NK026680 a novel suppressant of dendritic cell function prevents the development of rapidly progressive glomerulonephritis and perinuclear antineutrophil cytoplasmic antibody in SCGKj mice.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 54, No. 11, November 2006, pp 3707–3715 DOI 10.1002/art.22187 © 2006, American College of Rheumatology NK026680, a Novel Suppressant of Dendritic Cell Function, Prevents the Development of Rapidly Progressive Glomerulonephritis and Perinuclear Antineutrophil Cytoplasmic Antibody in SCG/Kj Mice Kan Saiga,1 Kazuhiro Tokunaka,2 Eiji Ichimura,2 Eriko Toyoda,2 Fuminori Abe,2 Minako Yoshida,3 Hiroshi Furukawa,3 Masato Nose,4 and Masao Ono3 dosage of 25 mg/kg once daily or 50 mg/kg once daily significantly suppressed 1) spontaneous mortality, 2) proteinuria and hematuria, 3) blood urea nitrogen levels, 4) glomerular damage characterized histopathologically, 5) IC deposition in glomeruli, 6) the development of pANCA and anti-DNA antibodies, and 7) lymphadenopathy. Conclusion. The newly identified DC inhibitor, NK026680, prevented the onset of RPGN, autoantibody production, and lymphadenopathy in SCG/Kj mice, suggesting a crucial role for DC function in these autoimmune phenotypes. NK026680 may be a potent immunosuppressive agent for the treatment of ANCAassociated renovascular disorders. Objective. NK026680 is a newly identified type of immunosuppressive agent that inhibits dendritic cell (DC) functions and consequently reduces the mortality of mice with experimental acute graft-versus-host disease. This study was undertaken to evaluate NK026680 suppression of DC functions in preventing development of rapidly progressive glomerulonephritis (RPGN) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) in SCG/Kj mice. Methods. Oral administration of NK026680 to SCG/Kj mice began when mice were 8–10 weeks old, before the onset of disease, and continued for 56 days. The efficacy of NK026680 was evaluated using the mortality of mice, the results of urinalysis, histopathologic evaluation for glomerular injury, and immunofluorescence staining for the detection of immune complex (IC) deposition in glomeruli, and by assessing lymphadenopathy and measuring autoantibody titers. Results. Oral administration of NK026680 at a Dendritic cells (DCs) play a central role in initiating and regulating immune responses through antigen presentation (signal 1), costimulatory signals (signal 2), and soluble factors (1,2). Several studies have demonstrated that blockade of the costimulatory signal from DCs markedly suppresses autoimmune diseases (3) and, conversely, that the prolonged presentation of self antigens by DCs exacerbates autoimmune diseases in animal models (4–7). These findings confirm the pathologic role of DCs in the pathogenesis of autoimmune diseases, suggesting that DC function is a potential target for autoimmune therapy. We therefore attempted to develop a pharmacologic approach to suppressing DC functions in vivo. Recently, we succeeded in isolating a new type of immunosuppressive agent, NK026680, by screening a large number of synthetic compounds. We demonstrated that NK026680 suppressed the up-regulation of class I Dr. Ono’s work was supported by Grants-in-Aid for Scientific Research (grant 16390113) from the Ministry of Education, Science, Sports, and Culture of Japan. 1 Kan Saiga, PhD: Tohoku University Graduate School of Medicine, Sendai, Japan, and Pharmaceutical Research Laboratories, Nippon Kayaku Company, Tokyo, Japan; 2Kazuhiro Tokunaka, MS, Eiji Ichimura, PhD, Eriko Toyoda, MS, Fuminori Abe, PhD: Nippon Kayaku Company, Tokyo, Japan; 3Minako Yoshida, MD, Hiroshi Furukawa, MD, Masao Ono, MD: Tohoku University Graduate School of Medicine, Sendai, Japan; 4Masato Nose, MD: Ehime University School of Medicine, Toon, Japan. Address correspondence and reprint requests to Masao Ono, MD, Department of Pathology, Tohoku University Graduate School of Medicine, 2-1 Seiryo, Aoba-ku, Sendai, Miyagi 980-8575, Japan. E-mail: firstname.lastname@example.org. Submitted for publication December 28, 2005; accepted in revised form July 26, 2006. 