitor quinapril suppresses inflammatory arthritis

N. Dalbeth1,2, J. Edwards3, S. Fairchild4, M. Callan1,2 and F. C. Hall2,4

1 Division of Medicine, Imperial College London, London, 2 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, 3 Nuffield Department of Orthopaedic Surgery, University of Oxford, Oxford and 4 University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK.

Correspondence to: N. Dalbeth, Department of Immunology, Imperial College London, Commonwealth Building, Hammersmith Campus, Du Cane Rd, London W12 0NN, UK. E-mail: n.dalbeth{at}ic.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. In addition to its vasoactive effects, angiotensin II has proinflammatory properties. Angiotensin-converting enzyme (ACE) inhibitors reduce the production of angiotensin II and could therefore act as anti-inflammatory agents. Here we investigated the capacity of the ACE inhibitor quinapril to modulate inflammatory arthritis.

Methods. We studied the effect of quinapril on disease activity in mice with collagen-induced arthritis (CIA). Mice received oral quinapril (10 mg/kg/day) at the time of arthritis induction (prophylaxis protocol) or at the onset of mild arthritis (therapy protocol). Concentrations of immunoglobulin G (IgG) subtypes specific for bovine Type II collagen and TNF-{alpha} were measured by enzyme-linked immunoassay.

Results. Quinapril significantly diminished the activity of CIA when given as prophylaxis or therapy (prophylaxis protocol, P<0.001; therapy protocol P = 0.002). Antigen-specific IgG2a antibodies were reduced by 52% (P = 0.02) in the quinapril prophylaxis protocol. Suppression of arthritis by quinapril was associated with reduced articular expression of TNF-{alpha} by 68% (P = 0.01) in the prophylaxis protocol and 27% (P = 0.06) in the therapy protocol. Quinapril therapy also inhibited expression of splenocyte TNF-{alpha} production following lipopolysaccharide (LPS) in vitro stimulation by 59% (P = 0.02). In parallel human in vitro experiments, ACE inhibition suppressed LPS-stimulated production of TNF-{alpha} by monocytes. In order to confirm that the action of quinapril occurred predominantly through suppression of angiotensin II, parallel experiments with the angiotensin receptor antagonist candesartan cilexetil demonstrated that this agent also inhibited disease activity in CIA.

Conclusions. These data suggest that angiotensin II is a mediator of chronic inflammation and that ACE inhibition may have therapeutic effects in human inflammatory arthritis.

KEY WORDS: Angiotensin II, Inflammation, Monocytes/Macrophages, Rheumatoid Arthritis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiotensin-converting enzyme (ACE) inhibitors have proven clinical efficacy in the treatment of congestive cardiac failure [1], ischaemic heart disease [2, 3] and renal disease [4]. ACE converts angiotensin I to angiotensin II, and catalyses the degradation of bradykinin and substance P. Although angiotensin II is known to be an important regulator of extracellular volume, vascular tone and blood pressure, it has become apparent that this octapeptide hormone also has a number of other effects and, in particular, autocrine and paracrine proinflammatory properties [5]. Monocytes/macrophages and dendritic cells produce angiotensin II via ACE and express the angiotensin II type 1 (AT1) receptor [6–8]. Angiotensin II signalling through the AT1 receptor leads to activation of the transcription factor nuclear factor {kappa}B (NF-{kappa}B) [9–11] with subsequent production of proinflammatory cytokines and chemokines, reactive oxygen species and adhesion molecules. ACE inhibitors prevent NF-{kappa}B activation [12], and inhibit monocyte and dendritic cell production of proinflammatory cytokines such as TNF-{alpha}, IL-1 and IL-6 [13, 14]. These inflammatory pathways are critical in the maintenance of disease in rheumatoid arthritis (RA) [15, 16], a chronic inflammatory disorder that affects 1% of the population worldwide.

