Download Local Activation of Dendritic Cells Leads to Insulitis and

Local Activation of Dendritic Cells Leads to Insulitis
and Development of Insulin-Dependent Diabetes in
Transgenic Mice Expressing CD154 on the Pancreatic
␤-Cells
Claus Haase,1 Kresten Skak,1,2 Birgitte K. Michelsen,1,3 and Helle Markholst1
The initial events leading to activation of the immune
system in type 1 diabetes are still largely unknown. In
vivo, dendritic cells (DCs) are thought to be the only
antigen-presenting cells (APCs) capable of activating
naı̈ve T-cells and are therefore important for the initiation of the autoimmune response. To test the effect of
activating islet-associated APCs in situ, we generated
transgenic mice expressing CD154 (CD40 ligand) under
control of the rat insulin promoter (RIP). RIP-CD154
mice developed both insulitis and diabetes, although
with different incidence in independent lines. We show
that activated DCs could be detected both in the pancreas and in the draining pancreatic lymph nodes. Furthermore, diabetes development was dependent on the
presence of T- and B-cells since recombination-activating gene (RAG)-deficient RIP-CD154 mice did not develop diabetes. Finally, we show that the activation of
immune cells was confined to the pancreas because
transplantation of nontransgenic islets to diabetic recipients restored normoglycemia. Together, these data
suggest that expression of CD154 on the ␤-cells can lead
to activation of islet-associated APCs that will travel to
the lymph nodes and activate the immune system, leading to insulitis and diabetes. Diabetes 53:2588 –2595,
2004
T
ype 1 diabetes is an autoimmune disease wherein the insulin-producing ␤-cells in the pancreas
are destroyed in a process mediated by cells of
the immune system. T-cells, B-cells, and antigenpresenting cells (APCs) have all been suggested to be
important for the pathogenesis of the disease. However,
despite increasing knowledge of the mechanisms of ␤-cell
destruction, the reasons for the initiation of the disease are
largely unknown. Several animal models of type 1 diabetes
From the 1Hagedorn Research Institute, Gentofte, Denmark; 2Pharmacology
Research 4, Novo Nordisk, Måløv, Denmark; and 3Early Projects, Novo
Nordisk, Virum, Denmark.
Address correspondence and reprint requests to Helle Markholst, Hagedorn
Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. E-mail:
[email protected].
Received for publication 15 March 2004 and accepted in revised form
16 July 2004.
APC, antigen-presenting cell; DC, dendritic cell; MHC, major histocompatibility complex; pBG, pBlueSKII-␤globin; RAG, recombination activating gene;
RIP, rat insulin promoter.
© 2004 by the American Diabetes Association.
2588
exist (1– 4), but the precise mechanism behind the initiation of the autoimmune response is not clear.
One of the important steps in the initiation of any
immune response is the conversion of immature dendritic
cells (DCs) to mature DCs. This can be mediated by many
different signals, including double-stranded RNA, bacterial
lipopolysaccharide, and CD40 ligation (5). Among these
signals, several studies (6 –11) have shown that triggering
of CD40 by CD154 (CD40L) can convert an immature and
tolerogenic DC to a mature and immunogenic DC.
The central role of DCs in the initiation of the immune
response suggests that these cells also may play an important role in the initial events leading to type 1 diabetes.
One possible mechanism, by which type 1 diabetes could
be initiated, is through activation of tissue-resident APCs
in situ by an inflammatory trigger. This would lead to
presentation of islet-antigens in the draining lymph nodes
and to initiation of autoimmunity. A relative deficit in
regulatory T-cells concomitant with the expression of
major histocompatibility complex (MHC) molecules, allowing the development of an autoreactive T-cell repertoire, further contributes to the pathogenesis of type 1
diabetes in NOD mice, BB rats, and possibly also in human
type 1 diabetic patients (12,13). Although diabetes does
not normally develop in C57BL/6 or BALB/c mice, there is
evidence that DC activation in vivo can indeed lead to
insulitis and diabetes, although in some studies expression
of B7.1 on the ␤-cells is necessary to augment and amplify
the T-cell response (14 –16). These studies may also suggest that all autoreactive T-cells are not intrathymically
deleted in normal mouse strains, implying that these
T-cells can lead to autoimmunity if activated and expanded properly.
