Download Islet inflammation in human type 1 diabetes

Islet inflammation in human type 1 diabetes
Noel G. Morgan1, Pia Leete1 Alan K. Foulis2 and Sarah J Richardson1
1
Institute of Biomedical & Clinical Sciences, University of Exeter Medical School, Barrack Road, Exeter
EX2 5DW, UK.
2
GG&C Pathology Department, Southern General Hospital, Glasgow, UK.
Correspondence:
Prof Noel G Morgan
Institute of Biomedical & Clinical Sciences,
University of Exeter Medical School,
Barrack Road, Exeter,
EX2 5DW, UK.
[email protected]
Abstract
Type 1 diabetes is caused by the selective deletion of pancreatic β-cells in response to an assault
mounted within the pancreas by infiltrating immune cells. However, this apparently clear and
focussed annunciation conceals a stark reality in which the cellular and molecular events leading to
β-cell loss remain poorly understood in humans. This reflects the difficulty of studying these
processes in living individuals and the fact that, using pathological specimens, islet inflammation has
been analysed in fewer than 200 recent-onset cases of type 1 diabetes worldwide, over the past
century. Nevertheless, insights have been gained and the composition of the islet infiltrate is being
disclosed. This is shown to be primarily lymphocytic in nature, with populations of both CD8+ and
CD4+ T-cells displaying an autoreactivity against specific islet antigenic peptides. The T-cells are
often accompanied by influent CD20+ B-cells although new data imply that the proportions of these
individual cell types vary and that patients fall into at least two distinct categories having either a
hyper-immune (CD20Hi) or a pauci-immune (CD20Lo) phenotype. The overall rate of β-cell decline
appears to correlate with these two phenotypes such that hyper-immune patients lose β-cells more
quickly and tend to develop disease at an earlier age than those with the pauci-immune profile. In
the present article, we review the evidence which underpins our current understanding of the
aetiology of type 1 diabetes and, in so doing, highlight both the established features as well as areas
of on-going ambiguity and debate.
Introduction
Type 1 diabetes in humans is considered to have an autoimmune aetiology in which the insulinsecreting β-cells of the pancreatic islets are destroyed selectively by influent immune cells
responding to the aberrant presentation of β-cell antigens (1-7). However, this seemingly
straightforward summary conceals a considerable degree of ambiguity that still surrounds almost
every stage of the process. In the present article, we will attempt to highlight these ambiguities and,
in doing so, indicate where active research efforts are still required.
There are various reasons why, almost 100 years after the discovery of insulin, the molecular basis of
human T1D remains shrouded in mystery but, foremost among these, is the fact that the pancreas is
inaccessible to non-invasive investigation in humans. Currently there are no reliable ways to image
the organ such that the cellular and molecular events associated with islet inflammation can be
studied in living individuals, although efforts are continuing towards the achievement of this goal (8).
Thus, the limited information which is available has been derived from samples recovered postmortem from patients with T1D (9-11); from glands recovered at the time of organ donation (6, 1214) or from surgical biopsy samples recovered from living patients (15, 16). Unfortunately, even
among these valuable resources, there are important issues which militate against a full
understanding of the disease pathology. Firstly, access to the whole pancreas is only possible at the
end of a patient’s life and this is usually many years after disease diagnosis. Secondly, the bulk of the
pancreas plays a role in digestion and, during the post-mortem phase, the organ is often subject to
autolysis such that the state of preservation of the tissue may be less than optimal when it is
recovered. This may be offset by the rapid processing of an organ at the time of donation in heartbeating donors, but such glands are rarely available from patients with recent-onset T1D. Figure 1
illustrates the process of insulitis in individual islets from pancreases collected either at autopsy or
organ donation. The biopsy approach could, in principle, provide a route by which many of these
problems are overcome but, in practice, this also presents difficulties. Some of these are technical
given that the surgical or post-operative procedures can lead to unexpected complications, as has
happened recently in the DiViD study from Norway (16). In addition, this approach relies on the
assumption that the disease process will inevitably be captured in the biopsied region but this does
not necessarily hold true as there is strong evidence that T1D progresses in a focal manner. As such,
both temporal and spatial differences exist in the distribution of inflamed islets and, collectively,
these limit the amount of useful information that may be available from within the biopsied area.
On this basis, it will already be clear that a detailed study of the cellular aetiology of T1D in humans
is not a trivial matter and the quest to provide a more complete picture continues. In particular,
consortia around the world are collaborating to develop new collections of samples which build on
the extant historical collections such that the process can be studied in molecular detail (6, 12).
Nevertheless, we should emphasise that conclusions about the underlying pathology of human T1D
are drawn from the study of as few as 150 cases worldwide. These cases and their historical context
have been summarised very comprehensively in recent reviews by In’t Veld (17, 18) and will not be
rehearsed here, although we will draw on the available information to present a contemporary view
of the process of islet inflammation in human T1D. In stating this objective, we should also
emphasise that it is not our purpose to discuss in detail, data which have arisen from the study of
the various animal models of T1D. This is not because we consider that these cannot provide
important insights (they clearly do!) but, rather, because the available evidence implies that the
human disease is not recapitulated fully in any of the available animal models.
β-cell loss in type 1 diabetes
Early studies of the pancreatic gland recovered from patients with T1D identified the presence of
small, nucleated, cells around the periphery (and sometimes within) the islets of Langerhans (19-21).
These were correctly considered to be immune cells and it is now clear that such cells congregate in
the vicinity of the islets during disease progression. However, islets do not become inflamed in
synchrony; rather, the process follows a focal course. Thus, in patients studied soon after diagnosis,
regions of the pancreas can be identified in which normal, apparently healthy, islets are found,
whereas other regions exist (often located in close proximity) containing islets in which β-cell
destruction is complete (4, 9, 11, 22). Such islets are sometimes referred to as “pseudoatrophic” (18,
23) to denote the fact that, although β-cells are absent, they retain a normal complement of the
other islet endocrine cells. This raises an important point, in that immunological approaches have
been (and remain) the mainstay of pathological investigation and all conclusions about the loss of βcells depend on an analysis of the presence (or absence) of immunoreactive insulin as a means to
identify these cells. In studies of T1D pancreas, the loss of insulin immunoreactivity has almost
universally been interpreted as synonymous with β-cell ablation, but it should be understood that
this has rarely been proven conclusively. This is mainly because there are no other known
immunological markers that define human β-cells uniquely. Thus, it is theoretically possible that
degranulation of β-cells could be misinterpreted as cell ablation during histopathological analysis.
