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Departments of Endocrinology and Metabolism (D.J.G.,), Bone Marrow Transplantation and Cancer Immunobiology Laboratory (L.W., I.R., S.S.), Hebrew University-Hadassah Medical Center, Jerusalem 91120, Israel; the Oxford Diabetes Research Laboratories, Radcliffe Infirmary (J.v.d.B., A.C.), Oxford, United Kingdom OX2 6HE; and the Department of Biochemistry, Tohoku University School of Medicine (H.O.), Sendai 98–77, Japan
Address all correspondence and requests for reprints to: David J. Gross, M.D., Department of Endocrinology and Metabolism, Hadassah University Hospital, Jerusalem 91120, Israel. E-mail: gross{at}vms.huji.ac.il
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-interferon expression by the ß-cells induces lymphocytic
infiltration and ß-cell damage (5), and in NOD (nonobese diabetic)
mice (6, 7). Moreover, the fact that only a small minority of
individuals with islet cell antibodies, an indicator of autoimmunity
toward the ß-cell, proceed to develop diabetes (8, 9, 10) indicates that
other factors, namely ß-cell function and capacity for proliferation
and/or neoformation, might play a crucial role in the development of
the disease. The lack of significant proliferative capacity of mature
ß-cells constitutes a major impediment to the treatment of diabetes,
illustrated by the low success rates of various immunomodulatory
approaches in patients with newly diagnosed IDDM (11). In this setting,
the unsatisfactory outcome can be accounted for by the lack of a
critical ß-cell mass necessary to maintain normoglycemia despite
successful abrogation of the autoimmune process. We have previously shown that linomide, a novel immunomodulatory drug, prevents diabetes and insulitis in young female NOD, but only partially ameliorates diabetes in animals with advanced disease (12). In the latter condition, linomide can protect from diabetes, providing that the ß-cell mass is sufficiently augmented by islet isografting (13). In a recent study, Watanabe et al. demonstrate induction of ß-cell replication and amelioration of diabetes in pancreatectomized rats by Reg protein (14), Therefore, we reasoned that linomide treatment of female NOD with advanced disease combined with this alternative approach for ß-cell augmentation, Reg-induced ß-cell expansion, might result in an improved salvage rate from clinical diabetes in this model of autoimmune diabetes. We report herein our results with this approach.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment compounds
Linomide. Quinoline-3-carboxamide (linomide) was provided by
Dr. Terje Kalland, Division of Immunology, Pharmacia Upjohn (Lund,
Sweden). Freshly dissolved linomide was diluted to 0.5 mg/ml in normal
drinking water (nonacidified tap water) and given daily for 10
weeks.
Reg peptide. The human reg complementary DNA
encompassing the coding sequence was introduced into a Pichia
pastoris expression vector for production of recombinant human Reg
(rhReg) protein (15). rhReg containing yeast supernatant was
concentrated by precipitation with a 60% saturated solution of
ammonium sulfate. The precipitate was dissolved and then further
concentrated and desalted using an Intersep apparatus (Intersep
filtration systems, Berkshire, UK) with a 10-kDa cut-off membrane. A
dominant component of the protein concentrate migrates on 15% SDS-PAGE
at the same position as the rhReg in unprocessed yeast medium and after
ion exchange chromatography, and showed a band at 16.5 kDa that was
detected by a monoclonal antihuman Reg antibody on Western blot (Fig. 1
). Before injection, the partially
purified lyophylized protein was reconstituted at a concentration of 1
mg/ml in 50 mM acetic acid. For control experiments,
supernatant from non-Reg-expressing yeast was processed according to
the same protocol.