Given the central role of CD40/CD154 in DC activation,
we therefore hypothesized that expression of CD154 on
the surface of the ␤-cells could lead to the activation of
islet-associated APCs and subsequently to activation of the
immune system. We generated transgenic mice expressing
the CD154 molecule under control of the rat insulin
promoter (RIP), which mediates ␤-cell–specific expression (17). Remarkably, these mice exhibited a massive
infiltration by mononuclear cells in the islets of Langerhans and had severely impaired blood glucose regulation
that ultimately led to the development of diabetes. Disease
development was dependent on the presence of T- and
B-cells, since diabetes development was inhibited by
DIABETES, VOL. 53, OCTOBER 2004
C. HAASE AND ASSOCIATES
FIG. 1. A: DNA construct pBG-RIP-CD154 driving transgenic expression of CD154 cDNA under control of the RIP. PCR primers for
genotyping (␤ ⴙ ␥) and RT-PCR (␣ ⴙ ␥) are indicated, along with
expected band sizes. B: Transgenic CD154 is expressed in the pancreas
of 7-week-old RIP-CD154 transgene-positive (TGⴙ) mice, but not in
transgene-negative (TGⴚ) littermates. RT-PCR on cDNA from various
tissues using expression-specific primers (␣ ⴙ ␥ in A, 386 bp) and
␤-tubulin (156 bp). ⴚ, negative control; ⴙ, plasmid control (pBG-RIPCD154, 959 bp). C: Pancreatic frozen sections from 5-week-old RIPCD154 transgene-positive mice (L90) were stained for insulin and
CD154. Scale bar: 50 ␮m.
crossing the mice onto a recombination activating gene
(RAG)-deficient background. Our data suggest that local
activation and subsequent maturation of islet-associated
DCs by CD154 can lead to activation of the immune
system, which results in tissue-specific inflammation of
the islets of Langerhans and diabetes.
RESEARCH DESIGN AND METHODS
Murine CD154 cDNA was cloned by PCR using oligo-dT primed cDNA from
C57BL/6 mouse spleen that was prepared as described (18), using the primers
CD154, 5⬘-GCCACCATGATAGAAACATA-3⬘, and CD154 stop, 5⬘-TCAGAGTT
TGAGTAAGCCAA-3⬘. PCR products were cloned into the pCRII vector by TA
cloning (Invitrogen). Ligated products were sequenced and confirmed to be
the CD154 cDNA (according to GenBank no. NP035746). The CD154 cDNA
was subcloned by EcoRI digestion into the pBlueSKII–␤-globin (19) vector
(pBG), containing the exon 2-intron 2-exon 3 cassette from the ␤-globin gene
for efficient posttranscriptional processing in vivo. The obtained promoterless construct was termed pBG-CD154. The RIP was cloned from the
pJNL1-RIP-Tag (17) by digesting with KpnI and HindIII and was digested
similarly into the pBG-CD154. This construct was termed pBG-RIP-CD154. For
generation of transgenic mice, pBG-RIP-CD154 was digested by FspI and NotI,
yielding the transgenic construct depicted in Fig. 1A. This fragment was
purified and microinjected into (C57BL/6xCBA) F1 oocytes to generate
RIP-CD154 transgenic mice (MouseCamp; Karolinska Institute, Stockholm,
Sweden). Among 96 offspring, 14 carriers were identified on the basis of PCR
screening. Among the 14 offspring, all carriers that expressed the transgene
also had insulitis. Two lines (L60 and L90) were selected for further study and
backcrossed to C57BL/6 mice.
DIABETES, VOL. 53, OCTOBER 2004
Mouse breeding and glucose measurements. RIP-CD154 mice were maintained by backcrossing to C57BL/6 or RAG12M (RAG KO on C57BL/6
background) and were bred under specific pathogen-free conditions at
Taconic M&B (Ry, Denmark), and animals selected for further study were
housed at our Animal Unit (Novo Nordisk, Gentofte, Denmark). DNA from tail
biopsies was analyzed by PCR using the following primers: ␤, 5⬘-ATACTCTG
AGTCCAAACCGG-3⬘, and ␥, 5⬘-CTCCTCACAGTTCAGCAAGG-3⬘, and as an
internal control the primers for the Hes1 gene 5⬘-AGCCAGTGTCAACACGAC
ACC-3⬘ and 5⬘-TGTTAA GTGCATCCAAAATCAGTG-3⬘. To test for expression
of the transgene, cDNA was prepared from RNA from various tissues from
transgene-positive RIP-CD154 L60 and L90 as described (18). For detection of
the transgenic CD154 cDNA, the primers ␣, 5⬘-GAGGAGGCTTTTTTGGAGG
C-3⬘, and ␥ (see above) were used with the primers for the ␤-tubulin cDNA and
5⬘-ATCCTGGTACTGCTGGTACT-3⬘ and 5⬘-GAGCTGTTCAAGCGCATCTC-3⬘
as an internal control. Expected band sizes were 386 bp for the spliced
transgenic cDNA, 959 bp for the unspliced transgenic cDNA (plasmid control),
and 156 bp for the ␤-tubulin cDNA.