One extension of this idea (which is increasingly being mooted as a possible cause of β-cell decline in
T2D (24-26)) is that β-cells might undergo a process of de-differentiation. In this view, the cells
would not necessarily be lost in significant numbers but, rather, they would lose insulin
immunopositivity and acquire a more “mesenchymal” phenotype associated with the expression of
stem cell markers such as Oct4, Ngn3 and vimentin. While this has not yet been evaluated in a fully
systematic way in the pancreases of patients with T1D (or, arguably, in human subjects with T2D)
our view from the examination of many pancreatic glands recovered from a wide variety of T1D
patients, is that the vast majority of islet cells stain positively for one of the relevant hormones
(either glucagon, somatostatin, pancreatic polypeptide or ghrelin). Hence, we do not find islets
containing large numbers of hormone-depleted cells. This implies that neither complete β-cell
degranulation nor β-cell de-differentiation is a frequent occurrence in human T1D and it seems
reasonable to conclude that immune-mediated β-cell ablation remains the primary mechanism of
loss. This does not mean, of course, that processes such as de-differentiation or selective
degranulation might not occur in some β-cells, but we are not persuaded that they could account for
the bulk of the apparent cell loss in T1D.
A further feature which suggests that the major mechanism of insulin depletion in T1D occurs by βcell loss is that the architecture is progressively altered in those islets displaying reduced (or absent)
β-cells. Islets frequently adopt a more condensed appearance upon light microscopic examination as
they lose β-cells and they are often smaller and less ovoid in appearance (as judged on sections of
pancreas; Figure 2). This feature is characteristic of the “pseudoatrophic” islets mentioned above
and these are taken to represent islets in which β-cell loss is complete and in which the remaining
cells have adopted a reorganised orientation to occupy the newly available space. Occasionally,
insulin-deficient islets may also adopt a more diffuse appearance suggesting that the demise of the
β-cells can also lead to a more generalised loss of cellular organisation [Figure 2c].
These considerations raise a further important matter which also remains largely unresolved. This
relates to the mechanism by which β-cell loss occurs in T1D and what subsequently happens to the
associated debris. It has been widely supposed that the primary mechanism of β-cell death is by
apoptosis (27-29) and that residual β-cell components are cleared rapidly by macrophages which
normally patrol and monitor the islet milieu (30). Such hypotheses are based mainly on evidence
arising from in vitro studies which show that pro-inflammatory cytokines (e.g. interleukin-1β;
interferon-γ, tumour necrosis factor-α) can promote β-cell apoptosis (31-35). Thus, it is concluded
that, if similar cytokines bathe the endocrine cells in inflamed islets, then enhanced β-cell apoptosis
is likely to ensue. Increased apoptosis might also derive from Fas-mediated β-cell toxicity (35-38) or
from enhanced endoplasmic reticulum stress (39) as has been suggested in some studies. The
concept of an apoptotic mode of β-cell death has received limited support in that, while apoptotic βcells have occasionally been identified in inflamed islets (40), they are not usually present in large
numbers, even in those islets which appear to be entering the most active phase of destruction. This
may simply reflect the efficiency of the phagocytic clearance mechanisms and the fact that, in any
pancreas sample, only a single point in time is being surveyed. Alternatively, it might mean that nonapoptotic mechanisms also operate. This could be the case if, for example, the release of granzymes
and/or perforin from influent immune cells contributes to β-cell loss (41-44). Extensive islet cell
necrosis might be expected to provoke a more intense immune response (since cellular contents will
more readily enter the extracellular space) and lead to widespread, less targeted, damage. This is
rarely seen upon histological examination of T1D pancreas but it remains unclear exactly how β-cells
die in human T1D.
Islet autoantibodies and type 1 diabetes
The definition of T1D as an autoimmune disease has been driven by the observation that many
(though not all) patients develop circulating antibodies to particular β-cell proteins (5, 45-49). These
include insulin itself (which is frequently the earliest antigen to elicit an antibody response) certain
secretory granule proteins (such as a protein phosphatase, IA2, and a specific zinc transporter, ZnT8)
and the cytosolic enzyme glutamate decarboxylase (GAD). Patients often develop antibodies to
multiple auto-antigens and there is an on-going debate as to whether these antibodies are truly
pathogenic or if they are generated secondarily, as markers of an underlying disease process. Most
evidence favours the latter interpretation although it should also be emphasised that individuals can
develop autoantibodies but never progress to clinical T1D. Thus, there is no absolute relationship
between the two. What is clear, is that the presence of multiple autoantibodies is strongly predictive
of diabetes development in susceptible individuals (50, 51) and that non-diabetic subjects with three
or more circulating autoantibodies have a greatly increased risk of progressing to T1D.
The inflammatory infiltrate in type 1 diabetes
Dogma (or, at least, accepted wisdom) states that the islets of patients with T1D are inflamed but, in
practice this has been observed much less frequently than might be imagined. Indeed, in one recent
review, it was noted that human insulitis is so rare that most pathologists will not witness a single
case during their entire careers as practitioners (18)! Nevertheless, there is abundant evidence from
the available pathological collections that insulitis does occur in human T1D albeit in a relatively low
(~10%) proportion of islets. This is partly because the inflammation dissipates once β-cells are lost
and that most pancreases of patients with type 1 diabetes contain a large number of insulindeficient islets. It may also reflect the focal nature of the disease and the fact that, by contrast with
animals such as the non-obese diabetic mouse, the total number of immune cells infiltrating any
given islet is often rather modest. Thus, when islets are observed in pancreas sections (i.e. in two
dimensions) small numbers of immune cells may be excluded from view because they lie either
above or below the plane of the section. As a result, an islet might then be scored by an observer as
“non-inflamed” whereas, in reality, it contains a few immune cells elsewhere within its volume. It is
also evident that insulitis is seen most frequently in recent-onset T1D patients and that this
frequency declines with disease duration.
Despite these difficulties, both the number and the phenotype of the influent immune cells have
been studied in the inflamed islets of humans with T1D. Early experiments showed that the infiltrate
is predominantly lymphocytic in nature (52) [Figure 3a] and that it contains CD8+ T-cells [Figure 3c],
which are considered to be among the primary cytotoxic mediators (53). However, additional
immune cells are also present, including CD4+ T-cells [Figure 3d], B-cells [Figure 3b] and
macrophages [Figure 3c] (4, 53). In addition, the surrounding pancreatic exocrine tissue has recently
been reported to be enriched in both lymphocytes (54) and neutrophils (55) in T1D and the
suggestion made that these might contribute to disease progression. Neutrophils do not appear to
be present within (or near to) inflamed islets in large numbers and, hence, their role remains
ambiguous. In some studies NK cells have also been found in the islet infiltrate (13) although this is
not universally observed (53).
These considerations raise the issue of exactly how islet inflammation (“insulitis”) is defined. This is
important because, with attempts to increase the number of available organs for study, it has been
discovered that immune cell infiltration can occur in the pancreas in association with periods spent
by subjects in intensive care units, prior to organ recovery (56). This occurs independently of the
presence or absence of T1D and could lead to misdiagnosis of insulitis in pathological specimens.