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Linomide and Reg peptide administration to female NOD mice with advanced disease. Fourteen-week-old animals with both NGT and IGT were assigned to four treatment groups: a) control group, rhReg vehicle or processed non-rhReg-expressing yeast medium, ip; b) rhReg (1 mg/kg·day, ip, daily; six times a week; a dose shown to be effective in the pancreatectomized rat model) (14); c) linomide (0.5 mg/ml in drinking water) (12); and d) linomide (0.5 mg/ml, orally) and rhReg (1 mg/kg·day, ip, daily). The level of urinary glucose (Labstix, Bayer Diagnostics, Hampshire, UK) was followed on a biweekly basis. The onset of diabetes was determined after the appearance of glucosuria on at least two consecutive determinations. After 10 weeks of treatment, in view of the increased nondiabetes-related mortality noted in group d animals (see Results), the experiments were terminated. In the surviving animals, an additional IPGTT was performed, and the following day they were killed for histopathological evaluation and determination of pancreatic immunoreactive insulin (IRI) content.
rhReg administration to prediabetic female NOD. Five-week-old animals were treated as described for group b above from 5–12 weeks of age. Therapy was then discontinued and animals were followed to 40 weeks of age, at which time determinations were made as detailed above for mice with advanced disease. Untreated female mice served as controls. Control group animals and a group of rhReg-treated animals were killed and examined at 21 weeks of age.
Pancreatic islet histology. Pancreatic tissue was removed, fixed in 10% formalin in 0.9% saline, and embedded in wax. Two sets of five serial 5-µm sections were cut for immunolabeling at a cutting interval of 150 µm. Sections were immunolabeled for insulin (guinea pig antiinsulin dilution, 1:1000; ICN, Thame, UK) and glucagon (rabbit antipancreatic glucagon dilution, 1:2000) and detected with peroxidase-conjugated antiguinea pig (Dako, High Wycombe, UK) or peroxidase-conjugated antirabbit antisera (dilution, 1:50; Dako).
Morphometry. Areas of pancreatic islets (islet area), immunolabeled ß-cells (ß-cell area), and lymphocytic infiltrate into islets (area of infiltrate) were measured by camera lucida at a magnification of x600. Morphometric data were analyzed by IBAS (Kontron, Munich, Germany) for statistical analysis. Data were collected from all islets in both sets of sections (n = 2–20 islets). In the absence of insulin immunoreactivity, data were derived from adjacent sections labeled for glucagon. Clusters of more than three cells were considered to be an islet, and only infiltration associated with islets was included.
Pancreatic IRI concentration. Fragments (70–50 mg) of pancreatic tissue were weighed, sonicated in 1 ml ice-cold 1 M acetic acid, and gently rocked at 4 C for 16 h. The particulate fraction was then sedimented by centrifugation in a microfuge at maximal speed for 5 min. The supernatants were removed, and aliquots were dried down in a Speed-Vac apparatus (Savant Instruments, Hicksville, NY), reconstituted in 1 ml RIA buffer (PBS containing 0.1% RIA grade BSA), and assayed for IRI using a rat insulin RIA as previously described (16).
Statistical analysis. Statistical significance was determined for group comparisons with Mann-Whitney’s test and for survival curves by the log rank test. Survival curves in which reversal of the incidence of glucosuria was observed were compared by Fisher’s exact test at the end of the experiment.
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Results |
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, with NGT animals shown in A and IGT animals in B. The incidence of
diabetes in control animals (group a) was 60% and 86% for the NGT and
IGT groups, respectively. In groups c and d, treated with linomide or
with linomide plus rhReg injections, no NGT animals developed diabetes
over the 10-week period of the study; in NGT animals receiving rhReg
treatment alone (group b), partial protection was observed: 20%
developed diabetes compared with 60% in the control group (P <
0.03). In the IGT animals (Fig. 2B), mice in all groups rapidly
developed diabetes, attaining an incidence of 30–50% after 2 weeks of
treatment. After this time, a reversal of glucosuria was observed in
animals treated with combined rhReg and linomide (group d), with a
diminution of the incidence to 13% after 10 weeks of treatment
compared with 80% in the control group (P < 0.0001). This
contrasted with the steady increase in the incidence of diabetes in the
other groups treated with each agent alone (Fig. 2B). With the
exception of the combined rhReg and linomide group, all animals with
frank diabetes remained diabetic and succumbed to the disease within
4–6 weeks after the onset of glucosuria (data not shown). In view of
the experiments which indicated a partial protective effect of the
rhReg peptide alone in mice with less severe disease (group b, NGT) and
the deleterious effects of combined therapy (group d; see below), we
further investigated the impact of rhReg by treatment of prediabetic,
5-week-old animals with rhReg alone for a limited period (Fig. 3). Over 80% of control mice developed
diabetes by 21 weeks of age, whereas only 25% of the rhReg-treated
mice developed the disease at 40 weeks of age (P <
0.0001).