Presented data were obtained from generation N3 or later. No change in
phenotype has been observed during backcrossing to C57BL/6 or at the
different sites (Ry versus Gentofte). All animal experiments were conducted
according to Danish legislation and approved by the Danish Animal Inspectorate. Blood glucose was measured by tail vein blood sampling and analyzed
using the Medisense Precision Xtra Plus system. Maximum measurable blood
glucose level was 34 mmol/l, and mice were considered hyperglycemic when
blood glucose levels were ⬎11 mmol/l (which was the maximum measurement for nontransgenic controls) and diabetic when blood glucose was ⬎16.6
mmol/l for ⱖ2 consecutive weeks. Unless otherwise stated, samples were
taken from nonfasting mice.
Histology and immunostainings. For fluorescence histology on frozen
sections, tissue was snap frozen in Tissue-Tek on dry ice immediately after
dissection, cut into 5-␮m sections, and blocked using 5% normal goat serum
and 5% normal donkey serum for 20 min at 20°C. Staining with primary
antibodies was performed overnight at 4°C, using the following antibodies:
CD11c (HL3), CD4 (H129.19), CD8 (53– 6.7), CD19 (1D3), CD154 (MR1), and
MHC-II (M5/114.15.2), all from BD Pharmingen, and insulin was from Zymed.
All antibodies were diluted 1:100 –1:200 in PBS with 0.25% BSA. After washing
in PBS, second-step reagents were applied for 45 min at 20°C and included the
following antibodies: goat-anti– hamster-Cy3 (127-165-160), goat-anti–rat-Cy3
(112-165-167), donkey-anti– guinea pig-FITC (fluorescein isothiocyanate) (706095-148), and donkey-anti– guinea pig-biotin (706-065-148), all from The Jackson Laboratory. If necessary, a third-step reagent was streptavidin-AMCA
(7-amino-4-methylcoumarin-3-acetic acid) (016-150-084; The Jackson Laboratory), which was incubated for 30 min at 20°C. Controls included staining with
secondary antibody only or with isotype controls and were always negative
compared to experimental slides. For formalin-fixed sections, tissue was fixed
overnight in 4% formalin and transferred to 70% ethanol. Tissue was embedded
in paraffin, and 4-␮m sections were cut and stained for insulin (HUI-18; Novo
Nordisk) using Histostain-SP Bulk (Zymed). Staining was revealed using the
AEC (3-amino-9-ethylcarbazole chromogen) substrate kit (Zymed), and all
sections were counterstained with hematoxylin. Images were recorded by a
Hamamatsu C5810CCD cooled camera and processed in Adobe Photoshop.
Glucose tolerance tests. For C-peptide measurements (oral glucose tolerance test), mice were fasted for 6 h, briefly sedated with isoflurane, and 2 mg/g
body wt glucose at t ⫽ 0 min was injected via a mouse feeding needle. Blood
samples for C-peptide measurements were obtained in EDTA-coated tubes at
t ⫽ 30 min, and serum was prepared by centrifugation. C-peptide levels were
measured by Linco Diagnostics, using a radioimmunoassay. For an intraperitoneal glucose tolerance test, mice were fasted before an intraperitoneal
injection of 2 mg/g body wt glucose at t ⫽ 0 min. Blood glucose was measured
at the indicated times.
Islet transplantation. Islets were isolated from 6- to 10-week-old C57BL/6
mice by collagenase treatment of the pancreas, followed by handpicking of the
islets under a microscope. The islets were cultured for 6 –7 days at 37°C in
RPMI with 10% FBS, 11 mmol/l glucose, 2 mmol/l glutamine, 20 mmol/l
HEPES, and antibiotics. Before transplantation, viable islets were recounted
and transferred to medium containing 0.5% NUSerum. Diabetic transgenepositive RIP-CD154 or nondiabetic transgene-negative littermates (from N4
and N6 generation of backcrossing to C57Bl/6 mice) were anesthetized using
fentanyl/fluanison/midazolam (0.6, 19, and 9 ␮g/g s.c., respectively). For postoperative analgesia, buprenorfine and carprofen (0.15 and 5 ␮g/g s.c., respectively) were used. Each mouse received 300 –350 islets, which were placed
under the left kidney capsule.
