Am80, a retinoic acid receptor agonist, ameliorates murine vasculitis through the suppression of neutrophil migration and activation

Authors


Abstract

Objective

Vasculitis is characterized by leukocyte infiltration in the vessel walls, with destructive damage to mural structures. Retinoids are compounds that bind to retinoic acid receptors and exert biologic activities similar to those of vitamin A, including modulatory effects on cell proliferation and differentiation. This study was undertaken to examine the therapeutic effects of a synthetic retinoid, Am80, in a murine model of vasculitis induced by Candida albicans water-soluble fraction (CAWS).

Methods

Vasculitis was induced in BALB/c mice by intraperitoneal injection of CAWS. Neutrophils were depleted by injection of antineutrophil antibody–positive serum. Am80 was administered orally once daily. Vasculitis was evaluated histologically. Migration of labeled adoptively transferred cells was quantified. Chemotaxis was assessed by cell mobility analysis. Production of reactive oxygen species (ROS) and phosphorylation of MAPKs were measured by flow cytometry. Concentrations of elastase were measured by enzyme-linked immunosorbent assay.

Results

Administration of CAWS induced vasculitis in the coronary arteries and aortic root, with abundant neutrophil infiltration. Depletion of neutrophils reduced CAWS-induced vasculitis. Treatment with Am80 led to a significant attenuation of the vasculitis score and inhibition of the migration of transferred neutrophils into the site of vasculitis. In vitro, Am80 suppressed fMLP-induced chemotaxis of human peripheral blood neutrophils. ROS production and elastase release by stimulated neutrophils were reduced by AM80 treatment, and Am80 also inhibited phosphorylation of ERK-1/2 and p38 in neutrophils stimulated with fMLP plus lipopolysaccharide.

Conclusion

Am80 significantly suppressed CAWS-induced vasculitis. This effect was presumably exerted via inhibition of neutrophil migration and activation.

Vasculitis is defined by the presence of inflammatory leukocytes in vessel walls, with destructive damage to mural structures. Antineutrophil cytoplasmic antibodies are often detected in certain types of vasculitis, such as microscopic polyangiitis. The affected vessels vary in size, type, and location according to the type of vasculitic disease (1). Although the exact mechanisms underlying these disorders are unclear, activated polymorphonuclear cells (neutrophils) in the vascular endothelium are thought to play an important role in the pathogenesis of the vasculitides (2). Oral corticosteroids and immunosuppressive agents are commonly used for the treatment of vasculitis. However, in some cases the disease is refractory to these treatments, and immunosuppression often leads to significant clinical complications. Therefore, there is a need for a new low-cost therapy for vasculitis that is more effective and safer than currently used treatments.

An experimental mouse model of vasculitis has been developed in which the disease is induced by administration of Candida albicans water-soluble fraction (CAWS) (3–7). The experimental mice exhibit severe coronary arteritis accompanied by neutrophil activation and production of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6 (8). CAWS-induced coronary arteritis is considered to be an appropriate model for use in studies aimed at understanding the pathogenesis of arteritis as well as developing novel treatments.

Retinoid, a derivative of vitamin A, is a general term for compounds that bind to and activate retinoic acid receptors (RARs [RARα, RARβ, and RARγ]) and/or retinoid X receptors (RXRs [RXRα, RXRβ, and RXRγ]), members of the nuclear receptor superfamily (9). RARs are transcriptional regulators that bind to specific retinoic acid response elements present in the promoters of their target genes. Retinoids have important roles in cell proliferation, differentiation, and morphogenesis (10). They have also been reported to promote differentiation of Th2 and Treg cells and to suppress Th1 and Th17 differentiation (11). In addition, retinoids inhibit tumor necrosis factor α and nitric oxide production by murine peritoneal macrophages and human keratinocytes (12, 13). Furthermore, previous studies have indicated that retinoids inhibit neutrophil activation, including superoxide anion and protease release (14–17). Clinically, retinoids are used for the treatment of cutaneous inflammatory disorders such as psoriasis and acne (18, 19). Therefore, retinoids may have a beneficial effect in neutrophil-dominant diseases. All-trans-retinoic acid, the most notable endogenous retinoid, is a ligand for RARα, RARβ, and RARγ.

