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Laboratory for Experimental Medicine and Endocrinology (K.M.C., C.M., D.V., J.M.L., R.B.) and the Laboratory for Experimental Transplantation (M.W., L.O.), Gasthuisberg; Catholic University of Leuven, 3000 Leuven, Belgium
Address all correspondence and requests for reprints to: Dr. Roger Bouillon, Legendo, U.Z. Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. E-mail: Roger.Bouillon{at}Med.Kuleuven.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and higher levels of
interleukin-4 in the combination group. In conclusion, nonhypercalcemic
analogs of 1,25-dihydroxyvitamin D3 administered to NOD
mice when the autoimmune disease is already active can prevent clinical
diabetes when this therapy is combined with a short induction course of
an immunosuppressant such as CyA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A more specific immunomodulation aiming at restoring the immune
imbalance without interfering with the overall immune defense systems
is clinically more attractive (9, 10, 11). We have demonstrated that the
active form of vitamin D, 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] and its nonhypercalcemic
analogs, exert such immunomodulation and can prevent insulitis and
diabetes in the NOD mouse (12, 13, 14). Indeed,
1,25-(OH)2D3 and its analogs did not induce a
generalized immunosuppression, but provoked after long term treatment a
restoration of the defective regulator system in the NOD mouse. The
actions of this family of molecules on the immune system are
exerted via receptors for 1,25-(OH)2D3 that
are present in several immune cells, such as monocytes/macrophages,
activated T lymphocytes, and B lymphocytes.
1,25-(OH)2D3 and some of its analogs induce
differentiation of monocytes toward macrophages and stimulate their
activity (15, 16, 17). On the other hand, these substances inhibit T cell
proliferation and cytokine production, such as interleukin-2 (IL-2),
IL-12, and interferon- (IFN) (18, 19, 20, 21). These in vitro
effects are reflected in vivo by a protection against many
autoimmune diseases and a prolongation of allograft survival (12, 13, 14, 22, 23, 24, 25, 26).
In the NOD mouse, insulitis and diabetes incidence was lowered by 1,25-(OH)2D3 and its nonhypercalcemic analogs when treatment was started before the appearance of insulitis and, thus, the initiation of autoimmunity (12, 13, 14). The first group of subjects in whom active diabetes prevention would be applied, however, consists of prediabetic relatives of type I diabetic patients in whom the autoimmune attack is already ongoing. Therefore, before introducing the analogs of 1,25-(OH)2D3 for the treatment of such subjects, it has to be clarified whether these analogs can also arrest the progression to clinically overt diabetes. The aim of this study was to evaluate the therapeutic potential of one of these analogs, MC1288 [20-epi-1,25-(OH)2D3], both alone and in combination with a short induction course of CyA, in a model of spontaneous diabetes, the NOD mouse, when started after the autoimmune attack against the ß-cell, as reflected by the presence of insulitis. To prove the presence of this ß-cell attack before any treatment was started, pancreatic biopsies to demonstrate the presence of insulitis beyond any doubt were performed in each individual mouse. By themselves, these biopsies did not alter the incidence of diabetes in the controls and allowed for histological certainty about ongoing autoimmunity in treated mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental design and treatment regimen
Pancreatic biopsies were performed in female NOD mice at the age
of 70 days to histologically confirm the presence of insulitis in each
individual mouse. Biopsies were taken by resecting a small piece (3
mm3) of the pancreas of ether-anesthetized mice. Each
procedure was performed under sterile conditions, with a mortality rate
of 1%. Pancreas biopsies were fixed in Bouin’s solution and embedded
in paraffin. Serial sections were made and stained with hematoxylin and
eosin. Biopsies were scored for insulitis in a double blinded manner,
and at least seven different islets per biopsy were examined. The level
of lymphocytic infiltration in the islets was scored as follows: 0
= no lymphocytes in or around the islets; 1 = periductular
infiltrate; 2 = periislet infiltrate; 3 = intraislet
infiltrate; and 4 = intraislet infiltrate associated with clear
ß-cell destruction. The mean score for each pancreas was calculated
by dividing the total score by the numbers of islets scored. After the
surgical procedure, mice were rested for 15 days, and subsequently,
independent of the histological score, they were randomized into a
control group (n = 26) receiving the treatment vehicle only and
three treatment groups receiving, respectively, CyA (7.5 mg/kg·day)
from days 85–105 (n = 19), MC1288 (0.1 µg/kg·2 days) from
days 85–200 (n = 20), or the combination of these two regimens
(n = 20).