Fortunately, a comprehensive consensus definition of insulitis has recently been proposed (23). This
deals with various aspects of the pathological appearance of inflamed islets and concludes that
insulitis can be confirmed in a T1D patient if three or more islets are shown to contain 15 or more
lymphocytes in the peri-islet region and/or within the islet structure. This definition does not, of
course, define insulitis at the level of any given individual islet and this remains a discretionary
consideration on the part of individual investigators. In our hands, we have rarely seen individual
islets which contain more than 5 immune cells (lymphocytes or macrophages) on a single crosssection of the islet in non-diabetic subjects and, therefore, we have used this as an operational
threshold (53).
An analysis of the composition of the immune cell infiltrate in islets at apparently different stages of
β-cell destruction (as judged by the extent of residual insulin immunopositivity) has led to the
conclusion that CD8+ T-cells are the predominant cell type throughout the period of inflammation
(4, 53, 57). This is consistent with the notion that these cells ultimately mediate β-cell death.
Interestingly, however, the B-cell (CD20+) profile also changes during disease progression and, in an
initial study, was found to align closely with the migration of CD8+ T-cells (53). By contrast, the
numbers of CD4+ T-cells and macrophages (defined with anti-CD68) were much less variable during
the period of β-cell decline. This suggests the existence of a dynamic interplay between some (at
least) of the immune cell subsets during the progression of T1D and this remains to be understood.
Moreover, very recent data imply that these initial observations might represent an oversimplification and that the profile of insulitis is more variable (57). This variability is not simply
stochastic but, rather, two closely regulated patterns of insulitis can be defined. The first of these
mirrors the pattern reported by Willcox et al. (53) based on the analysis of insulitis in a cohort of 29
patients with recent-onset T1D, whereas the second is quite different. The main difference lies in the
number of influent CD20+ cells which, as noted above, can be extensive and mapped in parallel with
the influx of CD8+ cells or, as revealed in the more detailed recent study, can be minimal (or absent).
As a consequence, the two distinct profiles have been termed “CD20Hi” or “CD20Lo” to denote the
differential involvement of these cells [Figure 4]. In fact, even this may be something of a misnomer
since the absolute numbers of all immune cell subtypes are reduced among patients displaying the
CD20Lo profile and, in recognition of this, an alternative designation of “hyper-“ or “pauci-“ immune,
respectively, has also been suggested.
In both forms of insulitis (CD20Hi and CD20Lo) it is clear that CD8+ T-cells predominate and it is
likely, then, that these will mediate the demise of the β-cells in both situations. Importantly, the two
profiles also appear to reflect patient differences rather than being islet-specific (or lobular)
patterns. This has not been evaluated completely since, to do so, would require the analysis of all
inflamed islets across an entire pancreas and this would represent a gargantuan task. Nevertheless,
based on the more limited analysis of islets from different regions of a given pancreas, it appears
that T1D patients fall into one of the two categories. The factor that then differentiates the two is
the apparent rate at which the β-cells are killed. This cannot, of course, be deduced with complete
certainty but the pathological evidence can be reconstructed to make a strongly supportive case.
This is based on several notable features. Firstly, the number of residual insulin-containing islets in
patients displaying the CD20Lo profile is greater than in those who are CD20Hi [Figure 5]. This
implies that β-cell killing is less efficient in patients with the CD20Lo profile than in those who are
defined as CD20Hi. In support of this, the residual insulin-containing islets also tend to contain
greater numbers of β-cells in CD20Lo cases. Secondly, (and, perhaps, most strikingly) when a cohort
of patients was studied who were diagnosed with T1D within a fixed range of ages (between 0-20
years old), the group who displayed the CD20Hi profile were, on average, diagnosed 6 years younger
than those designated CD20Lo. Taken together, these results imply that two differing profiles of
insulitis exist in human T1D patients and that these are associated with either a more (CD20Hi) or a
less (CD20Lo) aggressive autoimmune attack on the β-cells.
The factors that determine which profile occurs in any given patient are entirely obscure (although
genetic influences are an obvious, but untested, possibility) but these new data offer an important
perspective on the design of clinical trials intended to slow or halt the progression of autoimmunity.
For example, it was proposed previously that the administration of an anti-B-cell therapy might be
effective in T1D patients and trials with rituximab (a monoclonal anti-CD20 antibody) (58, 59) and
have been undertaken. These trials involved a short term administration of rituximab followed by a
lengthy period of follow-up and they were, at best, only partially successful. At first sight, this is
disappointing but the newly available data on immune cell infiltration suggest that it might be
premature to close the matter without further consideration. In particular, it is noteworthy that the
mean age of disease diagnosis among the patients recruited to the rituximab trials was 19 years (58,
59) and, as such, it seems plausible that many of them might have displayed the CD20Lo profile of
insulitis (although this prediction cannot be verified since it is not possible to interrogate their
pancreas). If so, then anti-CD20 antibody is likely to have been minimally effective (assuming that its
efficacy reflects the targeting and deletion of CD20+ cells having access to inflamed islets rather than
simply those within the circulation). By contrast, administration of rituximab showed more (albeit
still limited) protection in the younger patients. Since such patients might be expected to
preferentially display the CD20Hi profile of insulitis, this may be indicative of an altered insulitic
profile in response to the anti-CD20 therapy; at least temporarily. Thus, it is conceivable that
renewed attempts to deplete CD20+ cells in a selected sub-group of (younger-onset) patients might
yield more striking results?
Since antibodies are generated from activated B-cells and there is evidence that B-cells may
comprise a significant proportion of the immune cell infiltrate in certain patients, it is important to
understand whether islet-recruited B-cells are actively secreting antibodies within the vicinity of
islets. During activation and differentiation into plasma cells, B-cells usually down-regulate the
expression of CD20 and up-regulate CD138 (60). However, islet infiltrating B-cells retain CD20 and
they are not labelled by antisera directed against CD138, a marker of terminally differentiated,
antibody-secreting, plasma cells (60). Thus, this population appears to play a role that is
independent of antibody synthesis and secretion. One possibility is that the B-cells may be acting as
local antigen-presenting cells within the islet milieu and that they collaborate to promote the β-celldirected cytotoxic activity of CD8+ T-cells (61).
Recent studies have attempted to assess the specificity of the infiltrating CD8+ cells using tetramer
staining in which the target antibodies are loaded with potentially autoreactive peptides derived
from islet antigens (62). This has revealed that the specificity of the total population of CD8+ cells is
relatively wide in that specific sub-populations appear to be reactive against a range of different
antigenic peptides. However, it was also revealed that the majority of such cells within the vicinity of
any given islet display a common autoreactive phenotype, suggesting that they may be clonal in
origin. Moreover, it appears that the influent cells target defined β-cell antigens, implying that they
are truly autoreactive (62). Whether there are also additional CD8+ cells present which target non-βcell antigens (such as virus proteins, for example) remains to be established.
One subtype of immune cells which has not received as much attention is the population of CD4+ Tcells present within insulitic infiltrates. This may be because their numbers do not alter dramatically
during the progress of β-cell destruction but it also remains unclear whether these cells display a Th1
or Th2 phenotype. In future studies, it will be important to determine the Th phenotype of these
cells and to examine whether this differs according to the CD20Hi or CD20Lo status. In important
recent work, CD4+ T-cells were grown out from the islets of a patient with type 1 diabetes and were
shown to be reactive against epitopes present within proinsulin in an HLA-restricted manner (63).
