вход по аккаунту


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
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.
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.
Submitted for publication December 28, 2005; accepted in
revised form July 26, 2006.
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
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.
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,
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
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.
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).
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, %
25 mg/kg
50 mg/kg
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)
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)
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)#
* 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).
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
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
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
(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).
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
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
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-
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
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-
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
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
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. Kotake for animal breeding assistance,
and Mrs. N. Fujisawa for secretarial assistance.
1. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991;9:271–96.
2. Banchereau J, Steinman RM. Dendritic cells and the control of
immunity. Nature 1998;392:245–52.
3. Kinoshita K, Tesch G, Schwarting A, Maron R, Sharpe AH, Kelley
VR. Costimulation by B7-1 and B7-2 is required for autoimmune
disease in MRL-Faslpr mice. J Immunol 2000;164:6046–56.
4. Ludewig B, Odermatt B, Ochsenbein AF, Zinkernagel RM, Hengartner H. Role of dendritic cells in the induction and maintenance of autoimmune diseases. Immunol Rev 1999;169:45–54.
5. Ludewig B, Junt T, Hengartner H, Zinkernagel RM. Dendritic
cells in autoimmune diseases. Curr Opin Immunol 2001;13:
6. Banchereau J, Fay J, Pascual V, Palucka AK. Dendritic cells:
controllers of the immune system and a new promise for immunotherapy. Novartis Found Symp 2003;252:226–35.
7. Hackstein H, Thomson AW. Dendritic cells: emerging pharmacological targets of immunosuppressive drugs. Nat Rev Immunol
8. Saiga K, Toyoda E, Tokunaka K, Masuda A, Matsumoto S,
Mashiba H, et al. NK026680, a novel compound suppressive of
dendritic cell function, ameliorates mortality in acute lethal graftversus-host reaction in mice. Bone Marrow Transplant 2006;37:
9. Kamesh L, Harper L, Savage CO. ANCA-positive vasculitis. J Am
Soc Nephrol 2002;13:1953–60.
10. Jayne D, Rasmussen N, Andrassy K, Bacon P, Tervaert JW,
Dadoniene J, et al. A randomized trial of maintenance therapy for
vasculitis associated with antineutrophil cytoplasmic autoantibodies. N Engl J Med 2003;349:36–44.
11. Hoffman GS, Kerr GS, Leavitt RY, Hallahan CW, Lebovics RS,
Travis WD, et al. Wegener granulomatosis: an analysis of 158
patients. Ann Intern Med 1992;116:488–98.
12. Kinjoh K, Kyogoku M, Good RA. Genetic selection for crescent
formation yields mouse strain with rapidly progressive glomerulonephritis and small vessel vasculitis. Proc Natl Acad Sci U S A
13. Miyazawa S, Saiga K, Nemoto K, Mae T, Hotta O. A repeat biopsy
study in spontaneous crescentic glomerulonephritis mice. Ren Fail
14. Neumann I, Birck R, Newman M, Schnulle P, Kriz W, Nemoto K,
et al. SCG/Kinjoh mice: a model of ANCA-associated crescentic
glomerulonephritis with immune deposits. Kidney Int 2003;64:
15. Xiao H, Heeringa P, Hu P, Liu Z, Zhao M, Aratani Y, et al.
Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin
Invest 2002;110:955–63.
16. Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA,
Nagata S. Lymphoproliferation disorder in mice explained by
defects in Fas antigen that mediates apoptosis. Nature 1992;356:
17. Murray LJ, Lee R, Martens C. In vivo cytokine gene expression in
T cell subsets of the autoimmune MRL/Mp-lpr/lpr mouse. Eur
J Immunol 1990;20:163–70.
18. Takahashi S, Fossati L, Iwamoto M, Merino R, Motta R, Kobayakawa T, et al. Imbalance towards Th1 predominance is associated
with acceleration of lupus-like autoimmune syndrome in MRL
mice. J Clin Invest 1996;97:1597–604.
19. O’Garra A, Steinman L, Gijbels K. CD4⫹ T-cell subsets in
autoimmunity. Curr Opin Immunol 1997;9:872–83.
20. Jacob CO. Tumor necrosis factor and interferon ␥: relevance for
immune regulation and genetic predisposition to autoimmune
disease. Semin Immunol 1992;4:147–54.
21. Baraldi PG, Cacciari B, Romagnoli R, Spalluto G, Klotz KN,
Leung E, et al. Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine
receptor antagonists. J Med Chem 1999;42:4473–8.
22. Sato Y, Shimoji Y, Fujita H, Nishino H, Mizuno H, Kobayashi S,
et al. Studies on cardiovascular agents. 6. Synthesis and coronary
vasodilating and antihypertensive activities of 1,2,4-triazolo[1,5a]pyrimidines fused to heterocyclic systems. J Med Chem 1980;23:
23. Nicolai E, Cure G, Goyard J, Kirchner M, Teulon JM, Versigny A,
et al. Synthesis and SAR studies of novel triazolopyrimidine
derivatives as potent, orally active angiotensin II receptor antagonists. J Med Chem 1994;37:2371–86.
24. Hall LM, Devine MD. Cross-resistance of a chlorsulfuron-resistant biotype of Stellaria media to a triazolopyrimidine herbicide.
Plant Physiol 1990;93:962–6.
25. Mozes E, Kohn LD, Hakim F, Singer DS. Resistance of MHC
class I-deficient mice to experimental systemic lupus erythematosus. Science 1993;261:91–3.