3707 3708 major histocompatibility complex (MHC), class II MHC, CD83, and CD86 on human monocyte-derived DCs during immunologic activation, and that it acted prophylactically in experimental acute graft-versus-host disease (GVHD) in mice (8). Moreover, the effectiveness of orally administered NK026680, and its lower toxicity compared with that of cyclosporin A, suggested that it might be of clinical benefit in vivo when used to treat autoimmune diseases, by targeting DC functions. Renovascular disorders with rapidly progressive glomerulonephritis (RPGN) and the presence of antineutrophil cytoplasmic antibody (ANCA) are associated with autoimmune mechanisms and share some of the characteristics of small-vessel systemic vasculitides, such as Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome (9). Histopathologically, the glomerular lesions in these diseases are characterized by crescentic glomerulonephritis (CGN), a hallmark of life-threatening and irreversible nephrosis. Since the early 1970s, the combination of cyclophosphamide (CYC) and corticosteroids has been the standard treatment of these vasculitides, and its efficacy has been demonstrated (10); however, in many cases, the disease recurs despite this treatment. Moreover, CYC causes hemorrhagic cystitis and increases the risk of secondary cancer, gonadotoxicity, and infection (11). In an attempt to address these concerns, we tested the in vivo efficacy of NK026680 for the prevention of CGN- and ANCA-associated renovascular disorder in SCG/Kj mice. SCG/Kj is a recombinant congenic strain of mice that was genetically segregated from (BXSB ⫻ MRL/Mp-Faslpr [MRL/lpr])F1 mice by selected brother ⫻ sister matings (12). BXSB and MRL/lpr mice are known to be autoimmune-prone due to the Y chromosome–linked autoimmune accelerator Yaa mutation in BXSB mice and the lpr mutation in MRL/lpr mice. In each generation, the litter with the severest GN in its parent generation, determined by histopathologic evaluation, was selected for further breeding. The resultant male SCG/Kj mice have only the lpr mutation but develop far severer CGN than either BXSB or MRL/lpr mice. They also develop rapidly progressive hematuria and proteinuria, which resemble clinical findings in human RPGN (12,13). Of particular note, SCG/Kj mice further develop perinuclear ANCA (pANCA) and systemic vasculitides of small vessels. These manifestations suggest that the disease pathogenesis in SCG/Kj mice is closely related to that in human ANCA-associated vasculitis. However, Neumann and colleagues (14) demonstrated massive deposits of immune complex (IC) in the CGN lesions of SAIGA ET AL SCG/Kj mice, indicating that the condition might not be representative of human ANCA-associated vasculitis, which is characterized by pauci-immune GN. Even if this is taken into account, SCG/Kj mice are useful for evaluating various therapeutic approaches in renovascular disorders with RPGN and for studying the mechanism of pANCA production. The findings of this study provide evidence for the significant role of DCs in the development of CGN and ANCA in SCG/Kj mice and support the use of NK026680 in the treatment of ANCA-associated vasculitis. MATERIALS AND METHODS Mice. SCG/Kj mice were maintained under specific pathogen–free conditions in the animal department of Nippon Kayaku Company. All mice used in this study were killed under anesthesia with ether. Animal care and experimental procedures were carried out according to national and international laws and policies. The study was conducted with the permission of local government authorities. Agents and treatment protocol. NK026680 was synthesized and characterized at Nippon Kayaku Company. NK026680 was suspended in 0.5% carboxymethylcellulose at a concentration of 2.5 mg/ml or 5 mg/ml for oral administration. The aliquots were stored at ⫺20°C until used. We began treating mice when they were 8–10 weeks old, before the onset of renal disease, and continued treatment for 56 days, unless otherwise noted. Symptoms of CGN in mice were confirmed by screening for