Furthermore, the renin–angiotensin system is disturbed in RA. Elevated ACE activity has been demonstrated in blood monocytes, nodules, synovial fluid and synovial tissue of patients with RA [17–20], and AT1 receptors are present in human synovial tissue [21].

There have been several small open-labelled trials of ACE inhibitors in patients with RA, with variable results [22, 23]. Moreover, the clinical benefits of captopril have been attributed to structural similarities with penicillamine due to its thiol residue [22]. We hypothesized that ACE inhibitors may have anti-inflammatory properties due to the decreased production of angiotensin II. In this case, non-thiol ACE inhibitors and angiotensin receptor antagonists would also have a therapeutic benefit in systemic inflammatory arthritis. Therefore, we studied the effect of the non-thiol ACE inhibitor quinapril and the angiotensin receptor antagonist candesartan in an antigen-specific experimental model of arthritis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice
Adult male DBA/1O1aHsd mice were obtained from Harlan (Bicester, UK) and housed in conventional facilities with temperature control and 12-h light/dark cycles. Mice were kept in cages containing wood shavings and fed standard rodent chow and drinking water ad libitum. All protocols involving live animals were subjected to ethical review by the Oxford Research Ethics Committee and a range of welfare score sheets and action plans were used to monitor and minimize the impact of the study on the animals.

Arthritis induction and assessment
Mice aged 8–10 weeks were immunized with 100 µg bovine collagen type II (bCII, MD Biosciences, Zurich, Switzerland) emulsified in complete Freund's adjuvant (CFA) intradermally at the base of the tail. Optimization experiments were performed to identify an effective arthritis induction regimen with minimum impact on the animals. In our facility, a single intradermal injection of 100 µg bCII/CFA produced CIA of severity comparable with that of the classical regime with a booster at around 21 days [24].

Arthritis onset and severity were assessed by assigning an arthritis score twice weekly. Each paw was assigned a score between 0 and 4: 0 = no arthritis, 1 = mild involvement in a single area (single interphalangeal joint, mid-foot or wrist/ankle); 2 = moderate/severe involvement in a single area (as above) or mild involvement in two or three areas; 3 = severe involvement in two areas or moderate involvement in several areas; 4 = severe involvement in all areas. The total paw score was also recorded with a possible maximum score of 16. All paw assessments were done by the same examiner, who was blinded to the treatment allocation. Paws were harvested post-mortem at the end of the protocols (hindpaws were sectioned 0.5 cm above the ankle joint and forepaws were sectioned at the proximal carpus) and paw weight was recorded.

Treatment protocols
Mice were assigned to study groups using computer-generated random numbers. Mice were assigned to usual drinking water or 10 mg/kg/day of quinapril (Parke Davis, New York, NY, USA) in drinking water. Quinapril was administered at a concentration of 60 mg/l in drinking water, based on a daily fluid intake of 15 ml/100 g body weight [25]. This non-thiol ACE inhibitor was selected because its metabolite, quinaprilat, has the highest potency of binding to and neutralizing tissue ACE [26]. The dose of quinapril was selected after a review of reported rodent studies [27–30]. Furthermore, we studied the physical activity scores of mice in 14-day dose-ranging studies and found that normal spontaneous activity scores were maintained by this dose. The pharmacological effect of this dose of quinapril was confirmed by measuring plasma ACE activity. Mice were assigned to either a prophylaxis protocol in which quinapril (n = 15) or water (n = 13) was given from the time of bCII/CFA injection, or a therapeutic protocol in which quinapril (n = 7) or water (n = 7) was given after arthritis development, at the time that the total paw score reached 4. In addition, the effect of drug withdrawal was studied; mice received quinapril (n = 9) or water (n = 7) from the time of arthritis induction in a similar fashion to the prophylaxis protocol. After 6 weeks, the quinapril was changed to drinking water alone and paw scores were recorded for a further 21 days.