Thus, these cells were shown to be truly autoreactive and it was suggested that they could be
involved in β-cell destruction (63). Few, if any, of the infiltrating CD4+ cells can be classified as Tregs
since they do not stain positively for the presence of FoxP3 (53); a marker which defines this class.
Recruitment of immune cells to inflamed islets
The fact that the number of infiltrating immune cells surrounding (and/or within) inflamed islets is
often modest provokes questions relating to the nature of the attractant that draws these cells to
the site and their origin. It is frequently assumed that the immune cells migrate to the islets mainly
from influent blood vessels and that they extravasate in the immediate vicinity of the islets (possibly
from efferent capillaries carrying blood to collecting venules leaving the islet milieu) but this has not
been formally proven. There is little evidence that the cells reach the islet by egress from vessels
which penetrate to the core of the structure since the morphology of the insulitis places the influent
cells mainly in the surrounding pancreatic parenchyma adjacent to islets (often close to one pole of
an islet) rather than within the islet per se.
It is commonly argued that immune cells are drawn to islets by a chemokine gradient elaborated
from the endocrine cells themselves and there is evidence that these cells can synthesise and
release a range of relevant chemokines such as CXCL10, MCP-1 and MIP-1α (64-68). If this is the
case, then it seems reasonable to deduce that this may represent one means by which the β-cells
become visible to the immune cells during an early phase in the development of T1D. A second
mechanism occurs via the up-regulation of MHC class 1 molecules on the β-cell surface which,
although not seen in all islets, nevertheless represents a striking histological feature in patients with
T1D (9, 69, 70). Thus, it seems possible that the primary “defect” in T1D may lie at the level of the βcells themselves rather than in the immune system, since a dysfunctional immune system which
aberrantly targets the β-cells should be largely independent of chemo-attractants and MHC-1 hyperexpression.
One obvious possibility that continues to attract attention as a means to explain why β-cells might
become the target for autoimmunity is that they are mounting an active response to an
environmental insult. This would then represent the primary “triggering” event which initiates
disease progression. Considerable effort is currently being invested to establish whether this trigger
could be a viral (particularly an enteroviral) infection since such a mechanism would provide an
obvious route by which islet autoimmunity might be initiated (71). Evidence of enteroviral infection
has been reported in human β-cells from patients with T1D (13, 71-73) and this can occur in islets
which display no signs of inflammation (74). Thus, it is possible that insulitis follows from the
establishment of an enteroviral infection and this idea represents a working hypothesis which is still
being pursued by many workers globally (71). Irrespective of the precise nature of the insult, it
seems probable that the extent to which β-cells become visible to the immune system is increased
as an early event in the development of T1D and that insulitis develops as a consequence of this.
As noted above, one of the consistent morphological observations arising from the study of human
insulitis is that the lesion tends to be diffuse and localised mainly at the periphery of each islet.
There are certainly occasions when immune cells can be detected among the endocrine cells within
the islet structure but, in the majority of islets, this is not seen. This arrangement implies that the
influent immune cells may be prevented from accessing the interior of the islet by the presence of a
physical barrier which limits access. In support of this, it is known that islets are surrounded by a
basement membrane which serves to delineate the islet structure and which, following their
isolation, allows islets to retain their three-dimensional organisation in vitro (75-78). The
composition of this structure has not been defined systematically but it is almost certainly comprised
of a complex mixture of proteins and proteoglycans, including collagens, hyaluronic acids and
heparan sulphates. Thus, in order to access the islet interior it is probable that this physical barrier
must be breached and an examination of the morphology of inflamed islets implies that this occurs
at specific points around the perimeter of the islet rather than as a more generalised dissolution of
the basement membrane components. One possibility is that, on arrival within the peri-islet region,
specific immune cells are induced to secrete relevant proteases and/or other hydrolases that
selectively degrade the basement membrane to facilitate the entry of the cytotoxic cells (79, 80). If
this is the case, then it might offer a therapeutic opportunity since the administration of inhibitors
that attenuate the activity of such enzymes could be effective in slowing the progression of insulitis.
The nature of the specific immune cell subset responsible for such hydrolase secretion has not been
established and immunolabelling of relevant subsets in human pancreas sections has not, so far,
revealed any unique cell type whose arrival appears to presage the breaching of the basement
membrane. Nevertheless, this possibility warrants further consideration, especially as there is
increasing evidence that certain proteoglycans are lost from islets during the progression of T1D.
Insulin secretion from islets isolated from patients with type 1 diabetes
It has been implicit in much of the above discussion that T1D is caused by net β-cell loss and many
researchers have extrapolated from empirical observations to argue that the clinical symptoms arise
when the extent of this loss reaches ~80% (i.e. when ~20% of the original β-cell number remains).
However, this has not been formally proven. Moreover, the newly developed dual phenotype
hypothesis [summarised in Figure 6] in which the inflammatory process is envisaged to progress by
one of two distinct mechanisms (the CD20Hi and CD20Lo phenotypes described above) leads to a
situation in which T1D occurs when differing proportions of residual insulin-containing vs insulindeficient islets remain. The reasons for this are unknown but one possibility is that T1D is not solely
attributable to the absolute loss of β-cells (which undoubtedly occurs in all patients) but also, as a
contributory factor, to a decline in the insulin secretory capacity of any residual β-cells [Figure 6].
This might be particularly true in patients with a CD20Lo profile of islet inflammation where the
decline in β-cell numbers occurs more slowly and insulin-containing islets are present in greater
proportion (relative to insulin-deficient islets) at diagnosis [Figure 5].
There are rather few data available which allow firm conclusions to be drawn in relation to these
arguments but it has been possible to examine insulin secretory responses in vitro from islets
isolated from at least three patients with T1D. One of these was a young woman who died at the age
of 19 having been originally diagnosed with T1D at age 6 (14). Based on the age profiles of CD20Hi
and Lo patients, it might be speculated that this patient would feature among the CD20Hi group at
the time of diagnosis, but this conclusion is necessarily speculative. Nevertheless, histological
analysis suggested that the majority of islets were insulin deficient at the time of death with only a
small proportion (~15%) retaining insulin immunopositivity. Such islets had reduced insulin and
elevated glucagon contents compared to controls, consistent with on-going, though still incomplete,
β-cell destruction. The islets displayed a strong insulin secretory response to glucose in vitro
suggesting that, at least upon isolation, there was no major secretory defect and that T1D had
resulted from insufficient β-cell mass in this patient.