Am80 is a specific ligand for RARα and RARβ but not for RARγ (20), and is characterized by higher stability, fewer side effects, and superior bioavailability, compared with all-trans-retinoic acid (20, 21). All-trans-retinoic acid and Am80 are used for the treatment of acute promyelocytic leukemia (21, 22). The present study was designed to determine the effects of Am80 on CAWS-induced vasculitis and on neutrophil migration and activation.

MATERIALS AND METHODS

Induction of CAWS-induced vasculitis, depletion of neutrophils, and treatment with Am80.

CAWS was prepared from C albicans strain IFO1385, using a previously described method (23). Six-week-old male BALB/c mice were purchased from Oriental Yeast Company. To induce vasculitis, CAWS (1 mg) was injected intraperitoneally into the mice in a volume of 0.2 ml once daily for 5 days (day 1 to day 5).

Neutrophils were depleted using a modification of a previously described method (24). Briefly, 0.2 ml of saline-diluted (1:10) rabbit anti–mouse neutrophil antibody–positive serum (Accurate) was injected intraperitoneally once daily from day 1 to day 5. Subsequently, an additional dose was injected once every other day to maintain neutropenia. The control group received normal rabbit serum (Accurate) in the same volume. As a therapeutic administration after vasculitis had developed, rabbit anti–mouse neutrophil antibody–positive serum was injected from day 8 to day 12, with an additional dose injected every other day.

Am80 was suspended in carboxymethylcellulose. Carboxymethylcellulose alone as vehicle or Am80 (1.0 mg/kg or 4.0 mg/kg) in carboxymethylcellulose was administered orally once daily from day 1 to day 35 (prophylactic administration) or from day 8 to day 35 (therapeutic administration). On day 36 the mice were killed and the hearts were harvested and examined histologically. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.

Immunohistochemistry.

Immunohistochemical analysis was conducted on OCT-embedded sections of frozen heart tissue. Briefly, 6-μm–thick cryostat sections were fixed in 4% paraformaldehyde for 10 minutes, and then the samples were rehydrated in 0.1% Triton X-100 in phosphate buffered saline (PBS) for 3 minutes. Endogenous peroxidase activity was blocked by incubation in 1.5% H2O2 in PBS for 15 minutes, followed by rinsing with PBS. Nonspecific binding was blocked by addition of 10% normal goat serum in PBS for 40 minutes. The sections were incubated overnight at 4°C with 10 μg/ml rat anti-mouse Ly-6G monoclonal antibody (mAb) (RB6-8C5; eBioscience), 5 μg/ml rat anti-mouse F4/80 mAb (C1:A3-1; Serotec), 5 μg/ml rat anti-mouse CD4 mAb (GK1.5; Cymbus Biotechnology), or normal rat IgG in antibody diluent (BD PharMingen). The samples were washed 3 times in PBS and then incubated for 30 minutes with 2.5 μg/ml biotin-conjugated rabbit anti-rat IgG (DakoCytomation) pretreated with 5% normal mouse serum to reduce nonspecific binding. After washing in PBS, the sections were incubated for 30 minutes with streptavidin–horseradish peroxidase. After washing in PBS, diaminobenzidine (DakoCytomation) was used for visualization. The sections were counterstained with hematoxylin for 10 seconds and washed in tap water for 5 minutes. F4/80-positive and CD4-positive cells were counted in 4 randomly selected fields examined at 200× magnification under light microscopy.

Histologic evaluation.

The fixed hearts were embedded in paraffin and sectioned. In order to observe the histologic changes in the coronary arteries and the aorta in detail, we prepared step sections 20 μm apart in a horizontal direction. The sections were stained with hematoxylin and eosin. For quantitative evaluation of vascular inflammation, each of 5 areas (3 aortic root areas and both coronary arteries) was scored as 0 (no inflammatory cell migration) or 1 (presence of panvasculitis). The severity of the arteritis in each mouse was defined as the sum of the scores of the 5 segments (maximum possible score of 5).

Migration of Ly-6G–positive splenocytes into the aortic wall.