proteinuria and hematuria before starting treatment (day 0). Histopathologic examinations. Tissue samples were fixed in 10% formalin buffered with 0.01M phosphate (pH 7.2) and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS). The severity of lesions in the kidney was determined by grading crescent formation, hyaline thrombi, perivascular cell infiltration, protein cast formation, and intracytoplasmic PAS-positive droplets. Crescent formation was graded on PAS-stained sections using a 4-point scale, as follows: 0 ⫽ no crescents; 1 ⫽ focal and cellular crescents; 2 ⫽ diffuse and cellular crescents partially admixed with fibrous crescents; and 3 ⫽ diffuse fibrous crescents. A cellular crescent was defined as the presence of ⱖ2 cell layers within the Bowman space. The presence of hyaline thrombi in the glomerulus was graded on PAS-stained sections using a 2-point scale, as follows: 0 ⫽ no thrombi, and 1 ⫽ presence of thrombi. Perivascular cell infiltration was graded on H&E-stained sections using a 3-point scale, as follows: 0 ⫽ no infiltration; 1 ⫽ focal infiltration in perivascular spaces, with the diameter of the lesion not exceeding twice the vascular diameter; and 2 ⫽ diffuse infiltration in the kidney, with the diameter of the lesion frequently exceeding twice the vascular diameter. Protein cast formation and intracytoplasmic PAS-positive droplets were graded on PAS-stained sections using a 4-point scale, as follows: 0 ⫽ no lesions; 1 ⫽ focal or slight lesions; 2 ⫽ focal and multiple lesions; and 3 ⫽ diffuse lesions. For immunofluorescence (IF) examination, we prepared 5-m frozen sections from snap-frozen mouse kidneys, DC INHIBITOR NK026680 PREVENTS CRESCENTIC GLOMERULONEPHRITIS then fixed them in acetone for 5 minutes. Fluorescein isothiocyanate (FITC)–conjugated rabbit antiserum to mouse IgG (ICN Immunobiologicals, Irvine, CA) or C3 (Miles, Naperville, IL) was used for detection. Examination of hematologic and serologic features and urinalysis findings. Blood cell counts were determined in heparinized blood samples from the infraorbital venous plexus using a standard hemocytometer (Celltac MEK-4500; Nihon Koden, Tokyo, Japan). We tested mice for proteinuria and hematuria twice weekly using a standard urinary indicator (Uropaper II; Eiken Chemical, Tokyo, Japan). A total protein concentration of ⬎1,000 mg/dl was regarded as an indication of severe proteinuria. A red blood cell count of ⬎20 cells/ml was considered to be an indication of hematuria. Blood urea nitrogen (BUN) levels in the serum samples collected from mice at autopsy were measured with Fuji Drichem Slide BUN-PII (Fujifilm Medical Systems, Tokyo, Japan). Myeloperoxidase (MPO)–ANCA was measured using the enzyme-linked immunosorbent assay (ELISA) kit for human MPO-ANCA (Medical and Biological Laboratories, Nagoya, Japan). We used peroxidase-conjugated anti-mouse IgG (Amersham Biosciences, Buckinghamshire, UK) in place of the detection antibody supplied in the human ELISA kit. Anti–single-stranded DNA (anti-ssDNA) and anti–doublestranded DNA (anti-dsDNA) in serum diluted 1:100 were measured using ELISA kits (Shibayabi, Gunma, Japan). Concentrations of the cytokines tumor necrosis factor ␣ (TNF␣), interferon-␥ (IFN␥), interleukin-5 (IL-5), IL-4, and IL-2 in sera were measured with the Mouse Th1/Th2 Cytokine Capture Beads Assay Kit (BD PharMingen, San Diego, CA). The presence of ANCA was detected by indirect IF methods using glass slides containing ethanol-fixed human neutrophils (Medical and Biological Laboratories) and mouse neutrophils, as previously described (15). The glass slides were incubated with serum diluted 1:100 with calcium and magnesium–free phosphate buffered saline (PBS) and were then stained with FITC-conjugated anti-mouse Ig (Dako Cytomation, Glostrup, Denmark). The level of ANCA was graded semiquantitatively based on fluorescence intensity, using a 4-point scale, as follows: 0 ⫽ no staining; 1 ⫽ faint staining; 2 ⫽ distinct staining, but not as intense as typical staining in untreated SCG/Kj mouse serum; and 3 ⫽ strong staining, comparable with or