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Blood glucose levels and IPGTTs in nondiabetic animals
To obtain better assessment of metabolic control, blood glucose
levels and, in some experiments, IPGTTs were determined at the end
point of experiments in nondiabetic animals.
Fourteen-week-old animal experiments. In the animals assigned
initially to the NGT group, the control group was glucose intolerant
after 10 weeks of treatment. In contrast, all treatment groups
maintained blood glucose levels not significantly different from those
at the inception of therapy (Table 1). In
the IGT animals, glucose levels were higher after 10 weeks in all
treatment groups, with the striking exception of the linomide plus
rhReg group (Table 1), in which the mean blood glucose levels did not
deteriorate (rhReg plus linomide vs. control,
P < 0.0005). IPGTT determinations were performed
before and after the 10-week treatment period in glucose-intolerant
mice. In the control group, all animals succumbed to diabetes. In the
Reg-treated (Reg alone and Reg plus linomide) groups, 6 of 10 animals
who did not progress to frank diabetes had no deterioration of glucose
tolerance at 24 weeks [blood glucose: 0 min values (mean ±
SEM), 8.2 ± 2 and 7.9 ± 2.7 mmol/liter; 60 min
values, 15.3 ± 6.2 and 10.2 ± 2.4 mmol/liter at 14 and 24
weeks, respectively]. Linomide treatment alone, however, did not
confer protection from diabetes in this group.
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Effects of the various treatments on pancreatic ß-cell area,
islet ß-cell area, and islet mononuclear infiltrate
At 24 weeks of age, visible islet ß-cells were present in 14 of
23 animals that were glucose tolerant at the beginning of the treatment
period (groups a–d, NGT), but in only 2 of 16 mice that were initially
glucose-intolerant (groups a–d, IGT). There was no significant
difference in islet size between the groups, and the ß-cell area in
the islets (ß-cell area per islet) was variable (groups a–d: NGT
range, 0–0.7 µm2; n = 23; groups a–d: IGT,
0.075–0.792 µm2; n = 16). Treatment with linomide,
rhReg, or both agents together did not alter the morphological
characteristics of the islets (infiltrate area per islet area, ß-cell
per islet area) in the initially glucose-intolerant mice (Table 2). However, combined treatment with
linomide and rhReg caused a 3-fold increase in the mean ß-cell area
per islet in group d (NGT) compared with that in the untreated controls
(P < 0.05), and a trend toward a higher ß-cell area
was also noted in group b (NGT) treated with rhReg only (Table 2). In
the prediabetic animals, a significant augmentation of ß-cell area
was seen in animals at 21 and 40 weeks of age (Table 2). The
appropriate control group for the treated animals at age 40 weeks is
made up of frankly diabetic animals (in which ß-cell area is nil;
data not shown), as all female NOD mice develop diabetes by this
age.
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). In the IGT mice,
however, the IRI content was very low in control mice that survived
until 24 weeks, as was the content in the linomide-treated mice (Fig. 4). In rhReg-treated IGT mice, IRI content, although quite variable,
was considerably higher, on the average, (rhReg vs.
linomide, P < 0.04; rhReg plus linomide vs.
linomide, P < 0.04; Fig. 4). In the 5-week-old
prediabetic mice treated with rhReg alone (Fig. 3
), pancreatic IRI at
41 weeks of age was augmented in seven of nine animals and was similar
to that in nondiabetic male NOD mice (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion by NOD splenic macrophages (Weiss, L., I. Reibstein, M.