To remove the transplanted tissue, the kidney with the transplant was
exposed using the same approach as above and the renal artery and vein
ligated in common. The kidney was removed in toto. Kidney histology was
performed after formalin fixation, as described.
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DIABETES IN RIP-CD154 TRANSGENIC MICE
FIG. 2. Formalin-fixed pancreata from RIP-CD154 mice stained for insulin. Sections from L60 are shown at 2 (A), 4 (B), 8 (C), and 18 (D) weeks.
Sections from L90 are shown at 2 (E and F), 4 (G), 8 (H), 12 (I), and 18 (J) weeks. Transgene-negative littermates from L90 are shown at 2 (K)
and 18 (L) weeks. Arrows point to very dense infiltrates in the pancreata of L90 transgenic mice (H and J). All images are representative of two
to five mice. Scale bar: 100 ␮m.
Flow cytometry. For flow cytometric analysis on pancreatic lymph node
cells, pancreatic lymph nodes were dissected from three to four transgenic
mice, and tissue was homogenized using a cell strainer (Falcon). Cells were
labeled with biotin-conjugated CD11c and phycoerythrin-conjugated CD40
antibodies, followed by streptavidin-APC (BD Pharmingen). All cells were
analyzed on a FACSCalibur, and data were analyzed using CellQuest software.
Events (40,000 –100,000) falling into a live gate based on forward and side
scatter characteristics were acquired.
Adoptive transfer. Spleen cells from recently diabetic transgene-positive
RIP-CD154 (L90) mice were isolated by homogenizing the tissue through a cell
strainer, followed by washing in ice-cold Hank’s balanced salt solution.
Erythrocytes were lysed, followed by two washes in ice-cold PBS. Living cells
(10 ⫻ 106; based on propidium-iodide/annexinV staining) were injected intraperitoneally in 200 ␮l PBS into 6- to 7-week-old RAG KO mice (RAG12M;
Taconic). Blood glucose was tested once weekly for at least 8 weeks.
RESULTS
Generation and phenotype of RIP-CD154 transgenic
mice. To test the hypothesis that activation of pancreatic
APCs in situ could lead to insulitis, we cloned the CD154
cDNA from mouse spleen cDNA and obtained ␤-cell
specific expression by placing the CD154 cDNA under the
control of the RIP (Fig. 1A). The specificity of the construct was tested by transient transfections into the insulinoma cell line Min6 and the fibroblast cell line NIH-3T3.
As expected, only Min6 cells expressed detectable levels
of the transgene, and this expression was dependent on
the RIP because expression was lost when this sequence
was omitted from the construct (not shown).
To obtain transgenic mice, the purified pBG-RIP-CD154
construct was injected into (C57BL6/JxCBA)F1 hybrid
oocytes, and founder mice were selected by PCR. Immunohistochemical staining of frozen pancreatic sections for
insulin and CD154 demonstrated that the transgene was
correctly expressed in vivo (Fig. 1C). Of 14 positive trans2590
genic founders, 10 mice expressed the transgene in varying
levels, and all of these mice also exhibited a mononuclear
infiltration of the islets of Langerhans (not shown). For
more detailed analysis, two lines designated L60 and L90
having a medium (L60) or strong (L90) expression of the
transgene in the pancreatic ␤-cells were selected (Fig. 1C).
No staining for CD154 was seen in nontransgenic littermates (not shown). To verify that the transgene was not
expressed in any other tissue, we performed RT-PCR on
RNA extracted from various organs, using primers specific
for the transgenically encoded CD154 (Fig. 1A). As expected, the transgene was only expressed in the pancreas
of transgene-positive RIP-CD154 mice (Fig. 1B). We therefore conclude that RIP-CD154 mice express CD154 selectively in the pancreatic ␤-cells.
Histological analysis of pancreatic infiltrates. Immunohistochemical staining of pancreatic tissue for insulin
revealed that both RIP-CD154 L60 and L90 had a massive
infiltration of the islets of Langerhans. In L60, the infiltration was visible as early as 10 days after birth and was
sustained throughout the life of the mouse (Fig. 2A–D).