Ly-6G–positive splenocytes from normal BALB/c mice were purified using MACS Microbead–coupled mAb and magnetic cell separation columns (Miltenyi Biotec). The cells were shown to be >95% pure. Most of the purified cells were polymorphonuclear, indicating that they were neutrophils. The purified Ly-6G–positive cells were labeled with CellTracker Orange 5-(and-6)-([4-chloromethyl] benzoyl amino) tetramethylrhodamine (CMTMR) according to the protocol supplied by the manufacturer (Molecular Probes). The CMTMR-labeled cells (1.6 × 107) were injected intravenously into the tail vein of mice with CAWS-induced vasculitis on day 15. The recipient mice were administered vehicle or Am80 (4 mg/kg) orally, 24 hours and 2 hours before and 22 hours after the transfer. Twenty-four hours after the transfer, the mice were killed, hearts were harvested and embedded in OCT compound, and horizontal 5-μm–thick sections were prepared. The total number of CMTMR-labeled cells that had migrated into the aortic wall was counted under fluorescent microscopy (Biozero). Two slides from each heart were evaluated independently by 2 observers, and the mean number of cells was recorded.

Expression of RARs in human peripheral blood neutrophils.

Neutrophils from peripheral blood of healthy human controls were obtained using Mono-Poly resolving medium (Dainippon), followed by positive removal of all contaminating cells with mAb against CD3, CD56, CD19, CD36, CD49d, and glycophorin A using a custom-made EasySep kit (Stemcell Technologies) as previously described (25). Total RNA was prepared from human peripheral blood neutrophils and synovial fibroblasts obtained from a patient with rheumatoid arthritis (26), and first-strand complementary DNA (cDNA) was synthesized using 2 μg total RNA. Polymerase chain reaction (PCR) was performed in a total volume of 50 μl containing 1 μl cDNA, 0.2 mM dNTP, 0.02 μM of each primer, 1× PCR buffer (Roche Molecular Systems), and FastStart Taq DNA polymerase (Roche Molecular Systems), with a thermal cycler (PTC-200; MJ Research). After the initial denaturing step (94°C for 5 minutes), amplification was performed for 35 cycles at 94°C for 60 seconds, 66°C for 30 seconds, and 72°C for 60 seconds. The final cycle was followed by an extension step of 5 minutes at 72°C. The sequences of the primers were as follows: RARα 5′-TGG-GTG-GAC-TCT-CCC-CGC-CA-3′ (sense), 5′-CCC-ACC-TCC-GGC-GTC-AGC-GTG-3′ (antisense) (product size 438 bp); RARβ 5′-CAC-TGG-CTT-GAC-CAT-CGC-AGA-CC-3′ (sense), 5′-GAG-AGG-TGG-CAT-TGA-TCC-AGG-3′ (anti-sense) (product size 500 bp); RARγ 5′-GGC-CTG-GGC-CAG-CCT-GAC-CTC-3′ (sense), 5′-CAG-CCC-CAG-ATC-CAG-CTG-CAC-G-3′ (antisense) (product size 537 bp); β-actin 5′-GTC-CTC-TCC-CAA-GTC-CAC-ACA-3′ (sense), 5′-CTG-GTC-TCA-AGT-CAG-TGT-ACA-GGT-AA-3′ (antisense) (product size 239 bp). PCR products were resolved by electrophoresis on 1.5% agarose gels (Takara Bio) containing ethidium bromide.

In vitro chemotaxis assay.

The purified human peripheral blood neutrophils were incubated for 2 hours at 37°C in RPMI 1640 medium (Sigma-Aldrich) with 10% fetal calf serum (FCS), without Am80 or in the presence of Am80 at 10−7, 10−6, or 10−5 moles/liter. Chemotaxis of the neutrophils was examined using an EZ-TAXIScan (ECI, Inc.) for quantitative measurement of cellular chemotaxis, as previously described (27). Neutrophils were aligned to one edge of a TAXIScan holder, and 10 nM fMLP was applied to the opposite compartment. Cell migration at 25°C was recorded every 30 seconds for 45 minutes. After the assay, the digital images were converted into videos for analysis, and the number of cells moving into the assay field was analyzed using TAXIScan Analyzer software (ECI, Inc.). Cell viability was measured using a Cell Counting Kit-8 according to the instructions of the manufacturer (Dojindo). The viable cell number of neutrophils treated with 10−5 moles/liter Am80 for up to 5 hours was >90% compared with that of vehicle-treated neutrophils, suggesting that culture with Am80 did not affect neutrophil viability. Concentrations of Am80 in the in vitro experiments were determined, according to the plasma concentration of Am80 administered to the mice (28).

