Presence of residual beta cells and co-existing islet autoimmunity in the NOD mouse during...

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ORIGINAL PAPER Presence of residual beta cells and co-existing islet autoimmunity in the NOD mouse during longstanding diabetes: a combined histochemical and immunohistochemical study Shiva Reddy Ryan Chau Chia Chai Jessica Astrid Rodrigues Tzu-Hsuan Hsu Elizabeth Robinson Received: 15 May 2007 / Accepted: 6 July 2007 / Published online: 22 September 2007 Ó Springer Science+Business Media B.V. 2007 Abstract During type 1 diabetes, most beta cells die by immune processes. However, the precise fate and charac- teristics of beta cells and islet autoimmunity after onset are unclear. Here, the extent of beta cell survival was deter- mined in the non-obese diabetic (NOD) mouse during increasing duration of disease and correlated with insulitis. Pancreata from female NOD mice at diagnosis and at 1, 2, 3 and 4 weeks thereafter were analysed immunohisto- chemically for insulin, glucagon and somatostatin cells and glucose transporter-2 (glut2) and correlated with the degree of insulitis and islet immune cell phenotypes. Insulitis, although variable, persisted after diabetes and declined with increasing duration of disease. During this period, beta cells also declined sharply whereas glucagon and somatostatin cells increased, with occasional islet cells co- expressing insulin and glucagon. Glut2 was absent in insulin-containing cells from 1 week onwards. CD4 and CD8 T cells and macrophages persisted until 4 weeks, in islets with residual beta cells or extensive insulitis. We conclude that after diabetes onset, some beta cells survive for extended periods, with continuing autoimmunity and expansion of glucagon and somatostatin cells. The absence of glut2 in several insulin-positive cells suggests that some beta cells may be unresponsive to glucose. Keywords Beta cells Á NOD mouse Á Diabetes Á Insulitis Á Glucose transporter-2 Introduction The onset of human type 1 diabetes represents the culmi- nation of a silent and prolonged pre-diabetic phase of immune-mediated beta cell destruction (Eisenbarth et al. 1987). At clinical diagnosis, most beta cells are destroyed and daily and life-long treatment of subjects with paren- teral insulin becomes mandatory. Soon after diagnosis of type 1 diabetes and commencement of insulin therapy, many patients experience a ‘‘honeymoon’’ phase, which can last for several months, when there is a marked reduction in daily insulin dose (Assan et al. 1990; Palmer et al. 2004). Thus, there may be some restoration of beta cell function and/or mass during the early stages of clinical diabetes and upon commencement of insulin therapy. Earlier studies on pancreatic histology and immunohis- tology showed that some beta cells remain in many subjects with recent onset and for variable periods in longstanding type 1 diabetes (Gepts 1965; Gepts and De Mey 1978; Foulis and Stewart 1984; Foulis et al. 1986; Lohr and Kloppel 1987). A subsequent study involving 2,431 patients who were greater than 18 years of age at onset of diabetes, showed that *15 and 33% of patients had stimulated C-peptide levels of >0.5 and 0.2–0.5 nmol/l, respectively, within the first 5 years of diagnosis (Palmer et al. 2004). These data confirm that some residual beta cell mass and function may still exist in longstanding type 1 diabetic subjects. Experimental studies with the ultimate aim of promoting beta cell regeneration in human type 1 diabetes are being pursued with renewed vigour. However, the limited avail- ability of suitable human pancreatic material sampled at defined time-points soon after onset of type 1 diabetes has S. Reddy (&) Á R. C. C. Chai Á J. A. Rodrigues Á T.-H. Hsu School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand e-mail: [email protected] E. Robinson Department of Epidemiology and Biostatistics, School of Population Health, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand 123 J Mol Hist (2008) 39:25–36 DOI 10.1007/s10735-007-9122-5

Transcript of Presence of residual beta cells and co-existing islet autoimmunity in the NOD mouse during...

ORIGINAL PAPER

Presence of residual beta cells and co-existing islet autoimmunityin the NOD mouse during longstanding diabetes: a combinedhistochemical and immunohistochemical study

Shiva Reddy Æ Ryan Chau Chia Chai Æ Jessica Astrid Rodrigues ÆTzu-Hsuan Hsu Æ Elizabeth Robinson

Received: 15 May 2007 / Accepted: 6 July 2007 / Published online: 22 September 2007

� Springer Science+Business Media B.V. 2007

Abstract During type 1 diabetes, most beta cells die by

immune processes. However, the precise fate and charac-

teristics of beta cells and islet autoimmunity after onset are

unclear. Here, the extent of beta cell survival was deter-

mined in the non-obese diabetic (NOD) mouse during

increasing duration of disease and correlated with insulitis.

