Type 1 diabetes mellitus as a polygenic multifactorial disease: immunopathogenic mechanisms of beta-cell destruction.

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Type 1 diabetes mellitus is a multifactorial autoim-
mune disease for which susceptibility is determined by
genetic, environmental and immunologic factors (1).
Genetic studies have shown that this is a polygenic di-
sease involving one major locus (IDDM1=HLA) and
an undefined number of loci with minor effect (2).
Among these,besides the IDDM2 (INS) locus that has
been conclusively shown to be implicated in the disea-
se, the strongest evidence so far available is that on the
IDDM12 locus.This corresponds to a region homolo-
gous to that harboring the IDD5 locus in the mouse
and it includes genes such as CTLA-4 and CD28
known to be involved in the regulation of the T-cell re-
sponse to antigenic stimulation.In particular,a microsa-
tellite on the 3’UTR region of CTLA-4 and a G-A di-
morphism in the first exon of the gene have been shown
to be both linked and associated with the disease.
Humoral and cellular autoimmunity in type 1 diabetes
A number of studies have shown that the de-
structive process in autoimmune diabetes specifically
directed against islet β-cells, can last for years before
the appearance of clinical symptoms and is characteri-
zed by the presence of circulating autoantibodies and
of T-lymphocytes directed against islet molecules (3).
The information so far available on the disease patho-
genetic process has clearly indicated that the progres-
sive autoimmune islet β-cell destruction is a T-
lymphocyte-mediated event. However, despite the
pathogenic importance of the T-cell response in type
1 diabetes, the target molecules of such a response re-
main largely uncharacterized, especially in humans.
The autoantigens so far identified in autoimmune dia-
betes have been discovered starting from disease-asso-
ciated autoantibodies that, although representing im-
portant tools for the prediction of future diabetes de-
velopment, have no direct pathogenic role since they
do not cause any islet damage (4). Interestingly, mole-
cules such as insulin and GAD have been shown not
only to be target of autoantibodies, but also of auto-
reactive T-lymphocytes both in man and in an animal
model of autoimmune diabetes such as the NOD
mouse. In addition to insulin and to GAD, molecules
expressed in insulin secretory granules and in mito-
chondria with a molecular weight around 38kD may
be important target autoantigens in the T-cell respon-
se associated with type 1 diabetes.As far as the patho-
genetic role of T-lymphocytes in diabetes pathogene-
sis is concerned, it has been elucidated, at least in part,
only in animal models of islet autoimmunity, while no
data is so far available in man, due to the impossibility
to isolate islet-infiltrating lymphocytes from human
pancreas. For the same reason, the available informa-
tion on the pancreatic islets at the disease onset in
man is still rudimentary, with data obtained mainly at
autopsy from patients who died soon after diagnosis.
Consequently, much of what we know about the etio-
logy and pathogenesis of autoimmune diabetes comes
from studies on small animal models (such as the
NOD mouse) which spontaneously develop a disease
with many of the features of the human disorder. In
the NOD mouse, a complex pathogenetic picture has
Type 1 diabetes mellitus as a polygenic multifactorial disease:
immunopathogenic mechanisms of beta-cell destruction
Francesco Dotta*, Cecilia Fondelli*, Umberto Di Mario**
* Diabetes Unit, Dept. of Internal Medicine, Endocrine and Metabolic Sciences and Biochemistry, University of Siena, Italy
** Dept. of Clinical Sciences – Endocrinology, University “La Sapienza”, Rome, Italy
(Available free-on line at: www.actabiomedica.it)
L E C T U R E
ACTA BIOMED 2005; 76; Suppl. 3: 14-18© Mattioli 1885

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Pathogenesis of type 1 diabetes mellitus
emerged (5), involving distinct phases and multiple
lymphocyte subpopulations. It has been suggested
that disease progression is regulated at two check-
points: 1) at 3-5 weeks of age, islet infiltration (insuli-
tis) begins;2) at 12-15 weeks of age,active destruction
of β-cells starts,leading rapidly to disease onset.As far
as the immune-cells involved in diabetes pathogenesis
is concerned, a consensus is emerging that CD8+ cel-
ls seem to initiate the process,while CD4+ lymphocy-
tes have a major role in the islet destructive process,
according to the results of adoptive transfer experi-
ments. However, the role of other cells clearly present
in the insulitis process, such as macrophages and den-
dritic cells, has to be determined: with regard to this,
it has been proposed that potentially pathogenic
CD4+ cells can interact with these Antigen Presen-
ting Cells (APC) and, as a result of such interactions,
activated T cells may directly kill the islet β-cells (e.g.