To confirm that the therapeutic effect of quinapril was due to suppression of the renin–angiotensin system, the effect of an angiotensin receptor antagonist was studied in parallel experiments. In these prophylaxis (n = 16) and therapeutic (n = 7) experiments, candesartan cilexetil (AstraZeneca, Mölndal, Sweden) was administered at a dose of 5 mg/kg/day (30 mg/l in drinking water).

Measurement of plasma ACE activity
Plasma ACE activity was measured by the continuous spectrophotometric assay of Holmquist et al. [31]. In this assay ACE hydrolyses the synthetic substrate furylacryloylalanylglycylglycine (Sigma, Poole, UK) to produce furylacryloylalanine and glycylglycine. The hydrolysis of the substrate results in a decrease in absorbance at 340 nm. The assay was performed on a Cobas Mira analyser (Dade-Behring, Liederbach, Germany).

Histology
In order to verify the clinical assessments, paw histology was studied. Hindpaws obtained from mice in the therapeutic protocol were harvested post-mortem, fixed in 4% buffered formalin, decalcified in 5% nitric acid for up to 48 h, routinely processed and embedded in paraffin wax. Sagittal sections (5 µm) were then stained with haematoxylin–eosin and toluidine blue. Specimens were assessed semiquantitatively for synovial hyperplasia, inflammatory cell infiltration and cartilage damage.

Detection of antibodies against bovine type II collagen
Mice were bled from the tail at the beginning and end of the protocols, and individual sera were analysed for anti-bCII antibodies by ELISA. Maxisorp plates (NUNC) were coated overnight at 4°C with 5 µg/ml ELISA-grade bCII (Chondrex, Redmond, WA, USA) or a standard of either mouse IgG1 or mouse IgG2a in duplicate. After washing and blocking steps, serial 2-fold dilutions of mouse serum were added to the bCII-coated wells and incubated at 4°C overnight. Second layer antibody, either alkaline-phosphatase labelled anti-IgG1 or anti-IgG2a (BD Biosciences), was added to washed plates at room temperature for 2 h. Detection was performed using p-nitrophenyl phosphate substrate (Sigma). The reaction was stopped by the addition of 3M sodium hydroxide and the emission at 405 nm was quantified using a Bio-Rad microplate reader. The IgG concentrations were calculated using Microplate Manager III software (Bio-Rad, Hercules, CA, USA) by comparison with the IgG isotype standard curve.

Detection of forepaw TNF-{alpha} levels
Murine TNF-{alpha} was detected by enzyme-linked immunosorbent assay (ELISA; BD Biosciences, Oxford, UK) using paired monoclonal antibodies according to the manufacturer's instructions. Forepaws harvested post-mortem were snap-frozen in liquid nitrogen and stored at –80°C until the time of the assay. Individual paws were initially cut into small sections using a scalpel blade and added to 500 µl of ELISA assay diluent [10% fetal calf serum (FCS) in phosphate-buffered saline]. The tissue was then homogenized and the resultant homogenate was added directly onto the ELISA plate in duplicate.

Splenocytes obtained from mice at the end of a therapeutic protocol were cultured in 96-well plates at 3 x 106 cells/well in complete medium (CM; RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin) or CM supplemented with 100 ng/ml lipopolysaccharide (LPS; Sigma). Cells were cultured for 4 h at 37°C in the presence of 5% CO2. The supernatant was then harvested and the TNF-{alpha} concentration of the supernatant was measured by ELISA as described above.

T-cell assays
At the end of the protocols, cells were isolated from the spleen and draining lymph nodes of the control and quinapril-treated mice. Cells were plated into 96-well U-bottomed plates at a concentration of 5 x 105 cells/well. Cells were cultured for 16 h in 200 µl of CM with or without 50 µg/ml bCII in the presence of 5 µg/ml Brefeldin A. Staphylococcal enterotoxin B (SEB) was used as a positive control. Cells were then harvested and stained for surface expression of CD4 and CD8, then fixed, permeabilized and stained for intracellular expression of interferon {gamma} and IL-4. All samples were analysed on a FACSCalibur using Cell Quest Software (both from BD Biosciences).