CyA was purchased from the Sandoz Co. (Basel, Switzerland), and MC1288 was a gift from Dr. L. Binderup (Leo Pharmaceuticals, Ballerup, Denmark). All drugs were suspended in arachis oil and administered ip. Mice undergoing only the surgical procedure did not show any deterioration in glucose tolerance or any increase in diabetes incidence compared with stock mice.
Evaluation of insulitis and diabetes
Diabetes was evaluated as described previously (12). Briefly,
mice were weighed weekly, and glucosuria was tested 3 times a week
starting at the age of 30 days using Clinistix (Bayer Diagnostics,
Basingstoke, UK). Glycemia was determined with a Glucocard (Menarini,
Firenze, Italy). Mice were considered diabetic when they had glucosuria
and a glycemia exceeding 230 mg/dl (13 mM) on 2 consecutive
days. Mice becoming diabetic before the age of 85 days were excluded
from the study. At diabetes diagnosis or at 200 days of age, mice were
killed by ether inhalation and cervical dislocation. Pancreases were
removed and fixed in Bouin’s solution. Hematoxylin- and eosin-stained
serial sections were evaluated for insulitis by 2 independent
investigators. A mean of 30 islets/pancreas were screened for
insulitis. The level of lymphocytic infiltration in the islets was
scored as described above.
Parameters of calcium and bone metabolism
Serum concentrations of calcium and osteocalcin, and bone calcium
content. At the age of 200 days or at diabetes diagnosis, blood
was taken by heart puncture. Total serum calcium was determined by
microcolorimetric assay (Sigma Chemical Co., St. Louis, MO), and serum
osteocalcin was determined by an in-house RIA that used mouse
osteocalcin as standard and a polyclonal guinea pig antimouse
osteocalcin antiserum (14). The within- and between-assay coefficients
of variation were 4 and 6%, respectively, and the sensitivity was 0.02
nmol/liter. The calcium content in tibia was measured as described
previously (14, 28).
Calcium and collagen cross-links in urine. At the age of 180 days, normoglycemic mice (n = 5–10) from each treatment group and control mice were housed for 24 h in metabolic cages, and urine was collected in 10-ml glass tubes containing 10 µl 6 N HCl. Calcium in urine was determined using the same microcolorimetric assay as that used for the determination of serum calcium. Collagen cross-links in urine reflect collagen breakdown and bone turnover and were determined as described previously (14, 29).
In vitro immune evaluation
At the end of the study, normoglycemic mice (n = 5) from
each treatment group and the control group were screened for signs of
generalized immunosuppression. Moreover, mononuclear cell subsets in
the spleen were analyzed by FACS analysis.
T cell suppression was evaluated by performing mixed lymphocyte reactions (MLR) and concanavalin A (Con A) proliferation tests. For MLR, spleens were removed aseptically, gently teased apart, and pressed through a steel mesh. After T cell enrichment by nylon wool passage, splenocytes of test animals (5 x 106/ml; 5 x 105 cells/well) were cocultured for 5 days with irradiated (30 Gy; 60Co source) splenocytes from C57Bl/6 mice as stimulators (5 x 106/ml; 5 x 105 cells/well) in RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.2% mercaptoethanol. Cells were cultured in triplicate in flat bottomed 96-well microtiter tissue culture plates (Nunc, Roskilde, Denmark). Cells were pulsed after 4 days with 1 µCi [methyl-3H]thymidine (Radiochemical Center Amersham, Aylesbury, UK), harvested 18 h later on glass filter paper, and counted. Proliferation values were expressed as counts per min.
