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*
Ontario Cancer Institute/Amgen Institute, Toronto, Ontario, Canada; and
Department of Pathology, University of Zürich, Zürich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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are involved
in this process, we generated nonobese diabetic (NOD) mice that are
deficient for TNF receptor 1 (TNFR1 or TNFRp55). Insulitis developed in
these mice similarly to that in normal control NOD mice, but
progression to diabetes was completely abrogated. Since this was
probably due to the complex immunomodulatory effects of TNF and
lymphotoxin signaled via TNFR1 on lymphohemopoietic cells, adoptive
transfer experiments with spleen cells from diabetic NOD mice were
conducted. It was found that the absence of TNFR1 in recipients delayed
diabetes induced by normal control and precluded diabetes induced by
perforin-deficient spleen cells. In a CD8+ T cell-mediated
model of diabetes, however, diabetes induced by adoptive transfer of
TCR transgenic lymphocytic choriomeningitis virus glycoprotein-specific
CD8+ T cells was not delayed by the absence of TNFR1 in
recipient mice. Together with the described expression patterns of
perforin and TNF in the mononuclear islet infiltrates of NOD mice,
these results indicate that two diabetogenic effector mechanisms are
delivered by distinct cell populations: CD8+ T cells lyse
ß cells via perforin-dependent cytotoxicity, whereas CD4+
T cells, macrophages, and dendritic cells contribute to diabetes
development via TNFR1-dependent ß cell
toxicity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is generally believed that at least two distinct effector mechanisms account for ß cell elimination in diabetes. The first results from the specific recognition of ß cells by cytotoxic perforin-secreting CD8+ T cells and requires direct cell-to-cell contact. This mechanism is supported by the ability of CD8+ T cell clones to induce diabetes (6, 7), by the isolation of ß cell-toxic CD8+ T cell lines and clones from NOD mice (8), by the expression of perforin in infiltrated islets from NOD mice (9), and by the significantly delayed and reduced incidence of diabetes in perforin-deficient NOD mice (10). Also, lymphocytic choriomeningitis virus (LCMV)-induced diabetes in a transgenic diabetes model system was completely prevented by disruption of the perforin gene (11). Fas-dependent T cell-mediated cytotoxicity may be implicated in ß cell elimination based on the observation that Fas expression on ß cells was up-regulated by IL-1ß incubation in vitro (12, 13), and that diabetes upon transfer of islet-reactive cloned CD8+ T cells was dependent on Fas (14).
A second proposed effector mechanism for ß cell destruction
postulates that islet-infiltrating leukocytes release soluble factors
that are selectively toxic for ß cells in a paracrine manner not
requiring direct cell-to-cell contact. This concept was suggested
mainly by the observation that CD4+ T cell clones promote
diabetes in the absence of CD8+ T cells, yet MHC class II
molecules are not expressed on ß cells (15, 16, 17). In vitro studies
with ß cells from rats have implicated several candidate cytokines:
IL-1, IFN-
, TNF-, IL-6, and nitric oxide were all reported to be
toxic to ß cells, and synergistic effects among several of these
mediators were described previously (18, 19, 20).
TNF and lymphotoxin (LT) are inflammatory mediators with
pleiotropic effects such as activation, proliferation, differentiation,
and death induction on many different cell types. They are mainly
produced by macrophages and T cells during a variety of autoimmune and
infectious diseases (reviewed in 21). Due to the ability of TNF to
induce cell death in many tumor cells and because of its expression in
islet-infiltrating leukocytes from NOD mice (22, 23, 24), significant
efforts to experimentally define a possible diabetogenic role of TNF in
the elimination of ß cells were undertaken; treatment of NOD mice
with neutralizing anti-TNF Abs inhibited the development of
insulitis and diabetes (25), and systemic treatment with TNF had
paradoxical effects depending on the age of the NOD mice at the start
of treatment (25, 26, 27). When TNF treatment was started at birth or at 2
wk of age, diabetes onset was accelerated. If TNF treatment was started
only at 4 wk, however, disease was delayed. Local expression of TNF as
a transgene in ß cells induced insulitis without progression to
diabetes (28) and tolerance toward a transgenic Ag expressed in ß
cells (29). These studies have shown that TNF has complex effects on
the development of the autoimmune response that are highly dependent on
the time point and the localization of its action, but failed to shed
light on its involvement in ß cell elimination at the level of
effector functions.
