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Modulation of an early and lethal autoimmune diabetes in transgenic mice by IL-4 overexpression and treatment with anti-IFN-γ antibody

HERRERA, Pedro Luis, et al.

HERRERA, Pedro Luis, et al . Modulation of an early and lethal autoimmune diabetes in transgenic mice by IL-4 overexpression and treatment with anti-IFN-γ antibody. Transgenics , 1999, vol. 3, p. 97-106

Available at:

http://archive-ouverte.unige.ch/unige:126448

Disclaimer: layout of this document may differ from the published version.

1 / 1

(2)

nusgenics,l999, Vol. 3, pp. 97-106 Rcprints available dircctly frcm the publisher PhotocopyiDg permitted by licensc only

O 1999 OPA (Overseas Publishers Association) N.V Published by liceDsc under lhe Harwood Acadcnric Publishcrs imprinl, part ofthe Gordon aùd Brcach Publishing Group PriDied in Malaysia

the

B-cells bear the

B7-1

(CD80) co-stimulator on their surface

[3].

Transgenic mice bearing one or the

other

transgene

do not

develop diabetes;

the

TNF transgenic mice do, however, display a severe insuli- tis affecting

all

their islets, but they conserve normal B-cell numbers [2]. In contrast, all double transgenic mice develop diabetes by 5 to 6 weeks

of

age (lo07o

of

more than 100 such mice obtained

in

successive breeding experiments were diabetic

by

the 6th week

Modulation of an Early and Lethal Autoimmune Diabetes

in Transgenic Mice by IL-4 Overexpression and Tfeatment with Anti-IFN-y Antibody

pEDRO LUIS HERRERAU*, LILIANE FOSSATIb, SHOZO ZUIb, DAVID M. HARLANc, JEAN-DOMINIQUE VASSALLIA, LELIO ORCIA ANd PIERRE VASSALLID

àDepartmefis of Morphology andb Pathologl,, University of Genevtt Metlical Sclrcol,

I

rue Michel-Servet, l2I

I

Genève 4, Stvitzerlantl and

-

c

lnnwtobiology Departnrcn; Naval Medical Research Institute, Betltesda, Marylcutd, USA (Receivetl Decetnber 23, 1998)

Transgenic mice constitutively expressing TNF and

87-l

tt'ansgenes in their islet B-cells, both under the control of insulin gene promoter, develop insulin-dependent diabetes mellitus (IDDM) at 5 or 6 weeks of age. We have previously shown that the diabetes can be ptevented by treatment of these mice with anti-CD8 rnonoclonal antibody [1], indicating the Th1-type nature of this disease. We now report thât increased expression of cilculating IL-4 in these mice (through a third transgene expressed in B lymphocytes [17]) or decreased use

ofIFNy

(through injection of anti-IFNy antibody during four weeks) retarded by several weeks or months the occurrence of this diabetes. The effect of these two procedures appeared to be cumulative, leading occasionally to complete protection from diabetes, but always with per- sistence of the insulitis. Diabetes in TNF /

87-l

transgenic mice is accompanied by destruc- tion of both B-cells and non B-cells. In contrast, histologic analysis of pancreata from IL-4 overexpressing mice revealed intact islet non B-cells. These data suggest that partial deviation fi-om a Th I to a Th2 l'esponse can modify both diabetes occurrence and islet lesions observed in this form of early severe autoimmune IDDM.

Ke1tw6fi"' mouse, transgenic, islet, IDDM, autoimmunity, immune deviation, IL-4, IFN-1

INTRODUCTION

We

have developed

a

transgenic mouse model

of

autoimmune diabetes that is characterized by an early age

of

onset, rapid progression, and complete pene- trance by crossing two different strains

of

transgenic mice. TNF

I B1-l

double transgenic mice

[l]

were

generated

by

crossing

mice

overexpressing

TNF

in their insulin-producing B-cells

[2] to

mice

in

which

* Mailing address: Dr. Pedro Herrera, Département de Morphologie, Centre Medical Universitaire, 1, rue Michel-Servet, CH-121 i

Genève 4, Switzerland, tel.: +41221O2 5225,fax +4122702 5260, e-mail: Pedro.Herrera@medecine.unige.ch 9'7

(3)

98 PEDRO LUIS HERRERA er a/.

of life), with the clinical signs of type I,

rnsu-

lin-dependent, diabetes

mellitus (IDDM)

including:

hyperglycemia (>> 300 mg/dl), polyuria, polydipsia, polyphagia, and weight loss.