For Con A stimulation, splenocytes (1 x 106/ml; 2 x 105 cells/well) from test mice were cultured in RPMI 1640 added with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.2% mercaptoethanol in triplicate in flat bottomed 96-well microtiter tissue culture plates (Nunc, Roskilde, Denmark) in the presence of Con A (6.25 µg/ml) for 72 h. [Methyl-3H]thymidine incorporation was measured after 18 h. Again values were expressed as counts per min.
For the FACS analysis, splenocytes (1 x 106/ml) were incubated for 30 min at 4 C with 10 µl fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies. The markers used were Thy1.2 (pan T cell), L3T4 (CD4), Lyt2 (CD8; Caltag, San Francisco, CA), IgM+IgG (B cell; Jackson ImmunoResearch Laboratories, West Grove, PA), CD11b (monocytes and macrophages), and ASGM-1 (natural killer cells; Wako Chemicals, Neuss, Germany).
Cotransfer experiments
To detect the existence of suppressor cells, cotransfer
experiments were performed. Naive 6- to 8-week-old male NOD mice, were
irradiated (7.5 Gy) and received 48 h later 20 x
106 splenocytes, iv, which were obtained from overtly
diabetic NOD mice (30). Twenty-four hours before the transfer of the
diabetogenic cells, 20 x 106 splenocytes from mice
treated with MC1288, CyA, or the combination therapy or control mice
were injected iv. Mice were tested for glucosuria twice weekly and were
considered diabetic following the above-described criteria.
Cytokine analysis by quantitative RT-PCR
Total RNA was extracted from fresh pancreatic tissue (free of
lymph nodes), which was taken from normoglycemic test mice from each
group (n = 5) at the end of the study, using the acid guanidinium
thiocyanate-phenol-chloroform method (31).
A constant amount of 4 µg target RNA was reverse transcribed with Superscript II RT (Life Technologies, Merelbeke, Belgium) at 42 C for 80 min in the presence of random primers.
For IL-2, IL-4, IL-10, IL-12, IFN, and ß-actin, real time
quantitative PCR was performed (32, 33). PCR reactions were performed
in the ABI Prism 7700 sequence detector, which contains a Gene-Amp PCR
System 9600 (Perkin Elmer, Nieuwerkerk a/d Ijssel, The Netherlands).
Reaction conditions were programed on a Power Macintosh 7200 (Apple
Computer, Cupertino, CA), linked directly to the 7700 Sequence
Detector. The assay uses the 5'-nuclease activity of Taq
polymerase to cleave a nonextendible hybridization probe during the
extension phase of PCR. The approach uses dual labeled fluorogenic
hybridization probes. One fluorescent dye serves as a reporter (FAM or
TET), and its emission spectra is quenched by the second fluorescent
dye, TAMRA. The nuclease degradation of the hybridization probe
releases the quenching of the FAM fluorescent emission, resulting in an
increase in peak fluorescent emission at 518 nm. The use of a sequence
detector (ABI Prism) allows measurement of fluorescent spectra of all
96 wells of the thermal cycler continuously during PCR amplification.
Therefore, the reactions are monitored in real time. The model 7700
software constructs amplification plots from the extension phase
fluorescent emission data collected during the PCR amplification.