through Fas/FasL interaction) or may activate the cy-
tocidal functions of macrophages.
Being type 1 diabetes a T-cell mediated disorder,
several studies have aimed at the characterization of
the cell-mediated immune response in islet autoimmu-
nity (6). Research protocols performed primarily in
NOD mice have attempted to determine whether the
repertoire of islet-infiltrating T cells exhibits Vα or Vβ
restriction. To date, no sign of such a restriction has
been found in islet-infiltrating lymphocytes; however,
it is of interest to note that a Vα13 restriction has been
reported in pathogenic CD4+ T-cell clones in the
NOD mouse.These clones are directed against the in-
sulin B-chain and display a novel Vα-sequence that
was termed Vα13.3. In man, a preferential Vβ7 usage
was detected in two cases,suggesting a potential role of
an unidentified superantigen in disease pathogenesis.
As far as the characterization of T-cell clones is con-
cerned, a number of such clones have been isolated
from islet-infiltrating T-cells in NOD mice.These clo-
nes have been shown to be directed either against insu-
lin or GAD or carboxypeptidase-H, and in some cases
are able to transfer disease in recipient animals. Usual-
ly, these are CD4+ clones with a pattern of cytokine
production of Th1-cells (IL-2, IFNγ and TNFα).
Most investigators now agree that both CD4+
(helper) and CD8+ (cytotoxic) T-cells are required for
maximum development of diabetes although B cells,
macrophages and dendritic cells have important
pathogenetic functions.In NOD mouse and BioBree-
ding (BB) rat, macrophages and/or dendritic cells are
the first cell types to infiltrate the pancreatic islets.
The presentation of β-cell autoantigens by macropha-
ges and/or dendritic cells to CD4+ T-helper cells, in
association with MHC class II molecules, is conside-
red the initial step in the development of type 1 dia-
betes. Activated macrophages and CD4+ release cy-
tokines, such as IL1-β, TNF-α and INF-γ, inducing
the migration of CD8+ peripheral T-cells to the infla-
med islets.The activated cytotoxic CD8+ T-cells then
damage β-cell by releasing perforin, granzyme and by
Fas-mediated apoptosis (7).
The relative importance of CD4+ and CD8+
T-cells in the pathogenesis of diabetes is still contro-
versial; some authors have proposed that CD8+ are
sufficient for priming and expansion of autoreactive
CD4+ T-cells, while others have shown that cloned
autoaggressive CD8+ T cells are able to cause diabetes
by themselves (8).
Apoptosis and type 1 diabetes
Apoptosis or Programmed Cell Death is a highly
regulated and evolutionary-conserved form of cell
death, which plays a crucial role in the development
and homeostasis of multicellular organisms (9). Dy-
sregulated apoptosis has been shown to represent the
basis of several diseases including cancer and autoim-
mune syndromes. In organ-specific autoimmune di-
seases the role of apoptosis is double, representing the
mechanism of target cell death and being involved in
the selection of T-lymphocytes, which are the media-
tors of autoimmune tissue destruction. Fas system is
involved in the elimination of normal activated pe-
ripheral T-cells. Mature lymphocytes constitutively
express Fas, activation by antigen induces Fas ligand
(FasL) expression: Fas-FasL interaction determines
the killing (by apoptosis) of Fas-expressing cells. Fas-
mediated death plays an important regulatory role in
autoimmune disorders of lpr (lymphoproliferation)
and gld (generalized lymphoproliferative disease) mi-
ce that show, respectively, mutations in Fas and FasL
genes. Initial findings hypothesized a fundamental ro-

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F. Dotta, C. Fondelli, U. Di Mario
le of death receptor Fas in β-cell destruction, confir-
med by observation that NODlpr did not develop dia-
betes or insulitis, nor it was possible to transfer diabe-
tes to NODlpr recipient.