Pancreata from female NOD mice at diagnosis and at 1, 2,

3 and 4 weeks thereafter were analysed immunohisto-

chemically for insulin, glucagon and somatostatin cells and

glucose transporter-2 (glut2) and correlated with the degree

of insulitis and islet immune cell phenotypes. Insulitis,

although variable, persisted after diabetes and declined

with increasing duration of disease. During this period,

beta cells also declined sharply whereas glucagon and

somatostatin cells increased, with occasional islet cells co-

expressing insulin and glucagon. Glut2 was absent in

insulin-containing cells from 1 week onwards. CD4 and

CD8 T cells and macrophages persisted until 4 weeks, in

islets with residual beta cells or extensive insulitis. We

conclude that after diabetes onset, some beta cells survive

for extended periods, with continuing autoimmunity and

expansion of glucagon and somatostatin cells. The absence

of glut2 in several insulin-positive cells suggests that some

beta cells may be unresponsive to glucose.

Keywords Beta cells � NOD mouse � Diabetes �Insulitis � Glucose transporter-2

Introduction

The onset of human type 1 diabetes represents the culmi-

nation of a silent and prolonged pre-diabetic phase of

immune-mediated beta cell destruction (Eisenbarth et al.

1987). At clinical diagnosis, most beta cells are destroyed

and daily and life-long treatment of subjects with paren-

teral insulin becomes mandatory. Soon after diagnosis of

type 1 diabetes and commencement of insulin therapy,

many patients experience a ‘‘honeymoon’’ phase, which

can last for several months, when there is a marked

reduction in daily insulin dose (Assan et al. 1990; Palmer

et al. 2004). Thus, there may be some restoration of beta

cell function and/or mass during the early stages of clinical

diabetes and upon commencement of insulin therapy.

Earlier studies on pancreatic histology and immunohis-

tology showed that some beta cells remain in many subjects

with recent onset and for variable periods in longstanding

type 1 diabetes (Gepts 1965; Gepts and De Mey 1978; Foulis

and Stewart 1984; Foulis et al. 1986; Lohr and Kloppel

1987). A subsequent study involving 2,431 patients who

were greater than 18 years of age at onset of diabetes, showed

that *15 and 33% of patients had stimulated C-peptide

levels of >0.5 and 0.2–0.5 nmol/l, respectively, within the

first 5 years of diagnosis (Palmer et al. 2004). These data

confirm that some residual beta cell mass and function may

still exist in longstanding type 1 diabetic subjects.

Experimental studies with the ultimate aim of promoting

beta cell regeneration in human type 1 diabetes are being

pursued with renewed vigour. However, the limited avail-

ability of suitable human pancreatic material sampled at

defined time-points soon after onset of type 1 diabetes has

S. Reddy (&) � R. C. C. Chai � J. A. Rodrigues �T.-H. Hsu

School of Biological Sciences, University of Auckland,

Private Bag 92019, Auckland, New Zealand

e-mail: [email protected]

E. Robinson

Department of Epidemiology and Biostatistics, School of

Population Health, Faculty of Medical and Health Sciences,

University of Auckland, Private Bag 92019, Auckland,

New Zealand

123

J Mol Hist (2008) 39:25–36

DOI 10.1007/s10735-007-9122-5

hampered our understanding of the precise fate of residual

beta cells immediately after this phase and during increasing

duration of disease. They have also precluded direct studies

on the molecular, cellular and immune events mediating

further beta cell death and possible beta cell regeneration.

In the non-obese diabetic (NOD) mouse model of human

type 1 diabetes, there is also a paucity of knowledge on the

precise fate of beta cell populations and islet immunopa-

thology at defined time-points after disease onset.

Information gained from such studies in the NOD mouse

would provide experimental platforms to assess the efficacy

of therapies aimed at beta cell regeneration and turnover and

thwarting recurrent autoimmunity. The present study has,

therefore, investigated the islet endocrine-inflammatory cell

pathology in diabetic NOD mice at specific time-points after

onset of disease. Immunohistochemical and histochemical

techniques were employed to examine changes in the num-

ber and distribution of the remaining beta cells and glucagon

and somatostatin cells. These results were correlated with the

degree of insulitis, the phenotypes of islet inflammatory cells

and expression of glucose transporter-2 (glut2).