In

these mice, disease progresses rapidly, such that the mice are infertile and

their longevity is

considerably reduced

(10 to

20 weeks). Histologically, the severe insulitis is accom- panied

by

rnassive destruction

of

insulin-producing cells.

An

identical model

of

diabetes has also been developed

by

others,

with similar

observations [4].

This

acute diabetes has been shown

to

be CD8+ T lymphocyte-dependent,

since anti-CD8 (but

not anti-CD4) antibody prevented its appearance [1].

During a normal immune response, CD4+

T

lym- phocytes differentiate

into two different

groups

of

effector cells, the so-called

Thl

and Th2 subpopula- tions,

which

are characterized

by

the production

of distinct

sets

of

cytokines and

by

the

fulfillment of

specific functions [5].

Thl

lymphocytes play a major

role in

delayed-type hypersensitivity responses by secreting

IL-2

and

IFN-y,

among

other

cytokines;

antibody-mediated responses are modulated

by

Th2 cells through the production

of IL-4, IL-5, IL-6

and

IL-10, in

particular.

lt is

considered, although less

well

established, that CD8+

T

lymphocytes can also differentiate into comparable categories

of

cytotoxic Tc1 and Tc2 cells

[6-8].

Different studies have sug- gested

that one

possible therapeutic approach for

autoimmune inflammatory

diseases,

like

IDDM,

could be the

induction

of

antigen-specific immune deviation by means of cytokine treatment [9]. As Thl and Th2 cells regulate each other

[5], Thl

cytokines down-regulating

the

emergence

of Th2 cells

and

vice-versa, it was

suggested

that

inflammatory autoimmune diseases,

in which Thl cells

appear to play a dominant role, could be treated

with

Th2{ype cytokines, mainly

IL-4,

and some successful results have been obtained

in

experimental animal models

[0].

There

is

indeed strong experimental evidence supporting the idea that the ratio between the release

of

IFN-y and of

IL-4

determines the degree

of

tissue damage mediated

by

auto-reactive

T

lymphocytes (reviewed

in [0]).

Lymphocytic co-stimulatory signals, such as those conveyed by the T lymphocyte receptor CD28 and its

B7-1 (CD80)

and

B7-2

(CD86) ligands, are neces-

sary

for

optimal activation

of

naive

T

cells: they also appear to regulate differentially Th cell commitment, so that selective involvement

of B7-l or B7*2

may be

critical for

the development

of

autoimmune dis- ease

[11].

Some experiments indicate that

the 87- I|CD29

pathway favors

Thl-type

responses, which are also favored

by low

antigen doses

[2]. In

this

context, transgenic expression

of B7-l on

p-cells

leads to autoimmune diabetes, considered to be a Thl

or Tcl type of

disease,

in

various types

of

double transgenic mice [1, 3,

4, l3l,

and accelerates diabetes onset

in

nonobese diabetic (NOD) mice

[4].

Never-

theless, there is no a simple view of "Th2 equals pro- tection"; for instance, NOD mice deficient in CD28 or transgenic

for IL-10

have a more rapid onset

of

the disease, and transfer

of

Th2-like cells rnay induce a more severe damage

in

the islets than

Thl-like

cells

1il, ls,

161.

In

the present

work

we have investigated whether the early diabetes onset and its rapid progression can be modulated

in

doubly transgenic TNF

/ 87-1

mice by altering the circulating cytokine balance. We show that constitutive

IL-4

over-expression

(in

mice bear- ing a third transgene), or that treatment with a neutral- izing anti-IFN-y antibody, can delay or even prevent the

IDDM

development.

MATERIALS AND METHODS

Mice

The production

of TNF I B7-l

transgenic mice has been described previously [1]. Transgenic mice con-

stitutively

expressing

IL-4 in

B-lymphocytes were obtained from Dr.

\ff.