Although the islet morphology was severely compromised
early in life, insulin-positive ␤-cells were easily detectable
as late as 18 weeks of age and such remaining insulinpositive cells also expressed CD154 (not shown). In L90,
insulitis also started early after birth (Fig. 2E), although
intact islets could also be found at this age (Fig. 2F). The
insulitis persisted and increased in severity (Fig. 2G–I),
and at 18 weeks the morphology of most islets was completely destroyed (Fig. 2J). However, also in this line,
insulin-positive islets could be found as late as 18 weeks of
age (i.e., after diabetes development). Interestingly, during
DIABETES, VOL. 53, OCTOBER 2004
C. HAASE AND ASSOCIATES
FIG. 3. Pancreatic frozen sections from 5-week-old RIP-CD154 transgene-positive mice (L90) stained for insulin and CD11c (A), CD4 (B), CD19
(C), and CD8 (D) or CD4, CD11c, and insulin (E). CD11cⴙ cells exhibit dendritic morphology (A and E, arrows) and T-cells are in close proximity
of ␤-cells (B, arrow). All images are representative of three to four mice of both L60 and L90. Scale bar: 100 ␮m and 25 ␮m (for inserts).
disease progression, areas with a very high density of
lymphocytes could be detected (Fig. 2H and J), probably
reflecting the generation of lymphoid structures in the
pancreas.
To determine the nature of the infiltrates, we performed
immunohistochemical staining of frozen pancreatic sections. This revealed that the cellular infiltrate of the islets
of transgene-positive RIP-CD154 mice contained large
numbers of CD11c⫹, CD19⫹, CD4⫹, and CD8⫹ cells, demonstrating that both DCs, B-cells, and CD4⫹ and CD8⫹
T-cells were present (Fig. 3A–D). Furthermore, both Tcells and DCs were seen in close proximity with the
insulin-producing ␤-cells (Fig. 3A and B, inserts), and
many DCs were visible in the islets, especially around the
␤-cells (Fig. 3E). It is possible that this accumulation of
DCs is due to CD154-CD40 signaling since this has been
shown (20) to promote DC survival in vitro. We did not
detect any differences with respect to the nature of the
infiltrating cells when comparing L60 and L90 (not shown).
RIP-CD154 mice have impaired glucose regulation
and develop diabetes. Blood glucose regulation was
analyzed in both L60 and L90. We found that mice from
both lines experienced several hyperglycemic incidents
(Table 1 and Fig. 4A and B), and some also developed
diabetes. In L60, the diabetes incidence was relatively low
TABLE 1
Hyperglycemic incidents in RIP-CD154 L60 and L90 mice
Hyperglycemic animals (blood
glucose ⬎11.0 mmol/l)
L60
L90
Diabetic animals (blood glucose
⬎16.6 mmol/l)
L60
L90
Transgene
positive
Transgene
negative
17/30 (57)*
18/18 (100)*
1/22 (5)
0/17 (0)
3/30 (10)
18/18 (100)
0/22 (0)
0/17 (0)
Data are n/total (%). *P ⬍ 0.001 versus transgene-negative littermates
(Fischer’s exact test).
DIABETES, VOL. 53, OCTOBER 2004
(10%, n ⫽ 30) and developed relatively late (earliest at 20
weeks of age), whereas over one-half of the transgenepositive mice (17 of 30) experienced at least one hyperglycemic incident (blood glucose ⬎11 mmol/l). In L90, all
transgene-positive mice (100%, n ⫽ 22) experienced several hyperglycemic incidents, and all mice developed
diabetes with an onset time between 8 and 18 weeks
(mean 11.8 ⫾ 2.7 weeks, n ⫽ 22) (Fig. 4C). Thus, both lines
exhibited a heavy infiltration of the islets, and diabetes
developed in both lines.
Because L90 had the highest incidence of diabetes, we
chose to further characterize this line. When challenged
with an intraperitoneal injection of glucose after fasting,
pre-diabetic transgene-positive mice (age 6 –7 weeks) had
a significantly impaired glucose tolerance when compared
with their transgene-negative littermates (Fig. 4D). Furthermore, the mice quickly became clinically affected, as
seen by an impaired growth rate starting at 10 weeks of
age (Fig. 4E). Finally, residual ␤-cell function, measured as
plasma C-peptide levels 30 min after oral glucose challenge in transgene-positive mice (n ⫽ 13) after diabetes
onset, was significantly reduced (P ⬍ 0.02, Mann-Whitney
test) compared with transgene-negative controls (n ⫽ 10)
(data not shown). Together, these results suggested that
␤-cell function was severely impaired in RIP-CD154 mice
and that the increase in blood glucose content was due to
an inability of the pancreatic ␤-cells to produce sufficient
amounts of insulin.