Materials and methods

Animals

An inbred colony of NOD mice was established at the Ani-

mal Resources Unit of the School of Biological Sciences,

The University of Auckland. This colony originated from a

nucleus of three breeding pairs obtained from Dr. A. Mer-

riman at the University of Otago, Dunedin, New Zealand.

The University of Otago colony was established from

breeding pairs obtained from the Jackson Laboratories, Bar

Harbor, ME, USA. The Auckland colony is maintained on a

standard autoclaved diet (Harlan Teklad Global 18% Protein

Rodent Diet, code 2018S, UK) and sterile water ad libitum.

The current rate of spontaneous diabetes is *80% among

females usually between the ages of 80 and 250 days. Dia-

betes in NOD mice is defined as the presence of a non-fasting

hyperglycaemic value >12 mmol/l on three consecutive

days. In non-diabetic NOD mice, the non-fasting blood

glucose levels ranged from 4 to 6 mmol/l. All animal studies

were conducted in compliance with the guidelines of the

University of Auckland Animal Ethics Committee.

Study groups

Female NOD mice obtained from several breeding pairs

were monitored for the development of diabetes from day

70 onward. Following diagnosis of diabetes, mice were

sacrificed either immediately or following 1, 2, 3 and

4 weeks of diabetes (without insulin treatment; 5 NOD

mice per time-point). In addition, four newly diabetic NOD

mice were maintained on 0.5–1 U of Protophane insulin

(Novo Nordisk, Bagsvaerd, Denmark) administered once

daily by intra-peritoneal injection for 28 days to achieve a

daily blood glucose value <10 mmol/l. The ages of mice

enrolled in the various study groups are shown below:

Mouse

number

Newly

diabetic

(Days)

1 week

of

diabetes

(Days)

2 weeks

of

diabetes

(Days)

3 weeks

of

diabetes

(Days)

4 weeks

of

diabetes

(Days)

Newly

diabetic

treated

with

insulin

(Days)

1 124 127 182 173 190 149

2 117 111 139 183 164 154

3 95 144 148 93 156 154

4 117 117 121 136 211 246

5 95 104 136 150 142 –

Non-diabetic NOD mice between the ages of 100 and

200 days (n = 5) and NOD mice at various stages after

onset of diabetes were killed and the entire pancreas, with a

small piece of the adjoining spleen, was removed (see

below). Each pancreas was then divided immediately into

halves, one of which was snap-frozen (splenic half) in is-

opentane cooled in liquid nitrogen while the other fixed in

Bouin’s solution (duodenal portion). Bouin’s fixed pan-

creata were embedded in paraffin and sectioned (5 lm).

Frozen pancreata were cryosectioned (7 lm), air-dried

briefly, fixed in cold acetone and stored at �20�C until

required for immunohistochemistry.

Primary antibodies

Guinea pig anti-ovine insulin serum was available in this

laboratory and is specific for beta cells (Reddy et al. 1988,

2005). Rabbit anti-glucagon serum was purchased from

Dako, Glostrup, Denmark and sheep anti-somatostatin

serum was from Guildhay Limited, Guidford, Surrey, UK.

Rat monoclonal antibodies to mouse macrophages (clone

CD11b) and to CD8 T cells (clone KT15) were purchased

from Serotec, Oxford, UK. Rat monoclonal antibodies to

mouse CD4 T cells (clone GK1.5) and CD3 T cells (clone

KT3) were gifts from Dr. H Georgiou, Walter and Eliza Hall

Institute of Medical Research, Melbourne, VIC, Australia.

Highly specific rabbit anti-mouse glut2 was kindly provided

by Dr. B Thorens, University of Lausanne, Lausanne,

Switzerland, as affinity purified IgG. This antibody has been

used in immunocytochemical staining for mouse pancreatic

glut2 and specifically stains mouse beta cells (Thorens 1992;

Reddy et al. 1998). The immunohistochemical specificities

26 J Mol Hist (2008) 39:25–36

123

of the antibodies employed for detection of macrophages

and T cells in mouse pancreatic sections have been reported

previously (Reddy et al. 1995, 2003). In the immunohisto-

chemical procedure, all primary antibodies were titrated to

give maximum immunohistochemical reactivity. Normal

sera from a variety of mammalian species were available in

this laboratory for use as controls and blocking reagents for

immunohistochemistry.