Human studies
Ethical approval for human studies was obtained from the Central Oxford Research Ethics Committee and donors provided written consent. Peripheral blood mononuclear cells (PBMC) were obtained from healthy human donors by Lymphoprep (Nycomed Pharma, Oxford, UK) gradient centrifugation. To study the effect of ACE inhibition on human monocyte responses to LPS, cells were preincubated with CM in the presence or absence of captopril (Sigma). Captopril was added at concentrations of 10 and 20 mM. This agent was used for in vitro experiments because, unlike quinapril, captopril is not a prodrug that requires in vivo metabolism to produce its active form. After 2 h, LPS was added to the samples and cells were cultured for 16 h in the presence of Brefeldin A. Cells were stained for surface expression of CD14, then fixed, permeabilized and stained for intracellular IL-1 and TNF-{alpha}. All samples were analysed on a FACSCalibur using CellQuest Software. Dot plots of 3000 events are shown in the figures.

Statistical analysis
All mouse experiments were repeated on at least two occasions, with at least three cages for each experimental condition. The results from all experiments were then pooled. The outcome measures for the prophylaxis experiments were incidence, onset speed (number of days elapsing between immunization and development of arthritis with a score ≥1), peak total and individual paw arthritis severity score, number of paws affected and mean area under the curve (AUC) for arthritis severity score plotted against time. For the therapeutic experiments, the outcome measures were peak total and individual paw arthritis severity score, number of paws affected and mean AUC for arthritis severity score plotted against time.

The AUC was calculated using the trapezium rule [32]. The AUC data from each treatment group was assessed for normality using the Anderson–Darling normality test (Minitab Software; State College, PA, USA). This indicated that the majority of AUC data sets were non-parametric. For non-parametric data, comparative statistics were performed using the Mann–Whitney test. Arthritis incidence data were analysed using the {chi}2 test and parametric data with two-tailed Student's t-tests. Unless specified, data are presented as mean±S.E.M.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Treatment with quinapril inhibited plasma ACE activity
In order to validate the pharmacological effect of quinapril in the mice, plasma ACE activity was measured. We tested plasma ACE activity in five control mice receiving drinking water alone and five mice receiving quinapril at a dose of 10 mg/kg/day for 14 days. Mice receiving quinapril had significantly lower plasma ACE activity than the control mice, confirming the pharmacological effect of quinapril on the murine renin–angiotensin system (Fig. 1).



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FIG. 1. Quinapril inhibits murine plasma ACE activity. Plasma ACE activity was measured in control mice receiving drinking water alone (n = 5) or mice receiving quinapril 10 mg/kg/day (n = 5) for 2 weeks. ***P<0.001. Values indicate mean±S.E.M.

 
The ACE inhibitor quinapril suppressed CIA
In order to determine whether quinapril has a suppressive effect in CIA, we initially studied this agent as prophylaxis, from the time of CFA/bCII immunization (Fig. 2A). There was no significant difference in the incidence or the time to onset of any evidence of arthritis (total paw score >0) in those mice treated with quinapril compared with water. However, quinapril significantly suppressed the severity of CIA in these mice; in the quinapril group the median maximum total paw score was 3 (range 0–8) compared with 10 (4–16) in the control group (quinapril vs control, P = 0.0001). Overall the mean (±S.E.M.) AUC for the quinapril-treated group was 4.1±1.1 compared with 14.5±2.0 for the control group (P = 0.0004). Further analysis showed that quinapril reduced both the number of paws affected and the maximum paw score of individual paws (Table 1). Paw mass was also reduced in the quinapril-treated mice (Table 1).