RIP-CD154 mice have activated DCs in both the
pancreas and in pancreatic lymph nodes. Expression
of CD154 on ␤-cells was expected to lead to activation of
tissue-resident APCs, followed by an increase of activated
DCs in the pancreatic draining lymph nodes.
We therefore analyzed the phenotype of DCs in the pancreas as well as in the pancreatic lymph nodes by immunohistochemistry and by flow cytometry. In the pancreas
of L90 transgene-positive RIP-CD154 mice, we found both
mature (CD11c⫹MHC-II⫹) as well as more immature DCs
(CD11c⫹MHC-II⫺/low) (Fig. 5A, panels a–d). The absolute
number of cells in the pancreatic draining lymph node of
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DIABETES IN RIP-CD154 TRANSGENIC MICE
FIG. 4. A and B: Blood glucose (BG) levels of RIP-CD154 transgene-positive (TGⴙ) mice: L60 (A, n ⴝ 30) and L90 (B, n ⴝ 18). Dotted lines indicate
the blood glucose levels of transgene-negative (TGⴚ) littermates. C: Diabetes development in L90. D: Impaired glucose tolerance (by
intraperitoneal glucose tolerance test [IPGTT]) in pre-diabetic RIP-CD154 mice (L90). *P < 0.05; ***P < 0.005. One representative experiment
of two is shown. E: Body weight development in RIP-CD154 (L90). **P < 0.01; ***P < 0.005.
transgene-positive mice was two- to threefold increased
(not shown), and the fraction of CD40⫹ cells was doubled
(from 9 to 18%) (Fig. 5B, top), possibly due to increased
signaling through the receptor. Among these cells, an increase of activated DCs could be detected (CD11c⫹
CD40⫹) (Fig. 5B, bottom), suggesting that activated DCs
migrate to the pancreatic lymphoid tissue after activation
in the pancreas.
Diabetes development in RIP-CD154 mice is dependent on T- and B-cells and can be rescued by islet
transplantation. To investigate whether T- and B-cells
were necessary for development of diabetes, we crossed
L90 onto a RAG KO background. As seen in Fig. 6A,
transgene-positive RAG⫺/⫺ mice did not develop diabetes, whereas transgene-positive RAG⫹/⫺ littermates developed diabetes with similar kinetics as their wild-type
transgene-positive counterparts (compare Fig. 6A and
4D). Furthermore, histological examination of pancreatic
tissue showed less destructive cellular infiltrates in the
islets of transgene-positive RAG⫺/⫺ mice than in transgene-positive RAG⫹/⫺ littermates (Fig. 6B). Immunohistochemical staining showed that the infiltrates in the
transgene-positive RAG⫺/⫺ mice consisted mainly of
CD11c⫹ cells (data not shown), suggesting that the infiltrating cells were DCs.
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In order to investigate whether activation of the immune
system was confined to the pancreas or if extra-pancreatic
␤-cells would also be destroyed, we engrafted wild-type
C57BL/6 islets under the kidney capsule of established
diabetic RIP-CD154 mice and monitored the development
of their blood glucose. In all RIP-CD154 mice (6 of 6 transgene positive), the transplanted islets could rescue diabetes, and graft function was sustained for up to 100 days
after transplantation (Fig. 6C). When the kidney with the
transplant was removed, the mice quickly became diabetic, showing that the normalization of blood glucose was
due to the transplanted islets (Fig. 6C, arrows). Immunohistochemical analysis confirmed the functionality of the
graft since islet integrity was not compromised (Fig. 6D).
Finally, to test whether diabetes could be readily transferred by spleen cells, we adoptively transferred 107 total
spleen cells from diabetic RIP-CD154 mice to RAG KO
mice (n ⫽ 12). The blood glucose of these mice was
monitored for 8 weeks, and during this period none of the
recipients developed either insulitis or diabetes (not
shown). Together, these data suggest that the autoimmune
process was confined to the organ where the DC activation
originally occurred and did not result in the generation of
␤-cell–specific, autoreactive T-cells in the spleen capable
of transferring disease.