A second patient (18 years old) who was studied immediately after disease diagnosis (81) also
retained insulin-positive islets which could be harvested following isolation but, in this case, no
insulin-secretory response to glucose (or forskolin) was evident, leading the authors to conclude that
islet function was “disproportionately impaired” (81). The third patient was a girl of 14 who died 8
months after diagnosis of T1D and whose insulin-containing islets were harvested and then studied
after various periods of tissue culture (82). In perifusion studies, these islets initially showed a
modest but much reduced secretory response to glucose when compared with those isolated from a
non-diabetic control subject. If the islets were maintained in culture for a longer period (17 days)
they recovered secretory function and became indistinguishable from controls in terms of the
magnitude of the response to glucose. Clearly, in total, these data are insufficient to allow firm
conclusions to be drawn but it may be significant that an insulin secretory defect was observed in
the islets of two of three T1D patients, suggesting that this could have contributed to the disease
phenotype in these individuals.
Endocrine cell proliferation in inflamed islets
A further area of interest and importance in T1D is the extent to which islet cells might attempt to
regenerate in the face of an autoimmune insult. It is widely understood that β-cell replication occurs
at only a very low rate beyond the immediate post-natal period, in humans (83). Indeed, in adults,
the rate of β-cell proliferation is vanishingly small and there seems little prospect, therefore, that
islet cells could be replaced at a sufficient rate to offset their decline during the autoimmune attack
leading to T1D. Nevertheless, it appears that islet cells are not inert in the face of this assault and
that they can respond by increasing their rate of mitosis. This was shown in both an older patient
(84) and in two studies of pancreas samples harvested post-mortem from patients with recent-onset
T1D, where it was revealed that islet cell proliferation was increased by as much as 10-fold above
that measured in controls (74, 85). More importantly, the increase was most evident in those islets
which were inflamed, implying that a component associated with the inflammatory process was
responsible for mediating the effect. Interestingly, the proliferative response was not unique to βcells but a similar increase was also noted in α-cells, suggesting that the endocrine cells were
responding to a general mitotic stimulus present within the islet milieu. It is reasonable to propose
that this stimulus might have been elaborated from one or more of the immune cell subtypes
present in the inflammatory infiltrate but this factor has not yet been formally identified or
characterised. In support of this conclusion, the rate of endocrine cell proliferation has also been
examined in the inflamed islets of two patients who died without a diagnosis of T1D but who were
immunopositive for multiple islet autoantibodies (86), suggesting that they might have been in a
“pre-diabetic” state. The pancreases of each of these individuals contained islets which displayed
enhanced rates of endocrine cell replication and, again, this correlated with the presence of
inflammation (86).
Clearly, it is of importance to identify the factor(s) responsible for mediating islet cell proliferation
during inflammation. Once identified, this might offer a novel therapeutic option to increase
endogenous β-cell mass in patients with T1D, if the relevant factor(s) could be administered in
combination with approaches that limit the autoimmune attack.
Conclusion
Despite the dearth of human samples in which the process of islet inflammation can be studied,
important advances have been made which have improved our understanding of the underlying
aetiology of human T1D. These provide clear cause for continued optimism that the illness will yield
its secrets and that new and improved therapeutic options will emerge. Arguably, these are most
likely to lead to a future in which T1D is preventable in susceptible individuals prior to disease onset
rather than curable in those who already have the disease. Either way, it is important that further
studies are undertaken to address the remaining areas of uncertainty.
As a final statement we also note that, in most analyses it has been assumed that, once initiated,
T1D progresses in a broadly uniform manner until β-cell destruction reaches a critical point at which
the clinical diagnosis is made. However, we have emphasised that the pathology reveals an
asynchronous progression at the islet level. This then raises the alternative possibility that islet
inflammation might wax and wane over time in a manner which is associated with alternating
periods of relapse and remission as the illness proceeds. Such a mechanism would be consistent with
the situation in various other autoimmune conditions. For the present, this idea remains only an
interesting hypothesis but, if it proves to have validity, then further windows of opportunity might
be identifiable during the different phases, when therapeutic intervention might be at its most
effective.
Acknowledgements
We are pleased to acknowledge financial support from the European Union's Seventh Framework
Programme PEVNET [FP7/2007-2013] under grant agreement number 261441. Additional support
was from a Diabetes Research Wellness Foundation Non-Clinical Research Fellowship and, since
2014, a JDRF Career Development Award (5-CDA-2014-221-A-N) to S.J.R. The research was also
performed with the support of the Network for Pancreatic Organ Donors with Diabetes (nPOD), a
collaborative type 1 diabetes research project sponsored by the Juvenile Diabetes Research
Foundation International (JDRF) and with a JDRF research grant awarded to the nPOD-V consortium.
Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are
listed at www.jdrfnpod.org/our-partners.php.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Atkinson, M. A. (2012) The pathogenesis and natural history of type 1 diabetes, Cold Spring
Harbor perspectives in medicine 2.
Boitard, C. (2012) Pancreatic islet autoimmunity, Presse medicale 41, e636-650.
La Torre, D. (2012) Immunobiology of beta-cell destruction, Advances in experimental
medicine and biology 771, 194-218.
Richardson, S. J., Morgan, N. G., and Foulis, A. K. (2014) Pancreatic pathology in type 1
diabetes mellitus, Endocrine pathology 25, 80-92.
Roep, B. O., and Tree, T. I. (2014) Immune modulation in humans: implications for type 1
diabetes mellitus, Nature reviews. Endocrinology 10, 229-242.
Pugliese, A. (2014) Advances in the etiology and mechanisms of type 1 diabetes, Discovery
medicine 18, 141-150.
Eisenbarth, G. S. (1986) Type I diabetes mellitus. A chronic autoimmune disease, The New
England journal of medicine 314, 1360-1368.
Di Gialleonardo, V., de Vries, E. F., Di Girolamo, M., Quintero, A. M., Dierckx, R. A., and
Signore, A. (2012) Imaging of beta-cell mass and insulitis in insulin-dependent (Type 1)
diabetes mellitus, Endocrine reviews 33, 892-919.
Foulis, A. K., and Stewart, J. A. (1984) The pancreas in recent-onset type 1 (insulindependent) diabetes mellitus: insulin content of islets, insulitis and associated changes in the
exocrine acinar tissue, Diabetologia 26, 456-461.
Gepts, W. (1965) Pathologic anatomy of the pancreas in juvenile diabetes mellitus, Diabetes
14, 619-633.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Kloppel, G., Lohr, M., Habich, K., Oberholzer, M., and Heitz, P. U. (1985) Islet pathology and
the pathogenesis of type 1 and type 2 diabetes mellitus revisited, Survey and synthesis of
pathology research 4, 110-125.
Campbell-Thompson, M., Wasserfall, C., Kaddis, J., Albanese-O'Neill, A., Staeva, T., Nierras,
C., Moraski, J., Rowe, P., Gianani, R., Eisenbarth, G., Crawford, J., Schatz, D., Pugliese, A., and
Atkinson, M. (2012) Network for Pancreatic Organ Donors with Diabetes (nPOD): developing
a tissue biobank for type 1 diabetes, Diabetes/metabolism research and reviews 28, 608-617.