Measurement of generated reactive oxygen species (ROS).

Human peripheral blood neutrophils were incubated for 5 hours at 37°C, without Am80 or in the presence of Am80 at 10−8, 10−7, or 10−6 moles/liter in RPMI 1640 with 10% FCS. Cells were stimulated with 1 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 10 minutes at 37°C, 1 μg/ml palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4; Calbiochem) for 90 minutes at 37°C, or 5 μg/ml lipopolysaccharide (LPS; Sigma-Aldrich) for 90 minutes at 37°C. The Pam3CSK4- and LPS-treated cells were then stimulated with 100 nM fMLP (Sigma-Aldrich) for 5 minutes. The neutrophils were subsequently incubated with 1 μM dihydrorhodamine 123 (DHR-123; Marker Gene Technologies) for 5 minutes and then washed with cold PBS. To estimate intracellular peroxide production, the fluorescence intensity of rhodamine 123, which is a product of cellular oxidation of DHR 123, in 10,000 cells was recorded using an Accuri C6 flow cytometer. Three independent experiments were performed in triplicate.

Measurement of elastase release.

Human neutrophils were incubated for 5 hours at 37°C, without Am80 or in the presence of Am80 at 10−8, 10−7, or 10−6 moles/liter in RPMI 1640 with 10% FCS. The cells were then incubated for 30 minutes with 10 μg/ml cytochalasin B (Sigma-Aldrich) and 1 μM fMLP. The concentration of elastase in the culture supernatant was measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the instructions of the manufacturer (Hycult).

Phosphospecific flow cytometry.

The neutrophils were stimulated with fMLP (1 μM) and LPS (1 μg/ml) at 37°C. At various time intervals, 1 × 106 cells were fixed in 3% formaldehyde, washed in PBS, and resuspended in ice-cold 90% methanol at 4°C for 30 minutes. The cells were then washed twice in PBS with 2% FCS before incubation with rabbit anti–phosphorylated ERK-1/2 antibody (9101; Cell Signaling Technology) or rabbit anti–phosphorylated p38 antibody (9211; Cell Signaling Technology) for 20 minutes at room temperature. Phosphospecific antibodies were detected by subsequent incubation with phycoerythrin-conjugated goat anti-rabbit antibody (2.5 μg/ml; Beckman Coulter). Cells were then analyzed by flow cytometry using an Accuri C6 flow cytometer. To evaluate the effect of Am80 on the phosphorylation of ERK-1/2 or p38, the cells were incubated with 10−6 moles/liter Am80 or control medium for 3 hours at 37°C before stimulation with fMLP and LPS. Unstimulated samples showed no increase in phosphorylation of ERK-1/2 or p38 throughout the experiment.

Statistical analysis.

All data are expressed as the mean ± SEM. The statistical significance of differences between groups was assessed by Student's t-test. P values less than 0.05 were considered significant.

RESULTS

Effects of neutrophil depletion on CAWS-induced vasculitis.

We induced vasculitis in BALB/c mice by administration of CAWS once daily for 5 days (days 1–5). On day 36, the mice were killed and the hearts were evaluated histologically. Although normal mice exhibited no inflammatory changes (Figure 1A), CAWS-injected mice showed inflammatory cell infiltration into the aortic root and coronary arteries (Figure 1B). Immunohistochemical analysis revealed massive invasion of Ly-6G–positive neutrophils in the vascular wall (Figure 1C). Moderate infiltration of F4/80-positive macrophages and CD4-positive T cells was also observed (Figures 1D and E).

Figure 1.