DIABETES, VOL. 53, OCTOBER 2004
C. HAASE AND ASSOCIATES
FIG. 5. A: Pancreatic frozen sections from 5-week-old RIP-CD154
transgene-positive (L90) mice stained for MHC-II (a and b) and CD11c
(c). The depicted areas are from endocrine pancreas, and the overlay
image of panels b and c is shown in panel d, where colocalization
appears yellow. Scale bar: 100 ␮m (a) and 50 ␮m (b–d). B: Flow
cytometric analysis of pancreatic lymph node cells from 6-week-old
RIP-CD154 (L90) transgene-positive (TGⴙ) and transgene-negative
(TGⴚ) mice. Lymph node cells were stained for CD11c and CD40. The
experiment was performed twice, with similar results.
DISCUSSION
In this study we show that transgenic expression of CD154
on pancreatic ␤-cells leads to a rapid development of
mononuclear insulitis, including DCs, T- and B-cells, and a
subsequent loss of ␤-cell function, resulting in the development of diabetes. The data are in agreement with the
hypothesis that CD154 can activate islet-associated APCs
(including DCs) in situ and demonstrate that transgenic
expression of CD154 on pancreatic ␤-cells can lead to
insulitis and diabetes.
The infiltration of the islets in the RIP-CD154 mouse was
massive, and the islet morphology in these mice was
severely disrupted. Both insulin- and glucagon-positive
cells were visible at the time of diabetes onset; however,
␤-cells were fewer, insulin staining was faint, and plasma
C-peptide was reduced in many of the diabetic mice (Fig.
2 and data not shown). Thus, the functional ␤-cell mass
was too small to keep the mice normoglycemic at the time
of diabetes onset.
Although diabetes developed in both transgenic lines
presented here, only a few mice from L60 became diabetic
despite massive insulitis. The reasons for this might be
that the expression of the transgene in L90 was stronger
and more uniform than in L60 (data not shown). Also,
DIABETES, VOL. 53, OCTOBER 2004
although we did not observe any change in phenotype,
both lines were backcrossed to C57Bl/6 for three to five
generations, and we cannot completely exclude the possibility that remaining CBA alleles may contribute to this
discrepancy. Regardless, the data demonstrate that diabetes could develop in two independent lines, although the
incidence and kinetics differed.
DCs were initially thought to be important primarily for
the development of antigen-specific immunity, but lately
an important role for DCs in the maintenance of peripheral
tolerance in the steady state has emerged (21,22). Thus,
many studies, including tolerance to ␤-cell– derived antigens (23), have shown that immature DCs induce tolerance (24 –28) by taking up antigens in situ and traveling to
the draining lymph nodes (29), where they regulate peripheral tolerance, e.g., by deletion of autoreactive T-cells
(30 –32). DCs are therefore important for sustaining tolerance in the steady state and for induction of immunity
during inflammation. We believe that expression of CD154
on the ␤-cells could trigger the conversion of immature
islet-associated DCs into mature DCs, thereby activating
the immune system.
If expression of CD154 on pancreatic ␤-cells leads to
activation of tissue-resident APCs, at least two conditions
must be fulfilled. First, DCs and macrophages must be
present in islets of normal nontransgenic mice. Indeed,
both DCs and macrophages can be detected in pancreatic
islets in wild-type mice (33), which is consistent with the
perception of DCs as sentinel cells present in virtually all
organs (5,21). Second, the receptor of CD40 must be
present on the APC. Surely, the important role of CD40CD154 interactions in the conversion of an immature to
mature DC is supported by many studies (6 – 8), and the
tolerogenic potential of DCs can be ablated by CD40
ligation (9 –11,34). The data presented here are therefore
consistent with the hypothesis that activation of DCs by
CD40 ligation can change the DC from a tolerogenic to an
immunogenic APC.
One other study (35) has investigated the effects of
activating DCs in situ by transgenic expression of CD154.