Dotta, F., Censini, S., van Halteren, A. G., Marselli, L., Masini, M., Dionisi, S., Mosca, F., Boggi,
U., Muda, A. O., Del Prato, S., Elliott, J. F., Covacci, A., Rappuoli, R., Roep, B. O., and
Marchetti, P. (2007) Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in
recent-onset type 1 diabetic patients, Proceedings of the National Academy of Sciences of
the United States of America 104, 5115-5120.
Walker, J. N., Johnson, P. R., Shigeto, M., Hughes, S. J., Clark, A., and Rorsman, P. (2011)
Glucose-responsive beta cells in islets isolated from a patient with long-standing type 1
diabetes mellitus, Diabetologia 54, 200-202.
Itoh, N., Hanafusa, T., Miyazaki, A., Miyagawa, J., Yamagata, K., Yamamoto, K., Waguri, M.,
Imagawa, A., Tamura, S., Inada, M., and et al. (1993) Mononuclear cell infiltration and its
relation to the expression of major histocompatibility complex antigens and adhesion
molecules in pancreas biopsy specimens from newly diagnosed insulin-dependent diabetes
mellitus patients, The Journal of clinical investigation 92, 2313-2322.
Krogvold, L., Edwin, B., Buanes, T., Ludvigsson, J., Korsgren, O., Hyoty, H., Frisk, G., Hanssen,
K. F., and Dahl-Jorgensen, K. (2014) Pancreatic biopsy by minimal tail resection in live adult
patients at the onset of type 1 diabetes: experiences from the DiViD study, Diabetologia 57,
841-843.
In't Veld, P. (2011) Insulitis in human type 1 diabetes: The quest for an elusive lesion, Islets 3,
131-138.
In't Veld, P. (2014) Insulitis in human type 1 diabetes: a comparison between patients and
animal models, Seminars in immunopathology 36, 569-579.
Lecompte, P. M. (1958) Insulitis in early juvenile diabetes, A.M.A. archives of pathology 66,
450-457.
MB, S. (1902) Ueber die beziechung der langerhans'schen inseln des pankreas zum diabetes
mellitus, Munch Med Wochenschr 49, 51-54.
M, v. M. (1940) Ueber "Insulitis" bei diabetes, Schweiz Med Wochenschr 21, 554-557.
Kloppel, G., Drenck, C. R., Carstensen, A., and Heitz, P. U. (1984) The B cell mass at the
clinical onset of type I diabetes, Behring Institute Mitteilungen, 42-49.
Campbell-Thompson, M. L., Atkinson, M. A., Butler, A. E., Chapman, N. M., Frisk, G., Gianani,
R., Giepmans, B. N., von Herrath, M. G., Hyoty, H., Kay, T. W., Korsgren, O., Morgan, N. G.,
Powers, A. C., Pugliese, A., Richardson, S. J., Rowe, P. A., Tracy, S., and In't Veld, P. A. (2013)
The diagnosis of insulitis in human type 1 diabetes, Diabetologia 56, 2541-2543.
Dor, Y., and Glaser, B. (2013) beta-cell dedifferentiation and type 2 diabetes, The New
England journal of medicine 368, 572-573.
Talchai, C., Xuan, S., Lin, H. V., Sussel, L., and Accili, D. (2012) Pancreatic beta cell
dedifferentiation as a mechanism of diabetic beta cell failure, Cell 150, 1223-1234.
White, M. G., Marshall, H. L., Rigby, R., Huang, G. C., Amer, A., Booth, T., White, S., and
Shaw, J. A. (2013) Expression of mesenchymal and alpha-cell phenotypic markers in islet
beta-cells in recently diagnosed diabetes, Diabetes care 36, 3818-3820.
Anuradha, R., Saraswati, M., Kumar, K. G., and Rani, S. H. (2014) Apoptosis of Beta Cells in
Diabetes Mellitus, DNA and cell biology.
Quan, W., Jo, E. K., and Lee, M. S. (2013) Role of pancreatic beta-cell death and inflammation
in diabetes, Diabetes, obesity & metabolism 15 Suppl 3, 141-151.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
Santin, I., and Eizirik, D. L. (2013) Candidate genes for type 1 diabetes modulate pancreatic
islet inflammation and beta-cell apoptosis, Diabetes, obesity & metabolism 15 Suppl 3, 7181.
Richardson, S. J., Willcox, A., Bone, A. J., Foulis, A. K., and Morgan, N. G. (2009) Isletassociated macrophages in type 2 diabetes, Diabetologia 52, 1686-1688.
Delaney, C. A., Pavlovic, D., Hoorens, A., Pipeleers, D. G., and Eizirik, D. L. (1997) Cytokines
induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells,
Endocrinology 138, 2610-2614.
Dunger, A., Augstein, P., Schmidt, S., and Fischer, U. (1996) Identification of interleukin 1induced apoptosis in rat islets using in situ specific labelling of fragmented DNA, Journal of
autoimmunity 9, 309-313.
Eizirik, D. L., and Mandrup-Poulsen, T. (2001) A choice of death--the signal-transduction of
immune-mediated beta-cell apoptosis, Diabetologia 44, 2115-2133.
Hadjivassiliou, V., Green, M. H., James, R. F., Swift, S. M., Clayton, H. A., and Green, I. C.
(1998) Insulin secretion, DNA damage, and apoptosis in human and rat islets of Langerhans
following exposure to nitric oxide, peroxynitrite, and cytokines, Nitric oxide : biology and
chemistry / official journal of the Nitric Oxide Society 2, 429-441.
Loweth, A. C., Williams, G. T., James, R. F., Scarpello, J. H., and Morgan, N. G. (1998) Human
islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin1beta and Fas ligation, Diabetes 47, 727-732.
Signore, A., Annovazzi, A., Gradini, R., Liddi, R., and Ruberti, G. (1998) Fas and Fas ligandmediated apoptosis and its role in autoimmune diabetes, Diabetes/metabolism reviews 14,
197-206.
Stassi, G., Todaro, M., Richiusa, P., Giordano, M., Mattina, A., Sbriglia, M. S., Lo Monte, A.,
Buscemi, G., Galluzzo, A., and Giordano, C. (1995) Expression of apoptosis-inducing CD95
(Fas/Apo-1) on human beta-cells sorted by flow-cytometry and cultured in vitro,
Transplantation proceedings 27, 3271-3275.
Yamada, K., Takane-Gyotoku, N., Yuan, X., Ichikawa, F., Inada, C., and Nonaka, K. (1996)
Mouse islet cell lysis mediated by interleukin-1-induced Fas, Diabetologia 39, 1306-1312.
Marhfour, I., Lopez, X. M., Lefkaditis, D., Salmon, I., Allagnat, F., Richardson, S. J., Morgan, N.
G., and Eizirik, D. L. (2012) Expression of endoplasmic reticulum stress markers in the islets
of patients with type 1 diabetes, Diabetologia 55, 2417-2420.
Meier, J. J., Bhushan, A., Butler, A. E., Rizza, R. A., and Butler, P. C. (2005) Sustained beta cell
apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet
regeneration?, Diabetologia 48, 2221-2228.