more intense than typical staining in untreated SCG/Kj mouse serum. All positive sera in this study showed the pANCA pattern. Fluorescence-activated cell sorting analysis. Peripheral blood cells and splenocytes (106 cells) were incubated at 4°C for 20 minutes with FITC- or phycoerythrin-conjugated antibody to CD3⑀ or CD45RA (B220) in the presence of Fc block (BD PharMingen). The cells were washed and then resuspended in PBS containing 5 g/ml propidium iodide for elimination of dead cells. The expression was analyzed on a FACScan (Becton Dickinson, Tokyo, Japan) with CellQuest software version 3.3. Statistical analysis. Differences in survival rates were evaluated by the log rank test. Differences in the incidence of severe proteinuria or hematuria, the grade of renal abnormality, and the pANCA titer were evaluated by Fisher’s exact test. Differences in BUN concentration, the weight of the spleen or axillary lymph nodes, and autoantibody titers were evaluated by Steel test. Differences in the plasma concentrations of 3709 cytokines were evaluated by Wilcoxon’s rank sum test. Differences in the percentage of CD3⫹,CD45RA⫹ T cells in spleen cells were evaluated by Student’s t-test. P values less than 0.05 were considered significant. RESULTS Reduction of rate of spontaneous mortality. We evaluated the effect of preventive treatment with NK026680 on the spontaneous mortality of SCG/Kj mice. Three groups of mice were treated daily for 56 days. One group received 0.5% carboxymethylcellulose vehicle only, one group received 25 mg/kg NK026680 orally once daily, and one group received 50 mg/kg NK026680 orally once daily. At the study end point (day 56), the percentage survival in the group treated with vehicle, the group treated with 25 mg/kg NK026680 daily, and the group treated with 50 mg/kg NK026680 Figure 1. Effect of NK026680 treatment on mortality and glomerular injury in SCG/Kj mice. Three groups consisting of 25, 10, and 15 mice, respectively, received oral treatment with 0.5% carboxymethylcellulose vehicle, 25 mg/kg NK026680, or 50 mg/kg NK026680 daily for 56 days. A, Survival rate during the treatment period. B, Incidence of proteinuria (urinary protein concentration ⬎1,000 mg/dl) during the treatment period. C, Incidence of hematuria (urinary red blood cell count ⬎20/ml) during the treatment period. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle (by log rank test in A; by Fisher’s exact test in B and C). 3710 SAIGA ET AL Table 1. Histopathologic changes in the kidneys of SCG/Kj mice after 56 days of treatment with vehicle or NK026680* BUN on day 28, mg/dl BUN on day 56, mg/dl Crescent formation grade (0–3 scale) Perivascular cell infiltration grade (0–2 scale) Intracytoplasmic PAS-positive droplets grade (0–3 scale) Hyaline thrombi, % Protein casts, % Vehicle 25 mg/kg NK026680 50 mg/kg NK026680 26.5 (24.4–33.5) 37.5 (32.0–72.4) 1.5 (1.0–3.0) 1.0 (1.0–2.0) 0.0 (0.0–0.0) 100.0 50.0 24.5 (21.1–26.1) 30.7 (26.1–31.8)‡ 0.5 (0.0–1.0)¶ 0.5 (0.0–1.0)¶ 0.0 (0.0–1.0) 44.4¶ 33.3 23.9 (23.0–27.1)† 20.6 (18.7–23.9)§ 0.5 (0.0–1.0)¶ 0.0 (0.0–0.5)# 2.0 (2.0–2.0)# 7.7** 23.1 * Except where indicated otherwise, values are the median (95% confidence interval) in 6 mice treated with vehicle (8 mice for blood urea nitrogen [BUN] measurements), 9 mice treated with NK026680 25 mg/kg/day (7 mice for BUN measurements), and 13 mice treated with NK026680 50 mg/kg/day. PAS ⫽ periodic acid–Schiff. † P ⬍ 0.01 versus vehicle, by Steel test. ‡ P ⬍ 0.05 versus vehicle, by Steel test. § P ⬍ 0.001 versus vehicle, by Steel test. ¶ P ⬍ 0.05 versus vehicle, by Fisher’s exact test. # P ⬍ 0.01 versus vehicle, by Fisher’s exact test. ** P ⬍ 0.001 versus vehicle, by Fisher’s exact test. daily was 32.0%, 70.0%, and 86.7%, respectively. Treatment with 25 mg/kg NK026680 daily or 50 mg/kg NK026680 daily significantly reduced mortality in SCG/Kj mice (Figure 1A) (P ⬍ 0.05 and P ⬍ 0.01, respectively, versus vehicle group, by log rank test). Improvement of renal dysfunction and abnormality. Treatment with 25 mg/kg NK026680 daily or 50 mg/kg NK026680 daily significantly reduced