In this study, expression of CD154 by a keratin-specific
promoter leads to activation of skin-associated Langerhans’ cells and to inflammation of the skin. Our study
supports the finding that local expression of CD154 can
lead to activation of the immune system and inflammation
of the organ expressing the transgene. Furthermore, both
studies support a pivotal role of DCs in the inflammatory
process, where activation of the DCs in the tissue is most
likely the initiation point. It is intriguing, however, that
expression of CD154 in the epidermis results in the
development of autoreactive T-cells capable of transferring the disease to nontransgenic recipients. In contrast,
expression of CD154 in the islet of Langerhans generates
an immune response that is confined to the pancreas,
since transplanted islets are not rejected and adoptive
transfer of spleen cells does not transfer the disease to
immunodeficient RAG KO mice. This discrepancy likely
reflects 1) the need for initial activation of DCs in the
islets, leading to secretion of ␤-cell cytotoxic and/or T-cell
chemotactic factors that are absent in both the transplantation and adoptive transfer model but are required for
development of insulitis and diabetes, and 2) the fact that
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DIABETES IN RIP-CD154 TRANSGENIC MICE
FIG. 6. A: RIP-CD154 (L90) was crossed onto a RAG KO background, and development of diabetes was monitored. B: Formalin-fixed pancreatic
sections from RIP-CD154 (L90) on a RAG KO background were stained for insulin and evaluated by microscopy. Scale bar: 100 ␮m. C: Diabetic
RIP-CD154 transgenic mice were transplanted with 300 –350 C57BL/6J islets under the left kidney capsule, and blood glucose (BG) levels were
measured weekly (n ⴝ 6). The kidney carrying the transplant was removed from three of the transplanted mice at the indicated times (arrows).
Dotted lines indicate the normal blood glucose levels of transplanted transgene-negative littermates. D: Formalin-fixed kidney sections were
stained for insulin and evaluated by microscopy. Scale bar: 200 ␮m (left) and 50 ␮m (right). TGⴙ, transgene positive; TGⴚ, transgene negative,
WT, wild type.
the size of the two target organs are quite different
(epidermis versus ␤-cells). In any case, RIP-CD154⫹ transgenic mice on a RAG⫺/⫺ genetic background do not
develop diabetes (Fig. 6A), which implies that T- and
B-cells are necessary for disease progression from mild
insulitis to insulin-dependent diabetes. Therefore, diabetes
development in this model is clearly dependent on adaptive immunity. An alternative explanation could be that the
transgene per se was disrupting the integrity of ␤-cell
function, thereby leading to diabetes; however, the fact
that RIP-CD154⫹ RAG⫺/⫺ animals fail to develop diabetes
documents that this is not the case. Rather, these animals
exhibited a milder cellular infiltration of the islets that only
consisted of CD11c⫹ DCs and had no apparent loss of
␤-cells. This is consistent with our hypothesis that expression of CD154 on ␤-cells activates local APCs, which in
turn are dependent on adaptive immunity for diabetes to
develop. We expected to be able to adoptively transfer
diabetes by injection of spleen cells from diabetic RIPCD154 mice into RAG KO recipients. However, the fact
that disease could not be transferred does not necessarily
contradict this hypothesis since similar experiments in
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other transgenic models of type 1 diabetes showed that
disease was not readily transferable (36).
Although artificial in nature, the RIP-CD154 transgenic
mouse may well illustrate early events in the development
of type 1 diabetes. It is highly likely that the first event
toward development of disease is activation of isletassociated DCs by some environmental trigger (e.g., a
virus). This trigger is probably transient in nature but
could potentially initiate an autoimmune reaction, provided that a sufficient number of autoreactive T-cells
and/or a deficiency in regulatory T-cells are present.
In conclusion, we have presented a transgenic mouse
model where the consequences of a constitutive activation
of DCs in the pancreas can be studied. The RIP-CD154
develops a form of insulin-dependent diabetes, which in
many ways resembles type 1 diabetes in human patients. It
is our hope that this model can serve to study the vast
effects of inducing DC maturation in vivo. It will be
especially important in protocols of organ transplantation
and tolerance induction to understand the mechanisms in
more detail.
DIABETES, VOL. 53, OCTOBER 2004
C. HAASE AND ASSOCIATES
ACKNOWLEDGMENTS
This study was supported in part by the Danish Ministry of
Science, Technology and Development (to C.H.), by a
Freja research grant from the Danish Research Agency (to
B.K.M, grant no. 5008-01-0003), and institutional funds
from Novo Nordisk. Hagedorn Research Institute is an
independent basic research component of Novo Nordisk.
The authors thank Trine Larsen for excellent technical
assistance; the employees of the Animal Unit, Gentofte, for
taking care of the animals and for assistance in animal
experiments; Drs. Lars Hornum and Dorthe Lundsgaard
for critically reading the manuscript; and Dr. Helle V.
Petersen for help in testing the transgenic construct in
Min6 transfections.
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