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FIG. 2. Quinapril reduces the severity of collagen-induced arthritis. Arthritis was induced in DBA/1 mice by immunization with bCII/CFA. (A) Prophylaxis experiments. Mice were randomized to receive water (filled squares, n = 13) or quinapril (open circles, n = 15) from the time of immunization with bCII/CFA. (B) Therapeutic experiments. Mice were randomized to receive water (filled squares, n = 7) or quinapril (open circles, n = 7) when the total paw score reached 4. Day 0 denotes the day of randomization. (C) Withdrawal experiments. Mice were randomized to water (filled squares, n = 7) or quinapril (open circles, n = 9) from the time of immunization with bCII/CFA. All mice received drinking water alone from week 6. Day 0 denotes the day of drug withdrawal. Values indicate mean±S.E.M.

 

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TABLE 1. Analysis of prophylaxis and therapeutic experiments

 
We wished to establish whether this suppressive effect was also present following the onset of CIA. Therefore, we repeated these experiments in mice with clinically detectable arthritis in a therapeutic protocol (Fig. 2B). Again, quinapril significantly and rapidly suppressed the severity of arthritis. This effect persisted throughout the treatment period. In the quinapril-treated group, the median total maximum paw score recorded at any time during the treatment period was 4 (4–10), compared with 12 (11–16) in the control group (P = 0.002). Overall the mean AUC for the quinapril-treated group was 12.3±2.4 compared with 36.2±3.3 in the control group (P = 0.002). Paw mass was also reduced in the quinapril-treated group (Table 1).

In order to examine whether the effect of quinapril was present only when mice were receiving the drug, we studied the consequences of quinapril withdrawal. In those mice that had received quinapril from the time of immunization with bCII/CFA, the severity of arthritis remained less after quinapril withdrawal, compared with those mice that had received water throughout the protocol (Fig. 2C). Even after 3 weeks of drug withdrawal there was a significant difference between paw scores in the control and quinapril-treated mice (P = 0.03). The mean total paw score in the quinapril withdrawal group increased from 2.1±1.1 at the time of drug withdrawal to 4.4±1.6 after 3 weeks of drug withdrawal (P = 0.06).

The apparent clinical effect of quinapril was confirmed by histological examination; paws from quinapril-treated mice had reduced inflammatory infiltrate, bone erosion and cartilage damage on histological examination (Fig. 3).



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FIG. 3. Clinical benefit of quinapril is confirmed on paw histology. Hindpaws harvested at the end of a therapeutic protocol (week 8) were examined histologically using haematoxylin–eosin and toluidine blue. Quinapril therapy was associated with reduced inflammatory infiltration and cartilage erosion compared with control arthritic mice. Representative slides of the tarsal bones are shown. Magnification x40.

 
Quinapril suppressed the production of antigen-specific IgG2a antibodies
We then wished to examine the mechanisms whereby quinapril suppresses arthritis activity. Serum levels of anti-bCII IgG subtypes from mice at the end of prophylaxis and therapeutic protocols were measured (Fig. 4A and B). In all groups there was a trend to reduction in antigen-specific humoral response. This reached statistical significance only for the IgG2a subtype in mice receiving quinapril in the prophylaxis protocol. These data suggest that ACE inhibition may influence antigen specific B-cell function when given from the time of antigen exposure.



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FIG. 4. The effect of quinapril on bCII-IgG subtype concentrations and TNF-{alpha} production. Individual sera taken at the end of the protocols (week 8) were analysed for (A) anti-bCII IgG1 and (B) anti-bCII IgG2a by ELISA. Antibody levels are expressed as µg/ml. n = 18 for prophylaxis and n = 14 for therapeutic protocols. (C) TNF-{alpha} levels were measured on forepaw homogenates by ELISA. n = 23 for prophylaxis and n = 14 for therapeutic protocols. (D) Splenocytes were obtained from mice (n = 7) in a therapeutic protocol (day 56) and cultured for 4 h in complete medium (CM) with or without LPS 100 ng/ml. The culture supernatant was harvested and TNF-{alpha} concentration was measured by ELISA. *P<0.05. Values indicate mean±S.E.M.