Mueller (Kôln, Germany)

[7];

transgene presence and expression was explored by FACS analysis

on

peripheral blood lymphocytes to detect

MHC

class

II

expression as described [17].

Blood samples were collected by orbital sinus bleed- ing after a 4-hour fasting period. Glycemia was mea-

sured using the haemoglukotest strips

from

Boehringer-Manheim (catalog

#

1119420).

For

neu- tralization of IFN-y, new-born mice were tolerized at birth by injecting

i.

p. 20

pl

of normal rat serum. Rat anti-mouse

IFN-y

neutralizing monoclonal antibody

R4642 [18]

was then administered intraperitoneally

(4)

CYTOKINE MODULATION OF DIABETES PROGRESSION 99

(i.p.), twice weekly at a dose of 0.25 mg, to mice from

2

weeks

to 4

weeks

of

age,

when the

dose was increased to 0.5 mg until the 6th week, at which point antibody treatment was discontinued. Murine recom- binant

IL-4

was obtained

from

Ono Pharmaceutical Co. Ltd., Osaka (lot #881213). 100 ng of

rIL-4

(1.65

x

106 U/mg) was given i.p. twice weekly, from birth until the 6th week of life.

Immunostaining

Pancreata

were fixed either in Bouin's or in

47o

paraformaldehyde, dehydrated and embedded

in

par- affin. Alternatively, the pancreata were frozen in cold methylbutane and kept subsequently

in liquid

nitro- gen. The four different anti-islet hormone antibodies used

for

immunofluorescence (anti-insulin, anti-glu-

cagon, anti-somatostatin, and

anti-pancreatic polypeptide), as well as those used to stain T (GK1.5;

H35-11.2) and B cells (p Ig

chains),

have

been described elsewhere

[2]. When

indicated, paraffin sections

were

stained

using the

aldehyde fuchsin merhod f

l9l.

Morphometry

and statistical analysis

Quantification of pancreatic islet endocrine cells was performed using three

3 to 8

week-old animals in each group, computing a minimum

of 3l

islets per hormone and experimental group. The analysis was

carried out using

sections

of splenic

pancreata, derived

from the

dorsal pancreatic

bud,

where the islets are

rich in

glucagon cells but poor

in

PP cells.

Data

from TNF / B7-l

double transgenics and non transgenic control mice were compared

by

ANOVA (data were transformed into their square roots in order to

fit

a normal distribution).

100

90

80

70

E .g

60

Eo,

950

Ët

o

Ê40

Lo

t30

lL4-TNF-87 mice, anti-lFN1-treated

anti-lFN1, either alone or combined

with lL-4

L

lL4-TNF-87 mice

I I

I

\

lo Ëo

:

Ê 20

10

0\t (, (t i- @ oNltTrô(o

FFfFFf

age (weeks)

FIGURE I Incidence of diabetes in TNF / 87-l doubly transgenic mice, and in IL-4 / TNF / B7-l tfiply transgenic mice. Doubly transgenic mice wele either untreated or injected with IL-4, with anti-IFN-y antibodies, or both. Triple transgenics were either untreated or treated with ant!IFN-1 antibodies

untreated or lL4{reated

(5)

100 PEDRO LUIS HERRERA er a/.

RESULTS

Generation of

triple

transgenic mice: TNF

/r_7-l

mice

secretinglL-4 from their

B lymphocytes

In pilot

studies,

two TNF / B1*l

doubly transgenic mice received

two

weekly

i. p.

injections

of

100 ng murine

rIL-4,

starting from birth. This dose has been shown to be protective

in NOD

mice [20]; however, these mice became diabetic as their control untreated transgenic littel'-mates, i.e., at

5-6

weeks of age. Con- sequently, transgenic rnice constitutively expressing

IL-4

under the control of the

IgH

enhancer-promoter

[l7]

were crossed

to

the

TNF

and

B7-l

transgenic mice. Double transgenic

IL-4 / TNF andlL-4 lBl-l

mice, which never develop diabetes, were then inter-

bred, and twenty-two triple

transgenic

mice

were obtained

for

exploration

of

diabetes occurrence.

All TNF i B7-l

double transgenic rnice obtained as a result of such cl'ossings, either females or males, were diabetic

at 5 or 6

weeks.