26. Jevnikar AM, Grusby MJ, Glimcher LH. Prevention of nephritis in
major histocompatibility complex class II-deficient MRL-lpr mice.
J Exp Med 1994;179:1137–43.
27. Blanco P, Palucka AK, Gill M, Pascual V, Banchereau J. Induction
of dendritic cell differentiation by IFN-␣ in systemic lupus erythematosus. Science 2001;294:1540–3.
28. Zvaifler NJ, Steinman RM, Kaplan G, Lau LL, Rivelis M.
Identification of immunostimulatory dendritic cells in the synovial
effusions of patients with rheumatoid arthritis. J Clin Invest
29. Santiago-Schwarz F, Anand P, Liu S, Carsons SE. Dendritic cells
(DCs) in rheumatoid arthritis (RA): progenitor cells and soluble
factors contained in RA synovial fluid yield a subset of myeloid
DCs that preferentially activate Th1 inflammatory-type responses.
J Immunol 2001;167:1758–68.
30. Tsark EC, Wang W, Teng YC, Arkfeld D, Dodge GR, Kovats S.
Differential MHC class II-mediated presentation of rheumatoid
arthritis autoantigens by human dendritic cells and macrophages.
J Immunol 2002;169:6625–33.
31. Schnabel A, Csernok E, Isenberg DA, Mrowka C, Gross WL.
Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus: prevalence, specificities, and clinical significance. Arthritis
Rheum 1995;38:633–7.
32. Ardiles LG, Valderrama G, Moya P, Mezzano SA. Incidence and
studies on antigenic specificities of antineutrophil-cytoplasmic
autoantibodies (ANCA) in poststreptococcal glomerulonephritis.
Clin Nephrol 1997;47:1–5.
33. Tse WY, Howie AJ, Adu D, Savage CO, Richards NT, Wheeler
DC, et al. Association of vasculitic glomerulonephritis with membranous nephropathy: a report of 10 cases. Nephrol Dial Transplant 1997;12:1017–27.
Bosch X, Mirapeix E, Font J, Borrellas X, Rodriguez R, LopezSoto A, et al. Prognostic implication of anti-neutrophil cytoplasmic
autoantibodies with myeloperoxidase specificity in anti-glomerular
basement membrane disease. Clin Nephrol 1991;36:107–13.
Short AK, Esnault VL, Lockwood CM. Anti-neutrophil cytoplasm
antibodies and anti-glomerular basement membrane antibodies:
two coexisting distinct autoreactivities detectable in patients with
rapidly progressive glomerulonephritis. Am J Kidney Dis 1995;26:
Morse HC III, Davidson WF, Yetter RA, Murphy ED, Roths JB,
Coffman RL. Abnormalities induced by the mutant gene lpr:
expansion of a unique lymphocyte subset. J Immunol 1982;129:
Maldonado MA, Eisenberg RA, Roper E, Cohen PL, Kotzin BL.
Greatly reduced lymphoproliferation in lpr mice lacking major
histocompatibility complex class I. J Exp Med 1995;181:641–8.
Mixter PF, Russell JQ, Durie FH, Budd RC. Decreased CD4CD8- TCR-␣ ␤ ⫹ cells in lpr/lpr mice lacking ␤ 2-microglobulin.
J Immunol 1995;154:2063–74.
Ohteki T, Iwamoto M, Izui S, MacDonald HR. Reduced development of CD4-8-B220⫹ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class
I molecules. Eur J Immunol 1995;25:37–41.
Giese T, Davidson WF. In CD8⫹ T cell-deficient lpr/lpr mice,
CD4⫹B220⫹ and CD4⫹B220- T cells replace B220⫹ doublenegative T cells as the predominant populations in enlarged lymph
nodes. J Immunol 1995;154:4986–95.
Giese T, Davidson WF. Chronic treatment of C3H-lpr/lpr and
C3H-gld/gld mice with anti-CD8 monoclonal antibody prevents
the accumulation of double negative T cells but not autoantibody
production. J Immunol 1994;152:2000–10.
Zhou T, Bluethmann H, Eldridge J, Berry K, Mountz JD. Origin
of CD4-CD8-B220⫹ T cells in MRL-lpr/lpr mice. Clues from a T
cell receptor ␤ transgenic mouse. J Immunol 1993;150:3651–67.
Prud’Homme GJ, Park CL, Fieser TM, Kofler R, Dixon FJ,
Theofilopoulos AN. Identification of a B cell differentiation
factor(s) spontaneously produced by proliferating T cells in murine lupus strains of the lpr/lpr genotype. J Exp Med 1983;157:
Murray L, Martens C. The abnormal T lymphocytes in lpr mice
transcribe interferon-␥ and tumor necrosis factor-␣ genes spontaneously in vivo. Eur J Immunol 1989;19:563–5.
Hammond DM, Nagarkatti PS, Gote LR, Seth A, Hassuneh MR,
Nagarkatti M. Double-negative T cells from MRL-lpr/lpr mice
mediate cytolytic activity when triggered through adhesion molecules and constitutively express perforin gene. J Exp Med 1993;
Без категории
Размер файла
458 Кб
development, prevent, progressive, antibody, glomerulonephritis, cells, cytoplasmic, dendriticum, antineutrophil, suppressants, perinuclear, scgkj, nk026680, mice, rapidly, novem, function
Пожаловаться на содержимое документа