Histologic findings in BALB/c mice with Candida albicans water-soluble fraction (CAWS)–induced vasculitis. A and B, Hematoxylin and eosin (H&E)–stained heart specimens obtained on day 36 from a normal mouse (A) and a mouse that was injected with CAWS once daily from day 1 to day 5 (B). NCC = noncoronary cusp; LCC = left coronary cusp; RCC = right coronary cusp; LCA = left coronary artery; RCA = right coronary artery. C–F, Identification of infiltrated cells in the aortic wall. The hearts of CAWS-injected mice were stained with Ly-6G (C), F4/80 (D), CD4 (E), or isotype control (F). All sections were counterstained with hematoxylin. G and H, H&E-stained heart specimens obtained on day 17 from a CAWS-injected mouse administered control serum (G) and a CAWS-injected mouse administered antineutrophil antibody–positive serum (H) from day 1 to day 5 and subsequently every other day. I, Mean ± SEM vasculitis scores in mice treated as described in G and H (n = 8 in the control serum–treated group; n = 11 in the antineutrophil antibody–positive serum–treated group). ∗ = P < 0.05 versus control serum–treated group. J, Mean ± SEM vasculitis scores in mice treated with control serum or antineutrophil antibody–positive serum beginning on day 8 (n = 12 in each group). NS = not significant. Specimens shown in A–H are representative of the respective treatment groups. Original magnification × 40 in A, B, G, and H; × 100 in C–F.

Next we analyzed the effect of neutrophil depletion on CAWS-induced vasculitis. Rabbit anti–mouse neutrophil antibody–positive serum was administered from day 1 to day 5, and an additional dose was subsequently injected every other day to maintain neutropenia. The number of peripheral blood neutrophils was decreased to 43% of baseline by day 17. The neutrophil depletion reduced inflammatory cell infiltration into the aortic root and coronary arteries compared to that observed in mice treated with control serum (Figures 1G and H). The vasculitis score was significantly lower in neutrophil-depleted mice (Figure 1I). Neutrophil depletion also reduced the number of F4/80-positive macrophages and CD4-positive T cells infiltrating into the vessel wall (mean ± SEM 43.0 ± 4.2 F4/80-positive macrophages per field and 29.0 ± 2.7 per field in control serum–treated and antineutrophil antibody–positive serum–treated animals, respectively, and 20.0 ± 1.8 CD4-positive T cells per field and 13.25 ± 1.5 per field, respectively; P < 0.05 for both). Neutrophil depletion beginning on day 8 (after the onset of vasculitis) did not significantly reduce the vasculitis score (Figure 1J).

Effects of Am80 on CAWS-induced vasculitis.

Next we examined the effect of Am80 on CAWS-induced vasculitis, since previous studies have suggested that retinoids inhibit the activation of neutrophils (14–17). Am80 (1 mg/kg or 4 mg/kg) was administered orally once daily from days 1 to 35. Prophylactic Am80 administration reduced inflammatory cell accumulation in the aortic root and coronary arteries by day 36 (Figures 2A–C) and significantly reduced the vasculitis score in a dose-dependent manner (Figure 2D). We also analyzed the therapeutic effects of Am80 (4 mg/kg) administered from day 8 to day 35. Vasculitis was already present on day 8 (Figure 2E). On day 36, the above treatment schedule also significantly suppressed the histologic features of vasculitis compared with vehicle (Figures 2F–H); however, the vasculitis score on day 36 was not decreased compared with that on day 8 (Figure 2G).

Figure 2.

Effect of Am80 on Candida albicans water-soluble fraction (CAWS)–induced vasculitis. A–C, Hematoxylin and eosin (H&E)–stained heart specimens obtained on day 36 from CAWS-treated BALB/c mice administered vehicle (A) or Am80 at 1 mg/kg (B) or 4 mg/kg (C) (n = 10 per group) once daily for 5 weeks, as prophylaxis. D, Mean ± SEM vasculitis scores in mice treated as described in A–C. ∗ = P < 0.05 versus vehicle-treated group. E–G, H&E-stained heart specimens from CAWS-treated BALB/c mice administered vehicle (n = 24) (E and F) or Am80 at 4 mg/kg (n = 25) (G) for 5 weeks beginning on day 8, as therapy for existing vasculitis. Specimens were obtained on day 8 (E) or day 36 (F and G). H, Mean ± SEM vasculitis scores in mice treated as described in E–G. ∗ = P < 0.05. NS = not significant. Specimens shown in A–C and E–G are representative of the respective treatment groups. Original magnification × 40.