Strikingly

however,

at

9 weeks, 18/22

lL-4 / TNF / B7-l triple

transgenic

mice

(82o/o) remained normoglycemic, ancl 12122

(54%) remained normoglycemic at 12

weeks (Figure

l). At

15 weeks,

only

three mice

(of

which two were fernales) were

still

normoglycemic, and two

of

them became diabetic at

6

rnonths. The last triple transgenic mouse (a fernale) remained normoglyce-

mic for the entire period of study,

I

I

months (Figure 1), when the animal was sacrificed

for

histo- logical analysis.

The insulitis

in

triple transgenic mice, even

in

the absence of diabetes, was

of

similar cellular composi- tion and severity as compared to that

of

TNF

I B1-I

double transgenic mice (Figure 2).

In

particular, the

CD4/CD8 ratio among islet-infiltrating cells

was comparable both in double and triple transgenics. His- tologic and immunofluorescent studies performed on TNF

/ B7-1

double transgenic animals

in

the weeks

following the

occunence

of

diabetes

showed

a decrease

of

the islet lymphocytic

infiltrate,

as com- pared

to

what

is

observed

at the time of

diabetes occulTence, accompanied by a massive reduction not

only of the

insulin-producing B-cells, as described previously,

but

also remarkably

of the

three other

endocrine cell types, the so-called non

B-cells

(Figure 3, panels

E to H;

Table

I).

Interestingly, the frequency of endocrine cells (of the four types) found outside the islets, i.e. present within the epithelium

of

the pancreatic ducts or isolated

in

the interstitial tis- sue, was increased (128 cells out of904 cells counted,

i.e.

147o) compared

to

what we observed

in

normal mice (0.6Vo; 29/5101).

In

contrast

to

these observa- tions, histological analysis of the diabetic IL-4 / TNF

/ B7-1 triple

transgenic mice showed that islet non-B

cells

remained abundant (Figure

3,

panels

I to L;

Table I). In addition, the pancreata of some

of

the

tri-

ple transgenic mice, including the mouse that escaped diabetes and had nolmal islet B-cell content, showed a marked

insulitis

and fibrosis,

with

ductal structures and adipose tissue replacing most

of the

exocline acini in several lobes (Figure 4, A to C).

Injections of

neutralizing

anti-IFN-y

antibody Nine TNF / B7-l double

transgenic

mice

were injected

i.p. either

anti-IFNy alone (three

mice)

or anti-IFNy and

rIL-4 in

combination

(six

mice) until

their 6th week. Both

treatments

yielded

sirnilar results: no diabetes was observed at 6 weeks, but all mice were diabetic

by

the 9th week (Figure

l).

The

histological appearance of the islets from the diabetic

mice

was

identical to

that

of

the untreated double transgenic diabetic mice,

with

disappearance

of

all four types ofendocrine cells.

Since treatment with anti-IFN-T antibody appeared to delay diabetes onset

in

the TNF

/ B1-1

mice, six IL-4 / TNF

/ B1-l

triple transgenic mice we1'e treated

with

anti-IFN-y antibody

following

the same proto-

col. At

the end

of

antibody treatment

(6

weeks

of

age),

all were

normoglycemic (Figure

l) and

one mouse, sacrificed at this time for histologic examina-

tion,

showed the presence

of

a lymphocytic insulitis

but

abundant B-cells (Figure

4, D

and

E). Of

the remaining

five

mice, three showed a late occurrence

of

diabetes and

two

remained normoglycemic until sacrifice, at

I

months (Figure 1); the two non diabetic mice carried

out

normal pregnancies

giving birth

to normal litters. Histological analysis of pancreata from

the

anti-IFNy-treated

mice, either

double

or

triple

(6)

CYTOKINE MODULAIION OF DIABETES PROGRESSION 101

FIGURE 2 8 to 9 pm-thick cryostat sections, stained by immunofluorescence, of pancreata from TNF / B7-1 doubly transgenic mice (4, C, and E), and IL-4 / TNF / B7-l triply transgenic mice (8, D, and F). Panels A and B show islets stained for CD4+ cells; CD8+ cells are labeled in C and D, whereas B cells are demonstrated in E and F. The ratio CD4+/CD8+ is sirnilar in doubly and triply transgenic mice. The islet shown in panel F contains only few peripheral B lyrnphocytes. All mice were 5.5 weeks-old: the TNF / B7-1 were ah'eady hyperglyce- mic, whereas the IL-4 / TNF / B7-1 was normoglycemic. Bar represents 50 ptm (same magnification in all panels) (See Color Plate V at the back of this issue)

transgenics, either normoglycemic or diabetic, always showed the presence of an insulitis (Figure 4, F), indi- cating that delay

or

prevention

of

diabetes

did

not

result

from

variation

in

the incidence

of

islet

infiltra-

t1on.