Islet histopathology and insulitis

Pancreatic sections from different stages of diabetes and

from adult non-diabetic female NOD mice were stained by

H&E, studied for their histological characteristics and

graded for insulitis on a scale of 0–4 (Charlton et al. 1989;

Reddy et al. 1999). In this method, islets devoid of any

mononuclear cells = 0; minimum focal islet infiltrate = 1+;

peri-islet infiltrate of <25% of islet circumference = 2+;

peri-islet infiltration and <50% intra-islet area = 3+; intra-

islet infiltration >50% of islet area = 4+. All slides were

coded and at least ten separate islets from different levels

of each pancreas were scored. The insulitis score (%) for

each study group was calculated as follows:

Sum of (1 · number of islets with 1+, 2 · number of

islets with 2+, 3 · number of islets with 3+, 4 · number of

islets with 4+) divided by 4 · total number of islets scored.

The ratio obtained was expressed as a percentage. The

insulitis score (%) for each study group was expressed as

the mean ± SEM.

Immunolabelling of islet endocrine cells

Each of the three serial paraffin sections per slide was

immunolabelled for either insulin, glucagon or somato-

statin, with minor modifications (Reddy et al. 1988, 2005).

Briefly, sections were de-waxed with xylene, rehydrated in

increasing concentrations of ethanol, washed in water and

equilibrated in phosphate-buffered saline (PBS), pH 7.4. In

the immunohistochemical protocol, PBS acted as the wash

buffer and as a diluent, unless stated otherwise.

Sections were blocked with 5% normal donkey serum and

incubated with either guinea pig anti-ovine insulin (1:1,000

in 5% normal donkey serum), rabbit anti-glucagon (1:100) or

sheep anti-somatostatin (1:800 in 5% normal donkey serum)

for 1 h at 37�C. Following washing, sections were incubated

with either donkey anti-guinea pig IgG-FITC (1:50, Jackson

ImmunoResearch Laboratories, West Grove, PA, USA,

insulin section), donkey anti-rabbit IgG-Texas Red (1:100,

Jackson ImmunoResearch Laboratories, glucagon section)

or donkey anti-sheep IgG-FITC (1:100, Jackson Immuno-

Research Laboratories, somatostatin section) for 1 h at 37�C.

Sections previously immunolabelled for insulin and

somatostatin were dual-labelled for glucagon (Texas Red

fluorochrome). Selected sections previously immunola-

belled for glucagon (Texas Red fluorochrome) were dual-

labelled for somatostatin (FITC fluorochrome). All sections

were mounted and prepared for microscopy.

For triple-immunolabelling, sections were immuno-

stained for somatostatin as above (by sequential incubation

with sheep anti-somatostatin serum and donkey anti-sheep

IgG-FITC as secondary antibodies). Sections were then

blocked with 10% normal sheep serum to saturate any

remaining sites on donkey anti-sheep IgG conjugated to

FITC. Sections were washed and incubated with guinea pig

anti-insulin (1:1,000) followed by addition of goat anti-

guinea pig IgG-Alexa 568 (1:500, Invitrogen, Eugene, OR,

USA). After washing, they were incubated with rabbit anti-

glucagon (1:100) followed by addition of goat anti-rabbit

IgG-Alexa 350 (1:100, Invitrogen). Sections were washed

and prepared for microscopy.

Enumeration of insulin, glucagon and somatostatin

cells

Insulin, glucagon and somatostatin cells were enumerated

in each islet section either directly with the microscope

using the 20· objective or after photomicrography of im-

munolabelled islets. The corresponding cross-sectional area

of the islet in each pancreatic section was determined with

the Axiocam software provided with the Zeiss fluorescence

microscope. The mean number of immunoreactive insulin,

glucagon or somatostatin cells per millimeter square islet

area was calculated.

Immunolabelling of mouse glut2

Selected pancreatic sections were immunostained for

mouse glut2 and insulin as reported previously, with minor

modifications (Reddy et al. 1998). Briefly, paraffin sections

were blocked with 5% normal donkey serum and incubated

with a mixture of anti-glut2 (1:1,000 in 0.1% bovine serum

albumin) and guinea pig anti-insulin (1:1,000) at 4�C for

16 h, followed by reaction with donkey anti-rabbit IgG-

biotin and then with streptavidin Alexa 568 (1:500, Invi-

trogen). Following washing, sections were reacted with

donkey anti-guinea pig IgG-FITC (Jackson ImmunoRe-

search) and prepared for microscopy.