Effects of Am80 on neutrophil migration.

We also analyzed the effect of Am80 on neutrophil migration into the inflamed aortic wall. CMTMR-labeled neutrophils were adoptively transferred into mice with CAWS-induced vasculitis. The recipient mice were treated with 4 mg/kg Am80 24 hours and 2 hours before and 22 hours after the transfer. This short-term administration of Am80 did not reduce the vasculitis score (data not shown). Twenty-four hours after the transfer, the number of cells that had migrated into the aortic wall was counted. In normal mice, no labeled neutrophils migrated into the aortic wall. In contrast, significant numbers of labeled neutrophils migrated into the aortic wall in the vasculitic animals. However, treatment with Am80 reduced this migration of neutrophils to the vasculitic site (Figures 3A and B).

Figure 3.

Inhibition of neutrophil migration by Am80. A, Labeled neutrophils were adoptively transferred into BALB/c mice with Candida albicans water-soluble fraction (CAWS)–induced vasculitis. Vehicle or Am80 at 4 mg/kg was administered orally to recipient mice (n = 6 per group) 24 hours and 2 hours before and 22 hours after the adoptive transfer. Twenty-four hours after the transfer, the animals were killed, and hearts were harvested and examined by fluorescent microscopy for migrated cells (arrows). Specimens shown are representative of the respective treatment groups. Original magnification × 80. B, Labeled cells in the aortic wall of mice treated as described in A were counted. Values are the mean ± SEM. ∗ = P < 0.05 versus vehicle-treated group. C, Total RNA was extracted from purified human peripheral blood neutrophils and synovial fibroblasts from a patient with rheumatoid arthritis, as a positive control. Expression of the retinoic acid receptors (RARs) RARα, RARβ, and RARγ was analyzed by reverse transcription–polymerase chain reaction. D, The purified neutrophils were incubated with vehicle or Am80 (10−7, 10−6, or 10−5 moles/liter), and then exposed to fMLP in a chemotactic chamber within an EZ-TAXIScan device. Single motile cells were tracked every 30 seconds for 45 minutes. The cell paths of the neutrophils treated with vehicle or Am80 at 10−5 moles/liter are shown. Results are representative of 3 independent experiments. E and F, Parameters of average migration velocity (E) and directionality (F) were measured. Values are the mean ± SEM from 3 video sequences of 22 cells in each treatment group. ∗ = P < 0.05 versus no Am80 treatment.

The effect of Am80 on chemotaxis of human peripheral blood neutrophils was also analyzed in vitro; synovial fibroblasts from a patient with rheumatoid arthritis were used as a positive control for RAR expression in this experiment (29). The purified human neutrophils expressed RARα, but not RARβ or RARγ (Figure 3C). After culture with Am80 for 3 hours, fMLP-induced cell movement was video-recorded (see Supplementary videos 1 [control] and 2 [Am80-treated], available on the Arthritis & Rheumatism web site at http:// onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131), and the path of each cell was observed (Figure 3D). We examined the speed of movement and direction of each cell. The migration speed of Am80-treated neutrophils was slower than that of untreated neutrophils (Figure 3E). Am80 also significantly reduced the directionality of the chemotaxis (Figure 3F).

Reduced ROS production from neutrophils after Am80 treatment.

We also assessed the effect of Am80 on ROS production from human peripheral blood neutrophils, based on the fluorescence intensity of rhodamine 123. After incubation with Am80 in various concentrations, the cells were stimulated with PMA, Pam3CSK4, or LPS. ROS production was measured by flow cytometry. Incubation with Am80 significantly inhibited production of ROS by the stimulated cells (Figures 4A and B).

Figure 4.