Both necrosis and apoptosis have been proposed
as cell death mechanisms in autoimmune diabetes,
with the latter being a highly regulated process initia-
ted by a variety of intra- and inter-cellular signals and
involved in homeostasis of target cells as well as of im-
munocompetent cells. Regardless from the cell type
involved,apoptosis is a multi-step process which starts
with an extracellular death signal (induction phase),
activating one or more signal transduction pathways
(effector phase) that converge to few final death
pathways (degradation phase). These final pathways
are usually executed by proteases called caspases and
by caspase-activated DNAse (CAD). Death receptors
play a central role in apoptosis initiated by death si-
gnals (10); among such receptors, those belonging to
the TNF receptor gene superfamily are the best cha-
racterized, and include Fas (CD95), TNF receptor 1
(TNFR1), death receptor 3 (DR3), DR4 and DR5.
Once activated by their specific ligands, these recep-
tors, through adapter molecules (FADD for Fas and
TRADD for TNFR1), activate down-stream caspases
that can determine the apoptotic death within hours.
In addition to death receptors and their downstream
caspases, other molecules, usually associated with mi-
tochondria, are actively involved in apoptosis regula-
tion. These include anti-apoptotic protein bcl-2 and
pro-apoptotic bax, whose relative ratio has a profound
influence on the commitment of a given cell to die or
survive;furthermore,release of cytochrome-c from the
mitochondria rapidly leads the cell to apoptotic death,
since once in the cytoplasm, cytochrome-c forms an
essential part of the “apoptosome” together with ca-
spase-9 and Apaf-1. In autoimmune diseases, Fas-
and TNFR-dependent pathways have been proposed
as the main mechanisms of target cell death.
Cytokines and β β-cell death
A great body of evidence (11) indicates that pan-
creatic β-cell destruction results from a disorder of im-
munoregulation and that cytokines secreted by infil-
trating cells (CD4+ and CD8+ T lymphocytes, B
lymphocytes, NK and macrophages) might have an
important role in the pathogenesis in Type 1 diabetes.
In NOD mouse, macrophages are the first inflamma-
tory cells to infiltrate the islets, where they may engulf
apoptotic β-cells. A current hypothesis is that the
pathogenic immune response is mediated by a Th1
subset of T cells, whereas the protective response is
mediated by a Th2 subset of T cells.Th1 and Th2 cel-
ls produce a different cytokine pattern. Th1 cells se-
crete IL-2, INF-γ and TNF-β and activate cell-me-
diated immunity, whereas Th2 cells secrete IL-4, IL-
5, IL-6, IL-9, IL-10 and IL-13 and stimulate humo-
ral immunity. Many studies support the hypothesis
that Th1-cytokines are β-cell destructive whereas
Th2-cytokines protect β-cell by suppressing Th1-cy-
tokines expression (12). As a matter of, both IL-4 and
TGFβ, when transgenically expressed in the pancrea-
tic β-cells of NOD mice, prevent autoimmune diabe-
tes.
In addition, macrophages produce IL1(α and β),
TNF-α and IFN-α that are referred as proinflamma-
tory cytokines. Correlation has been observed
between β-cell destructive insulitis and expression of
proinflammatory cytokines. Several cytokines poten-
tially involved in the pathogenesis of Type 1 diabetes
have been found to be expressed in the insulitis lesion
in NOD mouse,BB rat and man; although this obser-
vation does not directly prove the pathogenic role of
these cytokines. It has been observed that, IL1-β,
TNF-α and INF-γ,in combination,induce functional
and morphological damage β-cells in both rodent and
human islets, in a dose and time dependent manner.