(7)

102 PF.DRO LIIIS HERRERA et al.

FIGURE 3 5 pm-thick paraffin sections, stained by immunofluorescence, of pancreata from control (non transgenic) mice (A-D), TNF / B7 1 double transgenics (E-H), and IL-4 / TNF /

87-l

triply transgenic mice (I-L). A, E, and I, anti-insulin staining; B, F, and J, anti-glucagon staining; C, G, and K, anti-PP family staining; D, H, and L, anti-somatostatin staining. Overtly diabetic TNF / B7-l mice (E to H) show an almost complete loss of all islet endocrine cell types, wheleas diabetic IL-4 / TNF / B7-1 mice (I to L) have abundant non F-cells (J-L). Mice were 8 to 10 weeks-old. Bar represents 50 pm (same magnification in all panels) (See Color Plate VI at the back of this issue)

DISCUSSION

The present

work

shows that constitutive production

of IL-4 by

a transgene expressed

in B

lymphocytes

influences the early and severe autoimmune diabetes

resulting from

combined transgenic expression

of

TNF and

B7-1

by pancreatic B-cells, by modifying its course, incidence and even islet lesioned pattern. The

(8)

CYTOKINE MODULATION OF DIABETES PROGRESSION 103

FIGURE 4 5 pm+hick paraffin sections of pâncreata from IL-4 / TNF / B7-1 triply transgenic mice. A, B, and C are from a triple transgenic mouse that wâs normoglycemic at sacrifice, when I months-old. The islets appear heavily infiltrated and distorted (A, B), with abundant col- lagen deposition (gleen, in B), while many acini undergo ductular metaplasia (A, C), so that entire pancreatic lobes appear as fibrotic, with abundant adipocytes filling the interstitia (C). In C, adipocytes appear as large white holes, whereas exocrine acini are mostly replaced by altered ducts (small holes, also visible in A). B-cells are still abundant (8, a cluster of cells stained violet). D and E are consecutive sections

from a normoglycemic triple transgenic mouse killed when anti-IFN-y trcatment was ended (6 weeks-old), showing severe insulitis (D) together with abundant remaining B-cells (violet, in E). F, insulitis in an anti-IFN-T-treated triple transgenic mouse having escaped diabetes (10.5 month-old). A, C, D, and F are sections stained by the hematoxylin and eosin procedure, whereas B and E are colored by the aldehyde fuchsin (Gomori's) method, which allows identification of insulin-producing B-cells (violet) as well as of collagen and elastin fibers (green and violet, respectively). Bars represent 50 pm (magnification is the same for A, D, E, and F) (See Color Plate VII at the back of this issue)

influence of

IL-4

is increased when the effect of IFNy

is

simultaneously (but only transiently) decrcased, as the result of anti-IFNy antibody injection.

While

1007o

of

the TNF

I B1-l

double transgenic

mice

developed diabetes

by the

6th

week of

age (whatever the genetic background resulting from vari- ous crossings, and independently of gender), diabetes was uniformly delayed

by

weeks or months in triple transgenic mice, and even did not occur before sacri-

fice at almost one year of age in one case. The obser- vations are different

from

those made

with IL-4

in

NOD

mice,

which

were protected

by

administration

of

recombina\t

IL-4 [20]

(inefficient

in

the case

of

TNF /

B7-1

transgenic mice) and in which expression of an IL-4 transgene within the islets prevented insuli- tis, and thus diabetes

l2ll;in

contrast, the triple trans- genic mica

in

the present study had insulitis, even in the case that never developed diabetes.