Immunolabelling of CD4 and CD8 T cells and

macrophages

Immunolabelling of CD4 and CD8 T cells and macrophages

was carried out as reported previously (Reddy et al. 1999,

J Mol Hist (2008) 39:25–36 27

123

2003). In this procedure, serial frozen sections of pancreas

were re-fixed in cold acetone for 10 min, equilibrated with

PBS and incubated with 5% normal donkey serum for 1 h at

37�C. Sections were washed and incubated with rat

monoclonal antibodies to CD4 T cells (undiluted), CD8 T

cells (1:50) and macrophages (1:10) and incubated for 16 h

at 4�C followed by washing. They were then incubated with

donkey anti-rat IgG-Alexa 488 (1:250, Invitrogen) for 1 h at

37�C, washed and prepared for microscopy.

Dual-immunolabelling of insulin and immune cells

Serial frozen pancreatic sections were immunolabelled for

CD3 T cells and macrophages as described above. Sections

Fig. 1 Photomicrographs of

pancreatic sections from non-

diabetic NOD mice and NOD

mice at various stages after

diabetes onset, stained by H&E,

showing the varying extent of

insulitis. Islets from 2 to 3 mice

are shown for each group. The

ages of mice and the

corresponding blood glucose

values prior to removal of

pancreata are indicated in each

photomicrograph. Scale bar in

(r) is 50 lm and applies to all

remaining photomicrographs

0

10

20

30

40

50

60

70

80

90

100

Non-diabetic Newly-diabetic 1 week 2 weeks 3 weeks 4 weeks

Stages of diabetes

Mea

n in

sulit

is s

core

(%

)

Fig. 2 The mean percent insulitis scores (%) ± SEM at various

stages of diabetes in NOD mice (five animals per time-point)

28 J Mol Hist (2008) 39:25–36

123

were then reacted with guinea pig anti-insulin (1:1,000) for

16 h at 4�C followed by incubation with goat anti-guinea

pig IgG-Alexa 568 (1:500, Invitrogen) for 1 h at 37�C,

washed and prepared for microscopy.

Statistical analyses

Linear regression was used to investigate changes in in-

sulitis scores at various time-points after diabetes onset.

Poisson regression was used to investigate changes in the

number of beta, glucagon and somatostatin cells per unit

cross-sectional islet area.

Results

Insulitis and islet morphology

The degree of insulitis in NOD mice at various stages of

diabetes is shown in Fig. 1. Islets displayed considerable

variation in the extent of insulitis within each pancreas,

both before the onset of diabetes and subsequently.

However, at 4 weeks of diabetes, despite the persistence

of insulitis in some islets, a number were atrophied and

devoid of insulitis (Fig. 1p–r). The mean insulitis

scores ± SEM (%) at various stages of diabetes are

shown in Fig. 2. The insulitis scores showed a decrease

Fig. 3 Dual-label

immunohistochemistry of

pancreatic islets from non-

diabetic NOD mice, newly

diabetic and 1 week after

diabetes. Left panel: insulin

(green) + glucagon (red).

Middle panel: glucagon (red)

+ somatostatin (green). Right

panel: corresponding islets from

the left panel counterstained by

H&E. The stages of diabetes,

age of mice and blood glucose

levels immediately before

removal of pancreas are also

shown on the right. Note that in

(n) and (q) glucagon cells often

surround somatostatin cells.

Scale bar in (r) is 50 lm and

also refers to a–q

J Mol Hist (2008) 39:25–36 29

123

from the onset of diabetes until 4 weeks after onset

(p = 0.0031).