the rates of proteinuria and hematuria (Figures 1B and C). Treatment with 25 mg/kg NK026680 daily or 50 mg/kg Figure 2. Histopathologic evidence of the improvement of glomerulonephritis after oral treatment with NK026680. SCG/Kj mice were treated for 56 days with vehicle (A, C, and E) or 50 mg/kg NK026680 (B, D, and F). A and B, Periodic acid–Schiff (PAS) staining. Arrows show crescent formation in the glomerulus; arrowheads show intracytoplasmic PAS-positive droplets in the renal tubules (magnification ⫻ 200). C–F, Indirect immunofluorescence staining using fluorescein isothiocyanate for the detection of IgG (C and D) and C3 (E and F) in the glomerulus (magnification ⫻ 400). DC INHIBITOR NK026680 PREVENTS CRESCENTIC GLOMERULONEPHRITIS 3711 NK026680 daily also significantly suppressed the elevated level of BUN at the end of the treatment period (P ⬍ 0.05 and P ⬍ 0.001, respectively, by Steel test) (Table 1). Histopathologic and IF examinations at the end point revealed that vehicle treatment did not change glomerular manifestations, perivascular cell infiltration, or deposition of IgG and C3, all of which are characteristic of CGN in SCG/Kj mice (Figures 2A, C, and E), whereas treatment with 50 mg/kg NK026680 daily markedly suppressed all pathologic manifestations (Figures 2B, D, and F). Histopathologic grading demonstrated that treatment with 50 mg/kg NK026680 daily significantly suppressed crescent formation and hyaline Figure 3. Reduction of serum autoantibody concentration after oral treatment with NK026680 for 56 days. A, Representative indirect immunofluorescence (IF) staining pattern in serum from untreated SCG/Kj mice, indicative of the presence of myeloperoxidase– antineutrophil cytoplasmic antibody (MPO-ANCA). Perinuclear ANCA (pANCA) was detected on IF of ethanol-fixed human and mouse neutrophils (Neut). In contrast, no detectable staining was observed in sera from NK026680-treated SCG/Kj mice (magnification ⫻ 100). B, Plots of the fluorescence intensity grade, determined on a scale of 0–3. ⴱⴱ ⫽ P ⬍ 0.01 by Fisher’s exact test. C–E, Titers of MPO-ANCA (C), anti–single-stranded DNA (anti-ssDNA) (D), and anti–double-stranded DNA (anti-dsDNA) (E) in diluted serum samples were measured by enzyme-linked immunosorbent assay. The numbers of samples in the groups treated with vehicle, 25 mg/kg NK026680, and 50 mg/kg NK026680 were 8, 7, and 13, respectively. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle, by Steel test. Figure 4. Reduction of lymphadenopathy after oral treatment with NK026680. A and B, Weight of axillary lymphoid nodes (A) and spleen (B) in SCG/Kj mice that received oral treatment with vehicle, 25 mg/kg NK026680, or 50 mg/kg NK026680 daily for 56 days (n ⫽ 8, 7, and 13, respectively). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle, by Steel test. C, Lymphocyte profile in the spleens of SCG/Kj mice treated with vehicle or 25 mg/kg NK026680. Spleen cells were incubated with fluorescein isothiocyanate– and phycoerythrinconjugated antibodies to CD3⑀ or CD45RA, and then analyzed using fluorescence-activated cell sorting. Cells in the CD3⫹,CD45RA⫹ fraction were defined as a unique T cell subset in Fas-deficient mice. The mean ⫾ SD percentages of CD3⫹,CD45RA⫹ T cells in the spleen are shown for mice treated with vehicle (n ⫽ 8) and mice treated with 25 mg/kg NK026680 (n ⫽ 7). ⴱⴱⴱ ⫽ P ⬍ 0.001 by Student’s t-test. 3712 thrombi in the glomerulus and perivascular cell infiltration in the kidney (Table 1). In all mice analyzed that were treated with 50 mg/kg NK026680 daily (13 of 13) and in some mice analyzed that were treated with 25 mg/kg NK026680 daily (2 of 9), intracytoplasmic PAS-positive droplets were observed as focal lesions in the epithelial cells of proximal renal tubules (Table 1). PAS staining of these droplets was diminished after diastase digestion, suggesting that the droplets consisted of glycogen. We therefore measured plasma glucose concentrations at the end of the treatment period, but there was no significant difference in the plasma glucose concentration in the mice treated with vehicle (mean ⫾ SD 267.3 ⫾ 59.6 mg/ml) and the mice treated with 50 mg/kg NK026680 daily (mean ⫾ SD 250.5 ⫾ 100.2 mg/ml). Histopathologically, there was no evidence of degeneration of the tubular epithelial cells in which the droplets were observed. Suppression of autoantibody production. A previous study demonstrated the presence of MPO-ANCA in SCG/Kj mice (14). It was also shown that ANCApositive sera from SCG/Kj mice were detected as a pANCA pattern on ethanol-fixed human neutrophils. In this study, we collected sera at the end point from mice treated with vehicle or with 50 mg/kg NK026680 daily. To detect ANCA, we first performed an indirect IF on ethanol-fixed human and mouse neutrophils. Indirect IF of both the human and the mouse neutrophils showed the presence of pANCA with mouse antigen specificity in the sera collected from vehicle-treated mice (Figure 3A). Conversely, pANCA was not detectable in the sera from mice treated with 50 mg/kg NK026680 daily (Figure 3A). Semiquantitative measurement of the IF signal in human neutrophils confirmed the suppressive effect of NK026680 on the development of pANCA in SCG/Kj mice (P ⬍ 0.01 by Fisher’s exact test) (Figure 3B). ELISA for the detection of MPO-ANCA using human MPO again confirmed this effect (Figure 3C). The titers of anti-ssDNA and anti-dsDNA were significantly suppressed, in a dose-dependent manner, by treatment with NK026680 at a dosage of 25 mg/kg or 50 mg/kg daily (Figures 3D and E). Suppression of lymphadenopathy. SCG/Kj mice develop generalized lymphadenopathy (12). This is largely due to the lpr mutation, which causes defects in Fas-mediated apoptosis in lymphocytes, thereby resulting in the accumulation of abnormal T cells expressing both CD3 and CD45RA (16). We examined the effect of NK026680 treatment on lpr-related lymphadenopathy in SCG/Kj mice. Daily treatment with 25 mg/kg or 50 mg/kg NK026680 significantly suppressed lpr-related SAIGA ET AL Table 2. Change in plasma concentrations of cytokines after 56 days of treatment with vehicle or NK026680* TNF␣, pg/ml IFN␥, pg/ml IL-5, pg/ml IL-4, pg/ml IL-2, pg/ml Vehicle (n ⫽ 8) 50 mg/kg NK026680 (n ⫽ 13) 276.6 (139.1–1767.3) 71.7 (40.0–584.0) 67.3 (40.0–275.5) 59.7 (40.0–344.2) 51.8 (40.0–159.2) ⬍40.0 (40.0–40.0)† ⬍40.0 (40.0–40.0)‡ ⬍40.0 (40.0–40.0)‡ ⬍40.0 (40.0–40.0)‡ ⬍40.0 (40.0–40.0)‡ * Values are the median (95% confidence interval). Forty pg/ml is the lower limit of detection in these assays. TNF␣ ⫽ tumor necrosis factor ␣; IFN␥ ⫽ interferon-␥; IL-5 ⫽ interleukin-5. † P ⬍ 0.001 versus vehicle, by Wilcoxon’s rank sum test. ‡ P ⬍ 00.05 versus vehicle, by Wilcoxon’s rank sum test. lymphadenopathy in these mice (Figures 4A and B). Flow cytometric analysis of spleen cells of mice treated with vehicle or 25 mg/kg NK026680 daily revealed a significant decrease in the percentage of abnormal spleen T cell subsets (CD3⫹,CD45RA⫹) in the mice treated with 25 mg/kg NK026680 (P ⬍ 0.001 by Student’s t-test) (Figure 4C). Suppression of cytokine production. It has been shown that sera and organs with autoimmune phenotypes contain high levels of cytokines, especially such Th1-related cytokines as TNF␣ and IFN␥ (17–19). The cytokine level is known to correlate with the activity of organ and tissue damage in autoimmune diseases (20). We measured the concentrations of the cytokines TNF␣, IFN␥, IL-5, IL-4, and IL-2 in sera collected at the end point of the study from 8 mice receiving vehicle and from 13 mice receiving 50 mg/kg NK026680 daily. It was found that treatment with 50 mg/kg NK026680 significantly suppressed the circulating levels of TNF␣, IFN␥, IL-5, IL-4, and IL-2 (Table 2). DISCUSSION Using serial screening of a large group of synthetic chemical compounds, we discovered that NK026680 exerts a highly suppressive effect on human monocyte-derived DCs during the maturation process. NK026680 was found to be a derivative of triazolopyrimidine, which has a very unique chemical structure compared with established immunosuppressive agents. Three other derivatives of triazolopyrimidine have been used in other applications, as an antagonist