CT (threshold) values are calculated by determining the
point at which the fluorescence exceeds a threshold limit (usually 10
times the SD of the baseline). Primers were chosen with the
assistance of the computer program Primer Express (Perkin-Elmer,
Norwalk, CT) and were always located in two different exons (Table 1
). Amplification reactions (25 µl)
contained 1 µl complementary DNA (cDNA) sample; 2.5 µl 10 x
TaqMan Buffer A (10x = 500 mM KCl, 100 mM
Tris-HCl, 0.1 M EDTA, 600 nM passive reference
1, pH 8.3; Nieuwerkerk a/d Ijssel, The Netherlands), 200
µM deoxy (d)-ATP, dCTP, dGTP, and 400 µM
dUTP; 7 mM MgCl2; 0.625 U AmpliTaq Gold
(Nieuwerkerk a/d Ijssel); 0.25 U AmpErase uracil
N-glycosylase (Nieuwerkerk a/d Ijssel); and 150
nM of each primer. The reactions also contained the
corresponding detection probe (100 nM; Table 1). For the
generation of standard curves, plasmid clones containing a partial cDNA
sequence of IL-2, IL-4, and IL-10 were constructed. Briefly, total RNA
was extracted from spleens, and cytokine cDNA fragments were generated
by RT-PCR using the PCR primers as described above. The amplicons were
cloned into pGEM-TEasy (Promega, Leiden, The Netherlands). The length
of the amplicons was confirmed by restriction analysis. Serial
dilutions from the resulting plasmid clones were used as standard
curves, each containing a known amount of template copy number (34).
For the standard curves of the other cytokines and ß-actin, serial
dilutions of a known amount of a cDNA sample were used. The
CT values of each cytokine were plotted on these standard
curves to obtain the amount of copies present in the initial cDNA
sample. Each PCR amplification was performed in quadruplicate, using
the following conditions: 2 min at 50 C and 10 min at 95 C, followed by
a total of 40 two-temperature cycles (15 sec at 95 C and 1 min at 60
C). A normalization to ß-actin (housekeeping gene) was performed for
each sample. Gel electrophoresis was performed to confirm the correct
size of the amplicons and the absence of nonspecific bands.
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(TNF) was
assessed by a competitive RT-PCR method, described by N. Zhou et
al. (35). Briefly, serial dilutions of an internal RNA standard,
designed to be identical to the target messenger RNA (mRNA) except for
the addition of an internal 92-bp fragment, were reverse transcribed
together with the target RNA. After PCR amplification, the ratio of
amplified target to competitor PCR product was determined for each
sample. Values were expressed in picograms per µg total RNA.
Statistical analysis
Data were expressed as the mean ±SD except for
histological scoring of insulitis and the PCR results (mean ±
SEM). Statistical tests used were the 
2 test
for insulitis and diabetes incidence and cotransfer experiments, the
Mann-Whitney U test for immune and cytokine analysis, or Student’s
t test for histological scoring of insulitis, calcium, and
bone parameters. Significance was defined at the 0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). When comparing the
different treatment groups and limiting the analysis to animals
presenting with insulitis before treatment was started, the occurrence
of diabetes by 200 days of age was 87% (13 of 15) in the group treated
with CyA, 69% (11 of 16) in the group treated with MC1288, and 47% (7
of 15) in the group receiving MC1288 and CyA (P < 0.01
vs. CyA; Fig. 2). Considering
only animals that were insulitis free at the time of biopsy (day 70),
insulitis developed in almost all cases treated with CyA or MC1288
monotherapy (3 of 4 and 4 of 4, respectively). In contrast, only 2 of 5
animals (40%) treated with the combination therapy developed
insulitis (P < 0.05). Interestingly, none of these
latter 2 mice progressed to clinically overt diabetes. When analyzing
the severity of islet infiltration at the end of the study, control
animals had a mean insulitis score of 3.2 ± 1.3, comparable to
that of CyA-treated (3.6 ± 0.8; P = NS) and
MC1288-treated mice (3.2 ± 1.2; P = NS), but mice
treated with the combination therapy had a significantly lower mean
score of 2.1 ± 1.5 (P < 0.05 vs. all
other groups). Again when comparing the individual treatment groups and
when classifying the islets according to insulitis score, we noted that
the group treated with the combination therapy had significantly higher
levels of islets with a score of 0 (P < 0.05) and
significantly lower levels of islets with a score of 4
(P < 0.001). We also noted a trend toward higher
numbers of score 2 islets (periinsulitis) in this combination treatment
group (Fig. 3).