Graham, K. L., Sutherland, R. M., Mannering, S. I., Zhao, Y., Chee, J., Krishnamurthy, B.,
Thomas, H. E., Lew, A. M., and Kay, T. W. (2012) Pathogenic mechanisms in type 1 diabetes:
the islet is both target and driver of disease, The review of diabetic studies : RDS 9, 148-168.
Hahn, S., Gehri, R., and Erb, P. (1995) Mechanism and biological significance of CD4mediated cytotoxicity, Immunological reviews 146, 57-79.
Kreuwel, H. T., Morgan, D. J., Krahl, T., Ko, A., Sarvetnick, N., and Sherman, L. A. (1999)
Comparing the relative role of perforin/granzyme versus Fas/Fas ligand cytotoxic pathways
in CD8+ T cell-mediated insulin-dependent diabetes mellitus, Journal of immunology 163,
4335-4341.
Thomas, H. E., Trapani, J. A., and Kay, T. W. (2010) The role of perforin and granzymes in
diabetes, Cell death and differentiation 17, 577-585.
Jaberi-Douraki, M., Liu, S. W., Pietropaolo, M., and Khadra, A. (2014) Autoimmune responses
in T1DM: quantitative methods to understand onset, progression, and prevention of disease,
Pediatric diabetes 15, 162-174.
Lernmark, A., and Larsson, H. E. (2013) Immune therapy in type 1 diabetes mellitus, Nature
reviews. Endocrinology 9, 92-103.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
Pietropaolo, M., Towns, R., and Eisenbarth, G. S. (2012) Humoral autoimmunity in type 1
diabetes: prediction, significance, and detection of distinct disease subtypes, Cold Spring
Harbor perspectives in medicine 2.
Christie, M. R., Hollands, J. A., Brown, T. J., Michelsen, B. K., and Delovitch, T. L. (1993)
Detection of pancreatic islet 64,000 M(r) autoantigens in insulin-dependent diabetes distinct
from glutamate decarboxylase, The Journal of clinical investigation 92, 240-248.
Christie, M. R., Brown, T. J., and Cassidy, D. (1992) Binding of antibodies in sera from Type 1
(insulin-dependent) diabetic patients to glutamate decarboxylase from rat tissues. Evidence
for antigenic and non-antigenic forms of the enzyme, Diabetologia 35, 380-384.
Bonifacio, E., Krumsiek, J., Winkler, C., Theis, F. J., and Ziegler, A. G. (2014) A strategy to find
gene combinations that identify children who progress rapidly to type 1 diabetes after islet
autoantibody seroconversion, Acta diabetologica 51, 403-411.
Nokoff, N., and Rewers, M. (2013) Pathogenesis of type 1 diabetes: lessons from natural
history studies of high-risk individuals, Annals of the New York Academy of Sciences 1281, 115.
Bottazzo, G. F., Dean, B. M., McNally, J. M., MacKay, E. H., Swift, P. G., and Gamble, D. R.
(1985) In situ characterization of autoimmune phenomena and expression of HLA molecules
in the pancreas in diabetic insulitis, The New England journal of medicine 313, 353-360.
Willcox, A., Richardson, S. J., Bone, A. J., Foulis, A. K., and Morgan, N. G. (2009) Analysis of
islet inflammation in human type 1 diabetes, Clinical and experimental immunology 155,
173-181.
Rodriguez-Calvo, T., Ekwall, O., Amirian, N., Zapardiel-Gonzalo, J., and von Herrath, M. G.
(2014) Increased Immune Cell Infiltration of the Exocrine Pancreas: A Possible Contribution
to the Pathogenesis of Type 1 Diabetes, Diabetes.
Valle, A., Giamporcaro, G. M., Scavini, M., Stabilini, A., Grogan, P., Bianconi, E., Sebastiani,
G., Masini, M., Maugeri, N., Porretti, L., Bonfanti, R., Meschi, F., De Pellegrin, M., Lesma, A.,
Rossini, S., Piemonti, L., Marchetti, P., Dotta, F., Bosi, E., and Battaglia, M. (2013) Reduction
of circulating neutrophils precedes and accompanies type 1 diabetes, Diabetes 62, 20722077.
In't Veld, P., De Munck, N., Van Belle, K., Buelens, N., Ling, Z., Weets, I., Haentjens, P.,
Pipeleers-Marichal, M., Gorus, F., and Pipeleers, D. (2010) Beta-cell replication is increased
in donor organs from young patients after prolonged life support, Diabetes 59, 1702-1708.
Arif, S., Leete, P., Nguyen, V., Marks, K., Nor, N. M., Estorninho, M., Kronenberg-Versteeg, D.,
Bingley, P. J., Todd, J. A., Guy, C., Dunger, D. B., Powrie, J., Willcox, A., Foulis, A. K.,
Richardson, S. J., de Rinaldis, E., Morgan, N. G., Lorenc, A., and Peakman, M. (2014) Blood
and islet phenotypes indicate immunological heterogeneity in type 1 diabetes, Diabetes.
Pescovitz, M. D., Greenbaum, C. J., Bundy, B., Becker, D. J., Gitelman, S. E., Goland, R.,
Gottlieb, P. A., Marks, J. B., Moran, A., Raskin, P., Rodriguez, H., Schatz, D. A., Wherrett, D.
K., Wilson, D. M., Krischer, J. P., Skyler, J. S., and Type 1 Diabetes TrialNet Anti, C. D. S. G.
(2014) B-lymphocyte depletion with rituximab and beta-cell function: two-year results,
Diabetes care 37, 453-459.
Pescovitz, M. D., Greenbaum, C. J., Krause-Steinrauf, H., Becker, D. J., Gitelman, S. E.,
Goland, R., Gottlieb, P. A., Marks, J. B., McGee, P. F., Moran, A. M., Raskin, P., Rodriguez, H.,
Schatz, D. A., Wherrett, D., Wilson, D. M., Lachin, J. M., Skyler, J. S., and Type 1 Diabetes
TrialNet Anti, C. D. S. G. (2009) Rituximab, B-lymphocyte depletion, and preservation of
beta-cell function, The New England journal of medicine 361, 2143-2152.
Bataille, R., Jego, G., Robillard, N., Barille-Nion, S., Harousseau, J. L., Moreau, P., Amiot, M.,
and Pellat-Deceunynck, C. (2006) The phenotype of normal, reactive and malignant plasma
cells. Identification of "many and multiple myelomas" and of new targets for myeloma
therapy, Haematologica 91, 1234-1240.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
Hinman, R. M., and Cambier, J. C. (2014) Role of B lymphocytes in the pathogenesis of type 1
diabetes, Current diabetes reports 14, 543.
Coppieters, K. T., Dotta, F., Amirian, N., Campbell, P. D., Kay, T. W., Atkinson, M. A., Roep, B.