A

plausible

(9)

104 PEDRO LUIS HERRERA et al.

interpretation

of

these data

is

that the

Thl

autoim- mune response

(or

probably

Tcl,

since

it

does not

require the

presence

of CD4 T

lymphocytes

[])

develop within the TNF-induced insulitis because

of the

antigen plus

B7-1

stimulation has been "devi- ated" by the

IL-4

released by

B

lymphocytes present

in the insulitis [2],

towards

a Th2 or Tc2

type

of

response, thus favoring auto-antibody formation but decreasing

the

strength

of the T cell

cytotoxic response. This explanation remains nevertheless spec- ulative, since we could not demonstrate any shift in

the cytokine profile of islet infiltrating cells

(as

assayed by semi-quantitative RT-PCR from total pan- creas

RNA,

not shown),

nor in

the

Thl /

Th2 ratio

among the islet-infiltrating lymphocytes (as assayed

by

immunofluorescence on pancreatic sections using

newly

available

Thl-

and Th2-specific monoclonal antibodies, not shown). The effect

of

anti-IFNy anti- body treatment on this type

of

diabetes may have a comparable explanation, although excess

of IL-4

is more directly instrumental than decreased availability

of

IFNy

in

differentiation

of

stimulated naïve T lym-

phocytes toward a pattern favoring a

humoral response t5, 101. In the case of NOD mice, prolonged treatment

with

anti-IFNy antibody reduces the inci- dence

of

diabetes 122), and

in RIP-LCMV

transgenic mice bearing an IFNy null mutation, both insulitis and diabetes

do not

appear

[23]. Anti IFN-y

antibody

treatment did

appear

to

enhance

the

protective

anti-diabetic effect of IL-4 transgene expression in the

TNF / B7-1

transgenic mice, since

two

mice out

of

nine never developed diabetes.

A

potential explana- tion for the protective effect conferred by anti-IFNy is that the lack of IFNy may prevent an adequate expres- sion

of

MHC class

I

and

II

antigens on B-cells [24],

thus preventing lymphocyte stimulation,

without influencing the TNF-driven insulitis.

TABLE I Average numbel ol endocrine cells per islet section in TNF /

B7-1 and in IL-4 / TNF / B7-1 transgenic mice (See "Morythometry and Statistical Analysls" paragraph in the Methods section)

Instrlin

Glucctgon

PP

Sonnlo-

family

stûtitr

13 13*

10 22

We observed that elevated circulating

IL-4 in

the transgenic

mice not only

decreased diabetes inci- dence,

but also modified the

nature

of the

islet lesions. We were surprised to observe and now report

for

the

first time that all four

endocrine

cell

types were destroyed in the TNF /

B7-1

transgenic mice.

It

is thus the only

IDDM

model so far reported, either spontaneous

or

experimental,

in

which the islet non B-cells (i.e. those producing either glucagon, somato- statin

or

pancreatic polypeptide) are destroyed. This non p-cell destruction takes place

in

spite

of

the fact that in these transgenic mice only B-cells, and not the other islet cells, bear the

B7-1

molecule, and would therefore be predicted to deliver the required co-stim- ulatory signal

to

activate B-cell-specific

T

cells. One possible explanation is that non p-cell specific T lym- phocytes could be co-stimulated in

trans;

that is from a

neighboringBT-l

expressing p-cell. Indeed, CTL precursors

with TCR

displaying

affinity for

peptide presented by the various islet cells may be, because

of

the strong insulitis and local lymphocyte traffic result-

ing from

the local expression

of

the

TNF

transgene [2], in contact with both an islet non B-cell, that some of them may recognize, and

B7-l

molecule borne by adjacent B-cells, thus providing the required second signal (Figure 5). Strikingly, the presence

of IL-4

in triple transgenic mice, in particular by

B

cells known to be present

in

the lymphocytic

infiltrate [2],

modi- fies the pattern

of

islet cells remaining

in

the diabetic animals, since non B-cells were preserved. This can

be explained by a

decreased

cytotoxic

response resulting

from

the proposed

shift

towards

a

type 2 lymphocyte response discussed above.

In this

case,

only the cytotoxic

response expressed against the most antigenic cells, those expressing self-peptides together

with

the

B7-1

molecule (i.e., B-cells) would persist.