 
Since the synthesis of IgG2a antibodies is associated with Th1-type responses in mice, we hypothesized that ACE inhibition would decrease the production of Th1 cytokines by antigen-specific T cells. However, our experiments of splenocyte and draining lymph node T-cell cytokine production following stimulation with bCII or with SEB failed to demonstrate a significant difference between the quinapril and control groups (data not shown).

Quinapril inhibited local and stimulated TNF-{alpha} production
Previous reports have shown that ACE inhibitors can suppress the production of TNF-{alpha} by dendritic cells and monocytes. In order to determine whether the therapeutic effect of quinapril was due to inhibition of TNF-{alpha}, we measured TNF-{alpha} concentrations at the paw, the local site of inflammation in CIA. Paw TNF-{alpha} concentration was significantly reduced in those mice receiving quinapril as prophylaxis, compared with those receiving water. A similar trend was found in arthritic mice receiving quinapril in the therapeutic protocol (Fig. 4C).

Although these results suggested that quinapril may have a beneficial effect by inhibiting TNF-{alpha} production, the reduction of paw TNF-{alpha} in quinapril-treated mice may simply reflect the suppressed disease activity. We wished to provide more direct evidence that quinapril suppresses TNF-{alpha} production in inflammatory arthritis. Therefore, we examined the effect of LPS on TNF-{alpha} production using splenocytes from arthritic mice. On in vitro stimulation with LPS, splenocytes from quinapril-treated mice produced significantly less TNF-{alpha} than those from water treated mice (Fig. 4D).

Effects on human monocyte cytokine production
In order to identify whether these observations have clinical relevance in human subjects, we examined whether ACE inhibition could reduce human monocyte cytokine production following LPS stimulation. We cultured human PBMCs in LPS with or without preincubation with the ACE inhibitor captopril. Quinapril was not used in these experiments because, unlike captopril, it is a prodrug that requires in vivo metabolism for conversion into its active form. In these in vitro experiments, ACE inhibition significantly inhibited LPS-stimulated production of IL-1 and TNF-{alpha} in a dose-dependent manner (Fig. 5). These results suggest that autocrine production of angiotensin II is important in promoting human monocyte responses to other proinflammatory stimuli.



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FIG. 5. ACE inhibition reduces human monocyte production of TNF-{alpha} and IL-1. PBMCs from healthy human donors were preincubated in the presence or absence of captopril at indicated concentrations for 2 h. LPS (1 ng/ml) was then added and cells were cultured in the presence of Brefeldin A (5 µg/ml) for 16 h. These figures indicate cells within the CD14+ gate, and show intracellular staining for TNF-{alpha} and IL-1. This figure is representative of experiments from four donors.

 
AT1 receptor blockade also suppressed disease activity in CIA
In addition to the decreased synthesis of angiotensin II, ACE inhibitors influence the metabolism of other peptides, such as bradykinin and substance P. These peptides may also influence the inflammatory response. In order to confirm that the observed benefit of quinapril was due to angiotensin II suppression, we also tested the effect of the AT1 receptor antagonist candesartan on disease severity in CIA. This drug directly inhibits the interaction between the AT1R and angiotensin II. Candesartan significantly inhibited disease activity in both prophylaxis and therapeutic protocols (Fig. 6). When administered in a prophylaxis protocol, candesartan reduced the time to onset of arthritis from 17.2±1.9 days (control) to 30.4±3.5 days (candesartan) (P<0.01). Candesartan prophylaxis significantly suppressed the severity of CIA in these mice; in the candesartan group the median maximum total paw score was 3 (range 1–15) compared with 10 (4–16) in the control group (candesartan vs control, P<0.01). Overall the mean (±S.E.M.) AUC for the candesartan-treated group was 7.2±2.6 compared with 14.5±2.0 for the control group (P<0.05) (Fig. 6A).