to A3 adenosine receptor (21), as an antihypertension drug (22,23), and as an herbicidal agent (24); unlike NK026680, however, these 3 derivatives have little effect on the activation of human monocyte-derived DCs (data not shown). This is the first report to describe the use of DC INHIBITOR NK026680 PREVENTS CRESCENTIC GLOMERULONEPHRITIS NK026680 as preventive therapy for autoimmune disease in mice. It was demonstrated that NK026680 improved a broad range of autoimmunity-related features, such as autoantibody production, cytokine production, and lpr-related lymphadenopathy. These results demonstrate the critical role of DCs in the development of these autoimmune traits in SCG/Kj mice. We previously showed that NK026680 suppressed the expression of class I MHC, class II MHC, CD83, and CD86 on human monocyte-derived DCs following maturation with TNF␣ in vitro, and the excretion of IL-12 in culture, while causing little inhibition of mitogenic T cell activation (8). Although the toxicity of NK026680 to monocyte-derived DCs in vitro was not evident at NK026680 concentrations ⬍200 nM, the suppression of allogeneic T cell responses in mixed lymphocyte reactions was evident at a concentration of 15 nM. Orally administered NK026680 markedly reduced the mortality of mice with experimental acute GVHD without notable adverse effects, showing NK026680 to be more effective in vivo than cyclosporin A. NK026680 treatment was not effective after the third day of allogeneic T cell challenge, suggesting that NK026680 targets early events in DC function during the onset of acute GVHD. Our preliminary study also showed that NK026680 significantly suppressed primary antibody production demonstrated by plaque-forming assay, and that it suppressed delayed-type hypersensitivity in the footpad in response to the immunization of mice with sheep red blood cells (8). These immunologic properties of NK026680 may make it a highly potent immunosuppressive agent. Taken together with these earlier findings, the present results suggest a unique mode of pharmacologic action of NK026680 in DCs. This identification of the molecular target of NK026680 is highly promising with regard to the development of therapies. Previous studies have shown that DC function is necessary for the development of autoimmune diseases (4,5). It has been reported that experimental systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human monoclonal antibody to DNA that bears a common idiotype (16/6 Id). However, mice lacking class I MHC molecules did not develop any of the clinical manifestations of SLE after immunization with 16/6 Id (25). Class II MHC–deficient MRL/lpr mice did not develop autoimmune renal disease or autoantibodies, demonstrating that class II MHC expression is critical for the development of autoimmune nephritis (26). A previous study of MRL/lpr mice deficient in CD80 and CD86 demonstrated that both of these costimulatory molecules were required for onset of lym- 3713 phoid hyperplasia, elevation of autoantibody levels, and development of autoimmune renal disease in these mice (3). These data reveal the contribution of MHCs and accessory molecules associated with antigen presentation of DCs in the induction of experimental autoimmune diseases. The pathogenic roles of DCs have been demonstrated in human autoimmune diseases. A recent report described the abnormal function and differentiation of DCs associated with an IFN␣-dependent mechanism in SLE patients (27). The abnormal function of DCs in arthritic lesions is evident in patients with rheumatoid arthritis (28–30). These findings from studies of mice with congenitally defective DC function and of patients with autoimmune diseases allowed us to predict that the novel DC inhibitor, NK026680, would suppress autoimmune disease. The results of the present study demonstrate the possibility of a pharmaceutical approach to suppressing DC function in vivo. It has been reported that SCG/Kj mice spontaneously develop CGN, systemic vasculitides of small vessels, and MPO-ANCA, all of which are characteristic of human ANCA-associated