One of the mechanisms proposed (13) is that, as
observed in rodent islets, incubation of human islets
with a combination of IL1-β+TNF-α+INF-γ deter-
mines up-regulation, in β-cells and macrophages, of
the expression of inducible nitric oxide synthase
(iNOS) and the subsequent synthesis of the radical ni-
tric oxide (NO) as well as of other free radicals such as
peroxynitrite (ONOO-) and superoxide (O2-) that con-
tribute to β-cell damage. Nitric oxide impairs β-cell
function by blocking the enzyme aconitase and by in-
ducing DNA strand breaks. Previous reports showed
that NO could induce Fas expression on β-cells, so
that, they might be destroyed by T-lymphocytes.

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Pathogenesis of type 1 diabetes mellitus
A recent study has shown that iNOS-deficient
mouse islets, treated with a combination of IL1-β+
TNF-α + INF-γ, expressed Fas mRNA in response to
cytokines similarly to wild-type islets and the murine
NIT-1β-cell line. In addition, the accumulation of
Fas-specific mRNA, in islets following exposure to
proinflammatory cytokines,correlated directly with an
increased surface expression of Fas comparably in
wild-type, iNOS-deficient islets and NIT-1. This re-
sult shows that Fas mRNA transcription and Fas pro-
tein expression in murine islets are independent of
NO production.
On the basis of the involvement of death recep-
tors in β-cell destruction,several observations (14) ha-
ve pointed out that TNF-α could have a diabetogenic
role in the elimination of β-cell.TNF-α is an inflam-
matory cytokine principally secreted by macrophages
and activated CD4+ T cells during autoimmune and
infection diseases; it is able to induce cell death in
many tumor cells and can modulate insulitis in NOD
mouse.
A pivotal role of this cytokine in the disease pro-
cess comes from several observations:TNF is produced
in inflamed islet in NOD mouse; neutralizing anti-
TNF monoclonal antibodies prevented both insulitis
and disease; treatment with recombinant TNF
(rTNF) had different effects, depending on age of
NOD mouse. Administration of rTNF at birth acce-
lerated disease onset while the same treatment, in
adult, delayed it.
Similar results have been obtained by experiments
performed with transgenic NOD mouse expressing
TNF in pancreatic β-cells: the early expression of
TNF-α induced an early disease onset while late ex-
pression of this cytokine resulted to be protective.
These findings show that TNF has complex ef-
fects on the development of the autoimmune response.
Several studies suggest that TNF is directly toxic
to β-cells although, it seems that this cytokine poten-
tiates its toxic effect in synergy with IL-1β and IFN-γ
to β-cell elimination (15).
A recent report seems to clarify the role of TNF-α.
The authors have observed that TNFR1-deficient
NOD mouse developed insulitis and the proportion of
CD4+ and CD8+ was similar to that in normal con-
trol NOD, but more importantly, progression to dia-
betes was completely abrogated. Transfer of spleen
cells from diabetic normal control NOD mice into
TNFR1-deficient recipient, significantly delayed dia-
betes and transfer of perforin-deficient NOD spleen
cells into TNFR1-deficient recipients failed to induce
diabetes.
These experiments would highlight the diabeto-
genic role of TNF-α/TNFR1 system that acting in
synergism with perforin system is able to determine
β-cell death. In addition, these findings argue against
the involvement of Fas system in the beta cells elimi-
nation in Type 1 diabetes.
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Correspondence: Prof. Francesco Dotta
U.O. Diabetologia,
Viale Bracci 18, 53100 - Siena, Italy.
Tel: +39-0577-586269
Fax: +39-0577-586186
E-mail: fradotta@tin.it