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). Osteocalcin levels, indicative of
osteoblast activity and bone turnover, were normal in mice treated with
CyA or MC1288, but surprisingly they were significantly lower in the
combination therapy group (62.8 ± 5.9 vs. 82.8 ±
4.0 µg/liter in controls; P < 0.001; Table 2). Bone
calcium content was significantly lower in CyA-treated mice, indicating
bone decalcification (5.63 ± 0.49 vs. 6.6 ± 0.26
mg/tibia in controls; P < 0.01), whereas treatment
with MC1288 only had no effect on bone calcium (6.43 ± 0.35
mg/tibia; P = NS vs. controls). Reflecting
the low osteocalcin levels and decreased bone turnover, mice treated
with the combination therapy presented with a higher calcium content in
their tibiae (7.18 ± 0.26 mg/tibia; P < 0.05
vs. controls).
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). The urinary
excretion of collagen cross-links (pyridinoline and deoxypyridinoline)
was measured to evaluate the effect of the treatment regimens on bone
resorption (Table 2). Treatment with MC1288 increased the excretion of
pyridinoline significantly from 592 ± 74
nM/mM creatinine in control mice to
876 ± 76 (P < 0.001), whereas in NOD mice
treated with CyA or the combination therapy the values did not differ
significantly from those in controls. The excretion of
deoxypyridinoline was comparable in all mice.
Immune evaluation
To evaluate whether the treatment regimens (CyA, MC1288, and the
combination therapy) resulted in generalized immunosuppression, MLR-
and Con A-induced proliferation tests were performed at 200 days.
Lymphocytes taken from 200-day-old NOD mice treated with MC1288, CyA,
or the combination therapy proliferated to the same extent as controls,
suggesting that no long term generalized immunosuppression was present
(Fig. 4). FACS analysis and phenotyping
of T and B lymphocytes, monocytes/macrophages, and natural killer cells
in the spleen were performed as well. No significant differences were
present among the different treatment groups (Table 3).
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Cytokine analysis by quantitative RT-PCR
To gain more insight into the protective mechanism of MC1288 and
CyA, the cytokine mRNA levels in the pancreases of protected NOD mice
were examined. We did not include acutely diabetic mice, as this would
bias our results; more diabetic mice were present in the control group
and in the groups treated with monotherapy, and these have a more
destructive cytokine pattern (data not shown) (36). Even when
considering only normoglycemic animals, IFN mRNA levels in
pancreases of mice treated with the combination therapy were
significantly lower than those in pancreases of control mice
(P < 0.05) and mice treated with CyA alone
(P < 0.05; Fig. 5).
IFN is the main cytokine whose gene expression is found to correlate
with islet ß-cell destructive insulitis (36). mRNA levels of IL-4
were clearly higher in mice treated with the combination therapy, and
this increase was significant compared with those in the CyA group
(P < 0.05; Fig. 5). IL-4 mRNA expression is reported
to be more associated with nondestructive insulitis than with ß-cell
destructive insulitis and may act as a diabetes-protective cytokine
(34).