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FIG. 6. Angiotensin receptor blockade also suppresses disease activity in CIA. Arthritis was induced in DBA/1 mice by immunization with bCII/CFA. (A) Prophylaxis experiments. Mice were randomized to receive water (filled squares, n = 13) or candesartan cilexetil (open circles, n = 16) from the time of immunization with bCII/CFA. (B) Therapeutic experiments. Mice were randomized to receive water (filled squares, n = 7) or candesartan (open circles, n = 7) when the total paw score reached 4. Day 0 denotes the day of randomization. Values indicate mean±S.E.M.

 
In the therapeutic protocols, candesartan also significantly suppressed the severity of arthritis. Overall, the mean AUC for the candesartan-treated group was 23.1±4.1 compared with 36.2±3.3 in the water group (P<0.05) (Fig. 6B).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
These experiments demonstrate that both the non-thiol ACE inhibitor quinapril and the angiotensin receptor blocker candesartan have significant anti-inflammatory properties, sufficient to suppress the severity of collagen-induced arthritis, either as prophylaxis or as therapy in established disease. Administration of quinapril from the time of antigen exposure may influence the priming of the immune response, as indicated by the sustained effect after quinapril withdrawal and by the reduction in bCII-specific IgG2a antibodies. Our data also show that quinapril has clear effects on TNF-{alpha} production, a key cytokine in the pathology of human inflammatory arthritis [16]. Suppression of arthritis by quinapril is also associated with reduced expression of TNF-{alpha} within the joints of mice and with diminished expression of splenocyte TNF-{alpha} production following LPS stimulation in vitro.

There have been conflicting reports regarding the benefits of the ACE inhibitor captopril in the rat adjuvant model of localized arthritis [33, 34]. The clinical anti-inflammatory effects of captopril in murine and human disease have been attributed to its thiol residue. An open-labelled study of the non-thiol ACE inhibitor pentopril in 15 patients with active RA has reported lack of efficacy. Interestingly, even with this small sample size, a significant reduction in C-reactive protein was observed, suggesting some benefit on inflammatory disease [23]. However, this study was inadequately powered. By analogy with the recent trial which demonstrated a significant disease-modifying activity of atorvastatin in RA [35], assessment of the effect of ACE inhibition in RA requires a placebo-controlled double-blind study of approximately 100 RA patients, using validated outcome measures. The present study demonstrates significant anti-inflammatory effects with both a non-thiol ACE inhibitor and an angiotensin receptor antagonist, suggesting that direct influence on angiotensin II is responsible for the therapeutic efficacy of quinapril in the CIA model. Whether these agents also provide significant clinical efficacy in human disease requires further analysis through adequately powered and controlled clinical trials.

The dose of quinapril used in these experiments is within the range commonly used in rodent studies, and was not associated with any significant alterations in mouse physical activity scores, thereby excluding significant hypotension. However, this dose is clearly higher than the usual human doses of quinapril. Mice and humans exhibit several differences in both phase I and II hepatic metabolism of drugs [36]. Therefore, it is inappropriate to predict an anti-inflammatory dose of ACE inhibitors in humans, using relative body mass. However, it is conceivable that the drug doses required for a therapeutic benefit may not be tolerated in humans with RA, particularly in those with normal blood pressure at baseline.

It is interesting that a 37% reduction in plasma ACE activity was associated with such significant improvements in arthritis in those mice treated with quinapril. Studies of patients receiving ACE inhibitors have demonstrated that the clinical benefits of these agents are often not reliably estimated by circulating ACE activity [37]. ACE is primarily located in tissues, and animal models have indicated that inhibition of tissue ACE generally has a stronger correlation with the haemodynamic effects of ACE inhibitors than inhibition of ACE in plasma [38]. Of the available ACE inhibitors, quinaprilat (the active metabolite of quinapril) has the greatest potency of ACE binding and also highest tissue retention [26]. The ability to achieve high levels of ACE inhibition within tissue may explain the apparent discrepancy between plasma ACE activity and clinical benefits on arthritis in this study.