vasculitis (12). However, it is important to note that these mice display a massive deposition of IgG and C3 in glomeruli. This deposition does not correspond to human ANCA-associated vasculitis, which is consistently characterized by pauciimmune GN (14). Neumann et al (14) speculated that the preexisting deposition of IC in glomeruli might provide an inflammatory milieu, and ANCA might then accelerate the inflammation, leading to severe vasculitis and CGN in SCG/Kj mice. In addition, glomerular diseases characterized by IC deposition have been observed to be aggravated by ANCA in various clinical settings, including typical IC nephritides such as lupus nephritis (31), poststreptococcal glomerulonephritis (32), and membrane nephropathy (33), and anti– glomerular basement membrane nephritis (34,35). The suppressive effect of NK026680 on the emergence of ANCA may be beneficial in preventing the progression toward CGN in glomerular diseases associated with IC deposition. It has also been reported that lymphadenopathy in Fas-deficient strains of mice, including the SCG/Kj strain, is due to the accumulation of an aberrant T cell subset, so-called lpr–T cells (16,36). Previous studies have shown that removal of the thymus from neonate MRL/lpr mice as well as ␤2-microglobulin deficiency, which results in the abolishment of class I MHC presentation, markedly attenuated lymphadenopathy in Fasdeficient mice (37–40); moreover, it has been shown that anti-CD8 treatment improved lymphadenopathy in Fas- 3714 SAIGA ET AL deficient mice (41). These findings suggest a role of class I MHC presentation on DCs in the development of lpr–T cells. It is reasonable to conclude that NK026680 inhibits the emergence of lpr–T cells by suppressing DC function. Although the contribution of lpr–T cells to autoimmune diseases in Fas-deficient mice is under debate, it has been shown that these cells proliferate in vivo (42), excrete some cytokines (43,44), and become cytotoxic (45) under experimental conditions, suggesting that they have a role in the development of autoimmune disease. The efficacy of NK026680 in SCG/Kj mice in vivo may be partly due to the inhibition of lpr–T cell development. It should be noted that long-term administration of NK026680 resulted in the accumulation of PASpositive and diastase-digested droplets in the renal proximal tubules of the mice. This manifestation is observed in patients with hyperglycemia. It is possible that NK026680 directly or indirectly impairs systemic glycogen metabolism or the renal uptake of glycogen. In order to address this concern, in this study we examined plasma glucose concentrations in mice after daily treatment with 50 mg/kg NK026680 and found no difference in glucose levels between mice treated with vehicle and mice treated with NK026680. Moreover, in a preliminary study of long-term administration of NK026680 in rats, we have observed neither accumulation of PAS-positive droplets in the renal tubules nor pathologic changes in the pancreas (Saiga K, et al: unpublished observations). Thus, it appears at this point that the PAS-positive droplets occur only in mice; however, further studies are needed to clarify this. Our study demonstrated the efficacy of NK026680 in a preventive approach to autoimmune disease. Although it is still too early to suggest clinical application of NK026680, its remarkable effect in a model of severe, lethal experimental autoimmune CGN raises the possibility of clinical use in autoimmune diseases. Since DCs highly contribute to protective immunity against pathogenic microorganisms, however, the administration of DC inhibitors in vivo may compromise protective immunity and increase susceptibility to infectious diseases. Evaluating these concerns is the next important step in considering the possible clinical use of NK026680. ACKNOWLEDGMENTS We thank Mrs. T. Mae, Dr. K. Sakitama, Dr. K. Nemoto, and Dr. Y. Ichikawa for critical discussions, Mrs. K. Togashi and Mrs. H. Mashiba for technical assistance, Mr. Y. Ichikawa and Mr. K. 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