Effect of Am80 on reactive oxygen species (ROS) production and elastase release from neutrophils. A, Purified human peripheral blood neutrophils were incubated without Am80 or in the presence of Am80 at 10−8, 10−7, or 10−6 moles/liter and stimulated with phorbol 12-myristate 13-acetate (PMA) (1 ng/ml), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4) (1 μg/ml), or lipopolysaccharide (LPS) (5 μg/ml). ROS levels in the neutrophils were determined based on the fluorescence intensity of dihydrorhodamine 123 (DHR). Representative histograms are shown. B, Neutrophils were treated as described in A, and the relative mean fluorescence intensity (MFI) ratio was calculated ([MFI of cells treated with Am80/MFI of cells without Am80 treatment] × 100). C, The purified neutrophils were incubated without Am80 or in the presence of Am80 at 10−8, 10−7, or 10−6 moles/liter and stimulated with cytochalasin B (10 μg/ml) and fMLP (1 μM). Elastase concentrations in the culture supernatant were measured by enzyme-linked immunosorbent assay. Values in B and C are the mean ± SEM of triplicate wells from at least 3 independent experiments. ∗ = P < 0.05 versus no Am80 treatment.

Effects of Am80 on elastase release from neutrophils.

To determine the effect of Am80 on elastase release by human peripheral blood neutrophils, we treated the neutrophils with cytochalasin B and fMLP after culture for 5 hours with Am80. The concentration of elastase in the culture supernatant was analyzed by ELISA. Incubation with Am80 inhibited elastase release by the neutrophils in a dose-dependent manner (Figure 4C).

Inhibitory effects of Am80 on MAPK signaling.

Retinoids are reported to inhibit MAPK signaling (30–33). Accordingly, we analyzed phosphorylation of ERK-1/2 and p38 MAPK in neutrophils stimulated with LPS plus fMLP, using intracellular flow cytometry. Phosphorylation of ERK-1/2 and p38 was increased to the greatest extent after 1 minute and 10 minutes of stimulation, respectively (Figure 5A). Am80 attenuated the increased phosphorylation of ERK-1/2 and p38 in stimulated neutrophils (Figure 5B).

Figure 5.

Am80-induced inhibition of MAPK signals. A, Purified human peripheral blood neutrophils were stimulated with fMLP (1 μM) and lipopolysaccharide (LPS) (1 μg/ml) for 1, 10, or 30 minutes. Phosphorylation of ERK-1/2 and p38 was analyzed by flow cytometry. B, Neutrophils were treated with Am80 (10−6 moles/liter) and stimulated with fMLP and LPS for 1 minute to analyze phosphorylation of ERK-1/2 and for 10 minutes to analyze phosphorylation of p38. Results shown are representative of at least 3 independent experiments.

DISCUSSION

In the present study we demonstrated that treatment with Am80, a synthetic retinoid, ameliorated murine vasculitis induced by CAWS and also inhibited neutrophil migration into inflamed vessels. Am80 treatment resulted in marked suppression of chemotaxis, ROS production, and elastase release in human peripheral blood neutrophils in vitro. Neutrophils mainly infiltrated the site of vasculitis, and depletion of neutrophils suppressed murine vasculitis. Collectively, the results suggest that Am80 suppresses CAWS-induced vasculitis through inhibition of neutrophil migration and activation.

Neutrophils infiltrate the affected vessels in patients with certain vasculitides, such as microscopic polyangiitis and polyarteritis nodosa (34), and there has been ample evidence implicating neutrophils in the pathogenesis of vasculitis (35). In our vasculitis model mice, abundant neutrophils infiltrated the inflamed vessel wall, and neutrophil depletion by rabbit anti-mouse neutrophil antibody–positive serum as a prophylactic treatment ameliorated the vasculitis, suggesting that neutrophils play an important role in this model. However, the rabbit anti-mouse neutrophil antibody–positive serum depleted peripheral blood neutrophils by only 57%. This insufficient depletion of neutrophils may have resulted in the inefficacy of the therapeutic administration.