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were not
significantly different among any of the treatment groups (Fig. 6). IL-2 mRNA levels were very low in all
groups (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have tested the potential of MC1288 [20-epi-1,25-(OH)2D3], alone or in combination with a short induction course of CyA, to arrest the progression from the histological autoimmune attack to clinically overt diabetes in NOD mice when therapy was started after the autoimmune attack had begun. The presence of an active autoimmune attack was evidenced by the presence of insulitis in pancreatic biopsies taken before treatment was started. Treatment with the combination of MC1288 and CyA decreased diabetes incidence with 46% vs. control animals. A critical analysis of these data demonstrates that in mice presenting with insulitis at the time of starting therapy, i.e. the equivalent of prediabetic subjects, MC1288 in combination with CyA clearly prevented diabetes progression. An even clearer effect of the combination therapy was seen in mice without insulitis at the start of treatment, where only 40% of these animals eventually developed insulitis, and none of them developed diabetes. In contrast, almost all animals receiving one of both monotherapies developed insulitis, and most of them proceeded to diabetes. Together these data suggest that a combination of a nonhypercalcemic analog of 1,25-(OH)2D3 and a short course of a low dose of CyA can interrupt progression of disease, but is still most effective in interfering with the induction of autoimmunity itself. Additive effects between 1,25-(OH)2D3 and CyA are in accordance with findings in other models (37) and can be expected from the mechanism of action of the individual drugs. Although both directly suppress IL-2 secretion, 1,25-(OH)2D3 does so in a stage distally from calcineurin (38). The mechanism of protection in our model is clearly not generalized immune suppression, and cotransfer experiments with splenocytes from animals treated with CyA, MC1288, or the combination therapy did not reveal the presence of suppressor cells. This is in contrast to the findings in NOD mice that had been treated with 1,25-(OH)2D3 or its analogs alone from an early age (21 days) onward (12, 14). Recent data, however, suggest that also in this long term model the suppressor cells, although they are induced, are not the main mechanism in the protection against diabetes (39). Therefore, suppressor cells are unlikely to be the only explanation for the protection against diabetes achieved in our previous as well as in the present study.
Signs of local immunoregulation were, however, noted in the islets
themselves. Cytokine analysis performed on the pancreas of
normoglycemic mice showed significantly lower levels of IFN and
higher levels of IL-4 in protected mice treated with the combination
therapy. It has been demonstrated that IFN is the main cytokine
whose gene expression is found to correlate with islet ß-cell
destructive insulitis (36). Also the levels of IL-4 mRNA, a cytokine
that appears to be protective against diabetes in NOD mice, were
elevated locally in the islets of combination-treated mice (34, 40).
These data combined suggest a local immune shift induced by the
combination treatment. Note that we did not find a clear effect on
IL-12, the key cytokine in immune balance, probably because the
analysis was performed only at a later age (200 days).
Finally, all treatments were well tolerated. Mice treated with MC1288 alone had a bone calcium content comparable to that in control mice, whereas those treated with CyA had a significantly lower bone calcium content, confirming the findings in rats treated with CyA (41). The combination therapy, surprisingly, resulted in even lower bone turnover and higher calcium content of bone. These data together with observations made for other analogs of 1,25-(OH)2D3 eliminate the major problem of possible clinical use of the 1,25-(OH)2D3 analogs, namely the hypercalcemia and osteoporosis induced by 1,25-(OH)2D3 itself, and allow the conclusion that treatment with these substances, even over the long term, appears to be safe. Side-effects on systems other than calcium or bone homeostasis have not been described to date.
In conclusion, the approach of combining an acceptable, short induction course of a classical immunosuppressant with nonhypercalcemic analogs of 1,25-(OH)2D3 is promising because it can block the progression from autoimmune ß-cell attack toward overt clinical disease. Further investigations are necessary and might open new perspectives in the prevention of autoimmune diabetes.
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Footnotes |
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2 Recipient of a National Foundation For Scientific Research
predoctoral fellowship.
3 Recipient of a National Foundation For Scientific Research
postdoctoral fellowship.
Received March 12, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin D3 inhibits gamma-interferon
synthesis by normal human peripheral blood lymphocytes. Proc Natl Acad
Sci USA 84:3385–3391,25-dihydroxyvitamin D3. Clin Exp Immunol 94:412–417[Medline]
gene expression in pancreatic
islet-infiltrating mononuclear cells correlates with autoimmune
diabetes in nonobese diabetic mice. J Immunol 154:4874–4882[Abstract]
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) group, diabetes incidence was 74% (14 of 19), which
is comparable to diabetes incidence in control mice (
; 65%; 17 of
26). Treatment with MC1288 (•) did not alter diabetes incidence (14
of 20; 70%), but the combination therapy (
) reduced the incidence
of diabetes to 35% (7 of 20; P < 0.05
vs. untreated; P < 0.01
vs. CyA; P < 0.025
vs. MC1288).