Islet endocrine cell populations

Representative photomicrographs of islets dual-labelled for

insulin and glucagon or glucagon and somatostatin are

shown in Figs. 3–5. The corresponding islets from adjacent

sections stained by H&E are also included. In non-diabetic

NOD mice, a central islet core of numerous beta cells

surrounded by glucagon and somatostatin cells with usually

peri-islet insulitis was observed, although islets with

advanced insulitis showed more extensive beta cell loss

(Fig. 3a–f). A similar pattern of immunostaining for the

three endocrine cell types was present in newly diabetic

NOD mice. However, several islets showed intra-islet in-

sulitis, a marked loss of beta cells and more centrally

located glucagon and somatostatin cells (Fig. 3g–l). After

1 week of diabetes, the pattern of immunostaining for the

three endocrine cell-types with concurrent insulitis was

similar to newly diabetic mice, although some islets

showed varying degrees of beta cell loss (Fig. 3m–r). Some

islets were beta cell negative with glucagon cells located in

a ring-like pattern often surrounding a cluster of somato-

statin cells (Fig. 3q). A similar immunohistochemical and

histochemical pattern was observed at 2, 3 and 4 weeks of

diabetes (Fig. 4).

Fig. 4 Dual-label

immunohistochemistry of

pancreatic islets at 2, 3 and

4 weeks of diabetes. Left panel:

insulin (green) + glucagon

(red). Middle panel: glucagon

(red) + somatostatin (green).

Right panel: corresponding

islets from the left panel

counterstained by H&E. The

stages of diabetes, ages of mice

and blood glucose levels

immediately before removal of

pancreas are also shown on the

right. Scale bar in (r) is 50 lm

and also applies to a–q

30 J Mol Hist (2008) 39:25–36

123

The relative distribution of insulin, glucagon and

somatostatin cells, determined by triple-immunolabelling, is

shown in Fig. 5a–f. Dual-labelling for insulin and glucagon

showed that occasional endocrine cells within some diabetic

islets were positive for both insulin and glucagon (Fig. 5g, h).

Islets from insulin-treated NOD mice showed consid-

erable variation in the number and distribution of glucagon

cells and residual beta cells at the end of 28 days of

treatment, with several islets showing advanced insulitis

(results not shown).

The mean number of beta, glucagon and somatostatin

cells ± SEM per mm2 of cross-sectional islet area is shown

in Fig. 6. There was a decline in the mean number of beta

cells in diabetic NOD mice compared with non-diabetic

mice. There was no change in the number of beta cells from

onset until 4 weeks of diabetes (p = 0.21). However, there

was an increase in the number of glucagon cells during the

duration of the disease (p = 0.019). A similar increase was

also evident for somatostatin cells (p = 0.0009).

Glucose transporter-2 expression

Representative islets dual-immunolabelled for mouse glut2

and insulin from selected stages of diabetes are shown in

Fig. 7. Strong immunolabelling for glut2 was present in a

large proportion of beta cells in non-diabetic NOD mice

and to a lesser extent at onset of diabetes. Immunolabelling

for the transporter was absent in some insulin-positive cells

that were usually located adjacent to the region of insulitis

(Fig. 7d–f). From 1 week of diabetes and onward,

Fig. 5 Dual- and triple-label

immunohistochemistry of islets

after varying duration of

diabetes. a–c Insulin (red),

glucagon (blue) and

somatostatin (green); duration

of diabetes is indicated in each

micrograph. Islets

counterstained by H&E in d, e, fare the same as in a, b, c,

respectively. g Shows an islet

dual-labelled for insulin (green)

and glucagon (red). The arrowin g points to an islet cell co-

expressing insulin and

glucagon; also refer to inset in

g. h Shows an islet from an

NOD mouse with 3 weeks of

diabetes immunolabelled for

glucagon (red) and insulin

(green); arrows in h point to 2

cells which co-express glucagon

and insulin. Scale bar in f is

50 lm and also applies to a–e;

scale bar in g is 20 lm and in

inset = 10 lm; scale bar in his10 lm

0

1

2

3

4

5

Non-diabetic Newly-diabetic 1 week 2 weeks 3 weeks 4 weeksStages of diabetes

Mea

n n

um

ber

of

end

ocr

ine

cells

per

mm

2 isle

t ar

ea

Fig. 6 The mean number ± SEM of insulin cells (open bars),

glucagon cells (hatched bars) and somatostatin cells (solid bars)

per mm2 islet area at various stages of diabetes

J Mol Hist (2008) 39:25–36 31

123

immunolabelling for glut2 was largely absent, despite the

presence of cells which expressed insulin (Fig. 7g–o).