Accumulated neutrophils can injure vessels by producing ROS, inflammatory cytokines, and elastase. Previous studies have suggested that neutrophils have important roles in the pathogenesis of vasculitis and that retinoid directly inhibits the production of superoxide anion and release of proteolytic enzymes by human and rat neutrophils (14, 15, 36). However, the effects on neutrophils vary among the different retinoids (15). In the present study, we showed that Am80 suppressed neutrophil migration into inflamed vessels in vivo, and fMLP-induced chemotaxis in vitro. In the in vivo chemotaxis assay, the recipient mice were administered Am80 only 3 times during 2 days, and the Am80 treatment did not significantly reduce the vasculitis score. It is believed that neutrophils may survive for 1–2 days after infiltrating tissue (37). Specifically, neutrophils that migrate to sites of inflamed tissue have a prolonged lifespan and become resistant to apoptosis (38). Therefore, neutrophils that have already infiltrated the vessels could remain in the tissue during the 2 days of Am80 treatment. Moreover, Am80 also reduced ROS production and elastase release by neutrophils. Thus, Am80 appears to inhibit CAWS-induced vasculitis via inhibition of neutrophil migration and activation.

It was previously shown that retinoid inhibited the MKK-6/p38 and MEK/ERK signaling pathways (39). In the present study, we observed that Am80 suppressed the activation of ERK-1/2 and p38 MAPKs in human peripheral blood neutrophils. Since ERK-1/2 and p38 MAPK are implicated in the respiratory burst, adherence, exocytosis, and cell motility in neutrophils (40, 41), Am80 may hamper neutrophil activation via interference with those signaling pathways.

The pathogenic mechanisms of CAWS-induced murine vasculitis are still unclear. Arteritis could be induced by C albicans–derived substances (42, 43). Investigators in our group have found that CAWS is a useful compound to induce murine vasculitis and have extensively studied this model of the disease (3–8). CAWS is primarily composed of a complex of mannoproteins, such α-mannan, and β-glucan (5) that can bind to Toll-like receptors and/or C-type lectin receptors on neutrophils, macrophages, and dendritic cells (44). Injection of CAWS increases peripheral blood neutrophil and monocyte numbers and promotes activation of neutrophils and complement, which in turn release ROS, proinflammatory cytokines, and myeloperoxidase from neutrophils and soluble intercellular adhesion molecule 1 from endothelial cells in mice (8). These processes of neutrophil and endothelial cell activation may be involved in the development of CAWS-induced vasculitis.

It has been reported that retinoids regulate differentiation and activation of T cells and macrophages (11, 12), and these effects of Am80 on T cells and macrophages might also contribute to the inhibition of vasculitis. However, in this study we did not investigate the effects of Am80 on these cells in CAWS-induced vasculitis. Further studies should be carried out to investigate inhibitory influences of Am80 on T cells and macrophages.

Am80, in addition to all-trans-retinoic acid, has been approved in Japan as a therapeutic agent for acute promyelocytic leukemia (21, 22). We and other groups have shown that Am80 ameliorates experimental autoimmune myositis (45), experimental autoimmune encephalitis (46), collagen-induced arthritis (47, 48), and murine chronic graft-versus-host disease (49). In the present study we have demonstrated for the first time that retinoids, especially Am80, ameliorate experimental vasculitis, suggesting that Am80 can be potentially useful for the treatment of vasculitis. In further studies to examine the potential effects of Am80 on different vasculitides, the inhibitory effects of Am80 on neutrophils from vasculitis patients must be analyzed, and the effects compared among different vasculitides.

In conclusion, Am80 significantly inhibits vasculitis in a murine model, presumably through suppression of neutrophil migration and activation. This agent may have potential as a new mode of treatment in vasculitis.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Nanki had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. C. Miyabe, Y. Miyabe, Shudo, Isobe, Matsushima, Tsuboi, Miyasaka, Nanki.

Acquisition of data. C. Miyabe, Miura, Takahashi, Terashima, Toda, Honda, Yamagata, Ohno.

Analysis and interpretation of data. C. Miyabe, Honda, Morio, Suzuki, Tsuboi, Miyasaka, Nanki.

Acknowledgements

We thank Yoshiko Iwai, Masashi Ebisawa (Tokyo Medical and Dental University), and Timothy J. Wright (Otsuma Women's University) for helpful suggestions, and Noriko Tamura for excellent technical support.

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