O., and von Herrath, M. G. (2012) Demonstration of islet-autoreactive CD8 T cells in insulitic
lesions from recent onset and long-term type 1 diabetes patients, The Journal of
experimental medicine 209, 51-60.
Pathiraja, V., Kuehlich, J. P., Campbell, P. D., Krishnamurthy, B., Loudovaris, T., Coates, P. T.,
Brodnicki, T. C., O'Connell, P. J., Kedzierska, K., Rodda, C., Bergman, P., Hill, E., Purcell, A. W.,
Dudek, N. L., Thomas, H. E., Kay, T. W., and Mannering, S. I. (2014) Proinsulin specific, HLADQ8 and HLA-DQ8 transdimer restricted, CD4+ T cells infiltrate the islets in type 1 diabetes,
Diabetes.
Berg, A. K., Korsgren, O., and Frisk, G. (2006) Induction of the chemokine interferon-gammainducible protein-10 in human pancreatic islets during enterovirus infection, Diabetologia
49, 2697-2703.
Cardozo, A. K., Proost, P., Gysemans, C., Chen, M. C., Mathieu, C., and Eizirik, D. L. (2003) IL1beta and IFN-gamma induce the expression of diverse chemokines and IL-15 in human and
rat pancreatic islet cells, and in islets from pre-diabetic NOD mice, Diabetologia 46, 255-266.
Chen, M. C., Proost, P., Gysemans, C., Mathieu, C., and Eizirik, D. L. (2001) Monocyte
chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and
in interleukin-1 beta-exposed human and rat islet cells, Diabetologia 44, 325-332.
Grewal, I. S., Rutledge, B. J., Fiorillo, J. A., Gu, L., Gladue, R. P., Flavell, R. A., and Rollins, B. J.
(1997) Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets
produces monocyte-rich insulitis without diabetes: abrogation by a second transgene
expressing systemic MCP-1, Journal of immunology 159, 401-408.
Roep, B. O., Kleijwegt, F. S., van Halteren, A. G., Bonato, V., Boggi, U., Vendrame, F.,
Marchetti, P., and Dotta, F. (2010) Islet inflammation and CXCL10 in recent-onset type 1
diabetes, Clinical and experimental immunology 159, 338-343.
Foulis, A. K., Farquharson, M. A., and Meager, A. (1987) Immunoreactive alpha-interferon in
insulin-secreting beta cells in type 1 diabetes mellitus, Lancet 2, 1423-1427.
Harrison, L. C., Campbell, I. L., Allison, J., and Miller, J. F. (1989) MHC molecules and beta-cell
destruction. Immune and nonimmune mechanisms, Diabetes 38, 815-818.
Morgan, N. G., and Richardson, S. J. (2014) Enteroviruses as causative agents in type 1
diabetes: loose ends or lost cause?, Trends in endocrinology and metabolism: TEM.
Richardson, S. J., Leete, P., Bone, A. J., Foulis, A. K., and Morgan, N. G. (2013) Expression of
the enteroviral capsid protein VP1 in the islet cells of patients with type 1 diabetes is
associated with induction of protein kinase R and downregulation of Mcl-1, Diabetologia 56,
185-193.
Richardson, S. J., Willcox, A., Bone, A. J., Foulis, A. K., and Morgan, N. G. (2009) The
prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human
type 1 diabetes, Diabetologia 52, 1143-1151.
Willcox, A., Richardson, S. J., Bone, A. J., Foulis, A. K., and Morgan, N. G. (2011)
Immunohistochemical analysis of the relationship between islet cell proliferation and the
production of the enteroviral capsid protein, VP1, in the islets of patients with recent-onset
type 1 diabetes, Diabetologia 54, 2417-2420.
Bollyky, P. L., Bogdani, M., Bollyky, J. B., Hull, R. L., and Wight, T. N. (2012) The role of
hyaluronan and the extracellular matrix in islet inflammation and immune regulation,
Current diabetes reports 12, 471-480.
Otonkoski, T., Banerjee, M., Korsgren, O., Thornell, L. E., and Virtanen, I. (2008) Unique
basement membrane structure of human pancreatic islets: implications for beta-cell growth
and differentiation, Diabetes, obesity & metabolism 10 Suppl 4, 119-127.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
van Deijnen, J. H., Hulstaert, C. E., Wolters, G. H., and van Schilfgaarde, R. (1992) Significance
of the peri-insular extracellular matrix for islet isolation from the pancreas of rat, dog, pig,
and man, Cell and tissue research 267, 139-146.
Wang, R. N., and Rosenberg, L. (1999) Maintenance of beta-cell function and survival
following islet isolation requires re-establishment of the islet-matrix relationship, The
Journal of endocrinology 163, 181-190.
Korpos, E., Kadri, N., Kappelhoff, R., Wegner, J., Overall, C. M., Weber, E., Holmberg, D.,
Cardell, S., and Sorokin, L. (2013) The peri-islet basement membrane, a barrier to infiltrating
leukocytes in type 1 diabetes in mouse and human, Diabetes 62, 531-542.
Irving-Rodgers, H. F., Choong, F. J., Hummitzsch, K., Parish, C. R., Rodgers, R. J., and
Simeonovic, C. J. (2014) Pancreatic islet basement membrane loss and remodeling after
mouse islet isolation and transplantation: impact for allograft rejection, Cell transplantation
23, 59-72.
Conget, I., Fernandez-Alvarez, J., Ferrer, J., Sarri, Y., Novials, A., Somoza, N., Pujol-Borrell, R.,
Casamitjana, R., and Gomis, R. (1993) Human pancreatic islet function at the onset of type 1
(insulin-dependent) diabetes mellitus, Diabetologia 36, 358-360.
Marchetti, P., Dotta, F., Ling, Z., Lupi, R., Del Guerra, S., Santangelo, C., Realacci, M., Marselli,
L., Di Mario, U., and Navalesi, R. (2000) Function of pancreatic islets isolated from a type 1
diabetic patient, Diabetes care 23, 701-703.
Butler, P. C., Meier, J. J., Butler, A. E., and Bhushan, A. (2007) The replication of beta cells in
normal physiology, in disease and for therapy, Nature clinical practice. Endocrinology &
metabolism 3, 758-768.
Meier, J. J., Lin, J. C., Butler, A. E., Galasso, R., Martinez, D. S., and Butler, P. C. (2006) Direct
evidence of attempted beta cell regeneration in an 89-year-old patient with recent-onset
type 1 diabetes, Diabetologia 49, 1838-1844.
Willcox, A., Richardson, S. J., Bone, A. J., Foulis, A. K., and Morgan, N. G. (2010) Evidence of
increased islet cell proliferation in patients with recent-onset type 1 diabetes, Diabetologia
53, 2020-2028.
In't Veld, P., Lievens, D., De Grijse, J., Ling, Z., Van der Auwera, B., Pipeleers-Marichal, M.,
Gorus, F., and Pipeleers, D. (2007) Screening for insulitis in adult autoantibody-positive
organ donors, Diabetes 56, 2400-2404.