Intra-islet CD4 and CD8 T cells and macrophages

By immunohistochemistry, qualitative differences in the

number of CD4 and CD8 T cells and macrophages within

the islets of NOD mice were observed over the duration of

diabetes. In most islets with insulitis, CD4 and CD8 T

cells were more numerous than macrophages and were

distributed in the peri-islet and intra-islet areas (Fig. 8). At

4 weeks of diabetes, extensive insulitis consisting of

mostly CD4 and CD8 T cells and lesser numbers of

macrophages persisted in some islets (Fig. 8m–p). In non-

diabetic NOD mice and in mice with increasing duration

of diabetes, dual-labelling showed that some islets which

were positive for insulin had a mantle of extensive insu-

litis consisting of T lymphocytes and macrophages

(Fig. 9a–f, i, j). However, islets which were negative for

insulin showed minimal peri-islet T cells and macro-

phages (Fig. 9g, h).

Fig. 7 Dual-immunolabelling

of insulin (green; left panel) and

glut2 (red; middle panel at

various stages of diabetes. The

right panel shows the

corresponding islets

counterstained by H&E. In a–karrows point to cells which co-

express insulin and glut2, but

extremely weakly in k, while

arrowheads point to insulin

cells which do not express glut2.

Note that at 3 weeks after

diabetes onset (m–o), the

remaining insulin cells are

negative for glut2. Scale bar in

q is 50 lm and also applies to

a–n

32 J Mol Hist (2008) 39:25–36

123

Discussion

The sequential changes in the cellular and molecular

pathology of the islet endocrine-immune cell axis, includ-

ing the expression of locally produced proinflammatory

molecules, during human type 1 diabetes remain incom-

plete. The relative unavailability and practical and ethical

difficulties in obtaining rapidly cryopreserved pancreatic

samples from diabetic subjects have limited such studies.

To address some of these issues, the present studies were

undertaken in the NOD mouse and pancreata analysed at

defined time-points at and after diabetes onset. Surpris-

ingly, our studies showed that insulitis persisted in most

islets of NOD mice even after onset of disease and until

4 weeks. Daily treatment of diabetic NOD mice with

insulin did not result in the retraction of insulitis or influ-

ence beta cell number during protracted disease. However,

therapeutically administered insulin may not only control

hyperglycaemia but also act as an autoantigen and invoke

an autoimmune response. Pancreata from diabetic NOD

mice without insulin treatment demonstrated islet atrophy

accompanied by minimum peri-insulitis, particularly with

increasing disease duration. The islet infiltrate consisted of

mostly CD4 and CD8 T cells with lower numbers of

macrophages, a pattern also seen in adult prediabetic NOD

mice (Reddy et al. 1995). Retention of this characteristic

immunophenotype during an increasing duration after

diabetes onset suggests that the remaining beta cells may

continue to act as immune targets after diabetes onset,

although it is possible that some of the surviving beta cells

may be refractory to immune recognition and injury. In this

study, although detailed enumeration of immune cells and

the possible presence of islet-located dendritic cells were

not conducted, there were qualitative variations in the

number of intra-islet CD4 and CD8 T cells and macro-

phages. In earlier studies by others, pancreatic samples

from children with new onset type 1 diabetes also showed

insulitis that was often patchy, in a majority of cases and in

a majority of islets (Foulis and Stewart 1984; Foulis et al.

1986, 1991). In children with recent-onset disease, the islet

inflammatory cells consisted of mainly lymphocytes and a

smaller number of macrophages (Foulis et al. 1991). By

immunohistochemistry, pancreatic sections from a recent-

onset diabetic child showed predominance of CD8 T cells

and a lesser number of B cells, macrophages and IgG

deposits, a pathology that is different from islets of newly

Fig. 8 Immunohistochemical

staining of CD4 (first row) and

CD8 T cells (second row) and

macrophages (third row) at

various stages of diabetes in

three serial sections. Islets in d,h, l, p are adjacent sections

stained by H&E and show the

same islet as in a–c, e–g, i–kand m–o, respectively. Scalebar in p is 50 lm and applies to

all photomicrographs. The

stages of diabetes, ages of mice

and blood glucose levels before

removal of pancreas are

indicated above the

photomicrographs

J Mol Hist (2008) 39:25–36 33

123

diabetic NOD mice where CD4 T cells predominate

(Bottazzo et al. 1985; Reddy et al. 1995). More recently,

pancreatic biopsies obtained from recent-onset diabetic

subjects showed that the remaining beta cells were usually

associated with the presence of T cells within the islets and

that macrophages and dendritic cells sometimes persisted

even in islets that were devoid of beta cells (Uno et al.

2007). However, studies carried out in biopsies offer his-

tolopathological patterns seen in only a restricted number

of islets.