Zeira, S. Slavin, and D. J. Gross, manuscript in preparation), it is
likely that linomide-induced down-regulation of these and possibly
other cytokines that have been shown to adversely affect ß-cell
function (17, 18, 19) and/or insulin action (20, 21) underlies the improved
metabolic outcome with the combined therapy. The lack of visible
preservation of ß-cell area in the glucose-intolerant group probably
results from the paucity of islets in the examined pancreata and the
small number of animals available for analysis. In glucose-tolerant
14-week-old animals and 5-week-old prediabetic mice, preservation of
ß-cell area was apparent with rhReg treatment alone. Thus, in animals
with less severe disease, rhReg-induced ß-cell augmentation might
override the destructive autoimmune process and prevent clinical
diabetes. As modulation of ß-cell mass has been shown to determine
the susceptibility of NOD mice to autoimmune diabetes (22), it is
conceivable that Reg acts via ß-cell mass-dependent modulation of
autoimmunity. The presence of insulitis in the majority of
rhReg-treated animals, however, would suggest a more direct effect of
Reg on ß-cell mass. Reg is a C-type lectin originally cloned from a complementary DNA library prepared from regenerating islets (23). As Reg is also expressed in pancreatic acinar tissue and is identical to pancreatic stone protein and thread protein, its role in ß-cell neogenesis/replication has remained controversial. In a study by Miyaura et al., Reg expression appears to positively correlate ß-cell replication in the insulinoma-implanted NEDH (New England-Deaconess Hospital) rat model (24). On the other hand, Smith and co-workers found no such relationship in rats either partially pancreatectomized or glucose infused, both of which are known to induce ß-cell replication (25). Both groups failed to demonstrate Reg expression in pancreatic islets or ß-cell lines; in contrast, studies by others demonstrated Reg at both the protein and messenger RNA levels in replicating islets (26, 27, 28) and islets of remission in BB/Wor/Tky rats (29). Adopting the protocol of Watanabe et al. (14) for amelioration of diabetes in pancreatectomized rats, we found that rhReg ameliorates diabetes in female NOD mice, providing that the autoimmune process is adequately controlled. We also found a concurrent increase in islet ß-cell area, possibly a result of Reg-induced maturation of NOD islet ß-cell precursors (6). Although not examined directly, the presence of the increased ß-cell area in rhReg-treated, but not in linomide-treated, animals and the direct demonstration of Reg-induced ß-cell proliferation in rat islets (14) lend credence to our interpretation. Interestingly, endogenous expression of pancreatic Reg messenger RNA correlates with islet neogenesis in the Syrian golden hamster islet regeneration model (30) and more recently in an analogous model for rat islet regeneration (31). Reg has also recently been shown to have a mitogenic effect on a ductular pancreatic cell line (32), a potential source of ß-cell neogenesis (4). These observations together with the finding that pancreatic Reg expression correlates with a reduced severity of the disease in the NOD mouse with active diabetogenesis (33) indicate that Reg could conceivably mediate cross-talk between the exocrine and endocrine pancreas under conditions where induction of ß-cell mass expansion is needed.
Linomide, the immunomodulating agent used in this study, has pleotropic effects on the immune system (12) and appears to abrogate diabetes in the NOD mouse by reinduction of tolerance to self antigens without general immunosuppression (12). A disturbing effect of linomide observed in this study was the appearance of marked peritoneal adhesions in the groups treated with the combination of Reg and linomide. In these groups, deaths unrelated to diabetes occurred and obliged us to terminate the experiments earlier than originally planned. This mortality was probably due to the peritoneal pathology and could be a result of the proinflammatory effect of linomide observed in some experimental settings, such as collagen-induced arthritis (34, 35). We are currently examining alternative modalities of rhReg administration to avoid this complication.
In conclusion, we have demonstrated that abrogation of autoimmunity combined with induction of expansion/maintenance of ß-cell mass constitutes a potential therapeutic approach for treatment of IDDM. The exact nature of both linomide-induced immunomodulation and Reg-induced expansion of ß-cell mass in the NOD mouse remains to be determined.
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Acknowledgments |
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Footnotes |
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Received October 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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transgenic mice.
Development 118:33–46[Abstract]
-Cell neogenesis in an animal model
of IDDM. Diabetes 46:599–606[Abstract]
-induced insulin resistance in vivo and
its prevention by troglitazone. Diabetes 46:1678–1683[Abstract]
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