Our finding that quinapril has a significant anti-inflammatory effect in arthritis is supported by several studies examining the benefits of ACE inhibitors in other models of inflammation, including autoimmune myocarditis [39], chronic pancreatitis [40] and experimental allergic encephalomyelitis [41]. The mechanism of this anti-inflammatory effect is likely to be complex. Nahmod et al. have shown that dendritic cell differentiation and endocytosis, mixed lymphocyte reaction responses and in vivo antibody responses are promoted by angiotensin II and inhibited by blockade of the AT1 receptor [8]. These data imply that angiotensin II has a significant effect on the development of adaptive immunity. The inhibition of bCII-specific IgG2a antibodies and the sustained effect of quinapril prophylaxis following drug withdrawal in our experiments suggest that this drug may modulate priming of the immune response at the time of antigen exposure [42]. However, we have been unable to demonstrate alterations of T-cell cytokine production in the arthritic mice following quinapril administration.

The reduction of collagen-specific antibodies by quinapril prophylaxis may have direct therapeutic effects. Passive transfer of serum or CII-specific antibodies from rats with CIA can induce arthritis [43] and B cells are required for the development of CIA [44]. CII-specific IgG2a antibodies are also associated with the development of arthritis in collagen immunized mice [45]. However, it is unlikely that reduction in antigen-specific antibodies accounts entirely for the therapeutic effect of quinapril in CIA, as the striking benefits of quinapril in the therapeutic regimen were not associated with significant reductions in antibody levels.

In order to clarify whether the therapeutic benefits of quinapril are related to effects on angiotensin II, we analysed parallel experiments of the AT1 receptor antagonist candesartan cilexetil. These experiments demonstrate for the first time that blockade of the AT1 receptor has therapeutic effects on inflammatory arthritis. Thus, it is likely that the dominant effect of quinapril in CIA is also due to suppression of angiotensin II. Previous studies have shown that angiotensin II promotes the activation of the transcription factor NK-{kappa}B, and thereby the production of reactive oxygen species, prostaglandins, matrix metalloproteases, angiogenesis factors, adhesion molecules and inducible chemokines [5]. The mechanism of action of quinapril in CIA may involve these pathways. However, we have focused on TNF-{alpha} because this cytokine plays a key role in the pathogenesis of inflammatory arthritis [16]. These data clearly show that ACE inhibition modulates TNF-{alpha} expression in arthritic mice.

While the synovial disease of RA is readily apparent, it has become evident that patients with RA are also at increased incidence of cardiovascular disease [46–49]. The risk of accelerated atherosclerosis in RA is independent of traditional cardiovascular risk factors [47], and may be associated with systemic inflammatory activity [50]. The use of other drugs beneficial in cardiovascular disease has been studied in inflammatory arthritis. Recent studies have shown that that statins have immunomodulatory effects by inhibiting the development of the Th1 response [51], and that these drugs have therapeutic benefits in both collagen induced arthritis [52] and human disease [35]. There is some evidence that ACE inhibitors and statins have synergistic anti-inflammatory effects in vascular disease [53]. It will be interesting to examine this combination of drugs further in other models of inflammation, such as inflammatory arthritis.

In summary, these data suggest that suppression of angiotensin II production by ACE inhibition has a potential therapeutic effect in inflammatory arthritis. These drugs represent oral agents that may be capable of modulating TNF-{alpha} expression. Our data provide support for well controlled clinical trials of ACE inhibitors in RA, both for management of articular disease and also for RA-associated cardiovascular disease.


    Acknowledgments
 
The authors wish to thank Dr Colin Hetherington for assistance with animal care and Mr Keith Burling for measurement of plasma ACE activity.

No conflict of interest has been declared by the authors.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 8 July 2004; revised version accepted 6 August 2004.