At diabetes onset in the NOD mouse, there was a

marked decline in the number of beta cells which persisted

until 4 weeks after diabetes but this decline did not reach

statistical significance. It is unclear if beta cells from NOD

mice have the capacity to regenerate spontaneously after

diabetes onset or even in the prediabetic period. Any net

increase in beta cell turnover may be thwarted by ongoing

autoimmune destruction and glucose toxicity during clini-

cal disease and thus, could escape detection. In humans

with long-standing type 1 diabetes, recent evidence sug-

gests that there may be limited beta cell regeneration and it

has been proposed that concurrent activation of the apop-

totic pathway may result in net beta cell loss (Meier et al.

2005).

Our demonstration of an increase in the number of

glucagon and somatostatin cells after diabetes onset is

noteworthy. In a previous study, an increase in glucagon

cells was shown in the NOD mouse at onset of diabetes

Fig. 9 Dual-immunolabelling

of insulin (red) + CD3 T cells

(green), left panel and insulin

(red) + macrophages (green),

right panel, at various stages of

diabetes. The ages of mice and

the blood glucose levels

immediately before removal of

pancreas are also shown. Note

that most islets show insulin

staining surrounded by T cells

and macrophages. In g and h a

few peri-islet immune cells

surround an islet that is negative

for insulin. Scale bar in j is

50 lm and applies to c–i; scale

bar in b is 100 lm and applies

to a

34 J Mol Hist (2008) 39:25–36

123

(O’Reilley et al. 1997). However, in our study, this

increase as well as an increase in somatostatin cells were

more pronounced at 3 and 4 weeks of diabetes and

restricted to diabetic islets but not to pancreatic ductal cells

as reported previously (O’Reilley et al. 1997). The mech-

anisms which underlie the increase in glucagon and

somatostatin cell number are unclear. In the low dose

streptozotocin mouse model, there is an expansion of

glucagon cells at onset of diabetes also (Li et al. 2000). In

humans, there are conflicting reports regarding changes in

glucagon cell number after onset of type 1 diabetes (Gepts

and De Mey 1978; Stefan et al. 1982; Rahier et al. 1983;

Somoza et al. 1994).

The significance of some islet cells double-positive for

insulin and glucagon after onset of diabetes is unclear. We

have previously shown a similar co-expression in islets

from NOD mice rendered diabetic with cyclophosphamide

(Reddy et al. 2005). It appears that an extreme diabetic

environment may recapitulate some of the embryonic

developmental processes governing islet hormones (Bon-

ner-Weir 2000). During pancreatic development in the fetal

rat, transient co-expression of insulin and glucagon has

been observed in a proportion of pancreatic cells from day

12.5 of gestation (Hashimoto et al. 1988).

The absence of glut2 expression in insulin-positive cells

in the later stages of clinical diabetes implies that most

surviving beta cells may fail to release insulin in response

to glucose and thus, may not be fully functional. Several

studies have suggested that hyperglycaemia can impair

beta cell function due to glucotoxicity and it is likely that

this dysfunction may be present in NOD mice with long-

standing hyperglycaemia. Indeed, adult NOD mice exhibit

defects in insulin release in response to glucose but not to

arginine even before diabetes onset (Kano et al. 1986).

In conclusion, the present studies have clearly estab-

lished that some beta cells are present in the NOD mouse

for increasing periods following onset of the disease.

Whether they represent immune-resistant or newly formed

cells is not clear. A net increase in beta cell mass may be

limited due to ongoing autoimmunity which can invoke

beta cell death by apoptosis. Co-administration of specific

peptides to diabetic NOD mice has been shown to reverse

diabetes (Suarez-Pinzon et al. 2005). Short-term experi-

mental therapies aimed at protecting the limited number of

beta cells present immediately after diagnosis, promoting

their regeneration and blocking beta cell autoimmunity

may lead to the development of promising interventions

aimed at reversing established disease.

Acknowledgements We thank Beryl Davy and Lorraine Rolston for

expert histological support and Jacqueline Ross for valuable advice

on image preparation and Vernon Tintinger for assistance with the

careful maintenance of the NOD mouse colony. Financial assistance

from the Child Health Research Foundation, the Maurice & Phyllis

Paykel Trust, University of Auckland School of Biological Sciences

Summer Studentship Programme and the Auckland Medical Research

Foundation is gratefully acknowledged.

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