Finally, another alteration observed as the result

of

the expression of the IL-4 transgene was the pl'esence of fibrosis

in

the exocrine pancreas.

IL-4

is known to enhance the synthesis of extracellular matrix proteins by fibroblasts [25]. Peri-insular fibrosis has also been observed in transgenic mice producinglL-4 under the control of an insulin promoter [26].

Control TNF / B7_1

IL-4lTNF/87_1

25 19

2

3'

o 1

I9 l3

" p<0.001, CONTROL vs. TNF / B7-l double transgenics.

(10)

A

o o o o

|t o 9O

o

B

class I MHC protein

"anti a-cell"

T cell receptor

CD-28

"anti p-cell"

T cell receptor FIGURE 5 Drawings depicting how an "anti-non B-cell" CD8+ T lymphocyte (labeled "CTL- 1") might be, within the islets of TNF / B7-l doubly transgenic mice, simultaneously in contact with a non B-cell (only glucagon cells are represented) and with a B-cell, and be activated by both. T cells as the one labeled *CTL-2" would be responsible of the destruction of B-cells. Panel A was drawn from an electron micrography of an infiltrated islet

In conclusion, the TNF

lB7-l

transgenic mice rep- resent

an

unique model

of

autoimmune

IDDM,

in which the pathogenic mechanism

of T

cell-mediated

105

B-cell destruction can be more precisely studied and influenced, since these events take place

in a

very limited period of time. Thus, they may afford a better opportunity to study how changes in cytokine expres- sion may be used to alter the cause of diabetes, as has been done in the present study. They should also facil-

itate the

study

of islet cell

regeneration following

rapid and

massive destruction (reviewed

in

[27D, since an increased presence

of

extra-islet endocrine cells was found in these diabetic mice.

Acknowledgements

We are most grateful to

Mr

Jorge

Ritz for

handling

and breeding mice, to Miss Gissela Gallardo and Mrs Ileana Condacci

for

genotyping by PCR, and to Mrs Danielle Ben Nasr and

Mrs

Gyslaine Moussard

for

paraffin sectioning and staining. This

work

was sup- ported

in

part

by a

grant

from

the Fonds National Suisse pour la Recherche Scientifique, and

in

part by Naval Medical Research and Development Command

Grant

EW.0O95.OO3.|4I2.

The

RIP-mCD80 trans- genic

mice

described

in this

study were generated according to the principles set forth

in

the Guide for the Care and Use of Laboratory Animals (Kommittee

on Care and Use of Laboratorl, Animals

(1985)

Guide

for

the Care and Use of Laboratory Animals

(Natl. Inst. Health,

Bethesda),

DHHS. PubL

No.

(NIH) 86-23). The views expressed

in

this article are those of the authors and do not reflect the official pol-

icy or position of the

Department

of the

Navy, Department

of

Defense, nor the United States Gov- ernment.

References

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and B7-2 costimulatory molecules activate differentially the Thl lTh2 developmental pathways: application to autoimmune disease Therapy. Cell 1995; 80 (5): 707-18.

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[14] Wong S, Guerder S, Visintin

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et al.: Expression of the co-stimulator molecule B7-l in pancreatic beta-cells acceler- ates diabetes in the NOD mouse. Diabetes 1995; 44 (3):326-

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Wogensen L, Lee MS, Sarvetnick N: Production of interleu- kin l0 by islet cells accelerates immune-mediated destruction of beta cells in nonobese diabetic mice. J Exp Med 1994; 179 (4): 1379-84.

[16] Pakala SY Kuffer MO, Katz JD: T helper 2 (Th2) T cells induce acute pancreatitis and diabetes in immune-compro-

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Mueller W, Kuhn R, Rajewsky K: Major histocompatibility complex class

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[24] Campbell IL, Wong GH, Schrader JW, Hanison LC: Inter- feron-gamma enhances the expression of the major histo- compatibility class I antigens on mouse pancreatic beta cells.

Diabetes 1985; 34(ll): 1205-9.

[25] Postlethwaite AE, Holness MA, Katai H, Raghow R: Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4. J Clin Invest 1992;90 (4): 1479-85.

[26] Mueller R, Krahl T, Sarvetnick N: Tissue-specific expression

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JM:

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