The activities of TNF and LT are transduced by two distinct cell
surface receptors, TNFR1 (p55) and TNFR2 (p75). Both receptors are
ubiquitously expressed and can transmit activatory signals, such as
NF-
B activation, and promote cell death in many tumor cell lines.
TNFR1 has been shown to mediate the symptoms of toxic shock and to
provide macrophage-mediated resistance against Listeria
monocytogenes (30, 31). TNFR2 seems to play a minor role in these
situations (32), indicating that TNFR1 may be more important in
mediating TNF-induced signals, especially cell death, than TNFR2.
To clarify the role of TNF in autoimmune diabetes and in ß cell elimination, we have generated TNFR1-deficient NOD mice and studied their susceptibility to diabetes. Our results indicate that beside the previously identified perforin-dependent cytotoxicity, a second pathway requiring the presence of TNFR1 is involved in ß cell depletion during diabetes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD mice were initially obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were bred under specific pathogen-free conditions at the Ontario Cancer Institute (Toronto, Canada). The colony was routinely surveyed by necropsy of sentinel animals. Mice were free of ecto- and endoparasites and pathogenic bacteria and had no Abs against a comprehensive panel of common mouse viral pathogens. Control (318) and perforin-deficient mice (PKO-318) transgenic for an H-2 Db restricted LCMV-GP-specific TCR have been described previously and were backcrossed for at least six generations to the C57BL/6 strain (11, 33). Both strains express the transgenic TCR on 50–70% of CD8+ T cells. Control (RIP-GP) and TNFR1-deficient (TNFR10/0 RIP-GP) transgenic mice expressing LCMV-glycoprotein under the control of the rat insulin promoter in pancreatic ß cells have been described previously as well (34, 35). TNFR1-deficient RIP-GP mice were provided by P. Ohashi.
Breeding of TNFR1-deficient mice
The TNFR1 gene was originally disrupted by homologous recombination in 129/Ola-derived AB1 ES cells (30), and heterozygous mice were backcrossed for several generations with C57BL/6 mice. These mice were backcrossed for eight generations with NOD mice to generate TNFR1-deficient NOD mice. At the second and third backcross generation, mice were tested for homozygosity of H-2NOD (H-2 Kd) by cytofluorometry with H-2 Kb-specific Abs.
PCR analysis of Idd6-associated polymorphic chromosomal markers
The diabetes susceptibility gene Idd6 on chromosome 6 was tested with PCR for the polymorphic markers D6 Mit14 (36) (primers 5'-ATG CAG AAA CAT GAG TGG GG-3' and 5'-CAC AAG GCC TGA TGA CCT CT-3') and D6 Mit15 (37) (primers 5'-CAC TGA CCC TAG CAC AGC AG-3' and 5'-TCC TGG CTT CCA CAG GTA CT-3').
Assessment of insulitis
Sections from the head of the pancreas were prepared on five different levels, each 100 µm apart, and stained with hemotoxylin and eosin. Twenty to forty randomly chosen islets per mouse were semiquantitatively classified according to the severity of insulitis.
Measurement of blood glucose
The glucose concentration in blood obtained from a tail vein was measured using Haemo-Glucotest strips (Boehringer Mannheim, Mannheim, Germany). Diabetes onset was assumed when two consecutive blood glucose readings exceeded 17 mM.
Cytofluorometric analysis of pancreas-infiltrating lymphocytes
A sample of pancreas-infiltrating lymphocytes was obtained by forcing pancreata from 13-wk-old control and TNFR1-deficient NOD mice through a fine metal screen. After washing once, lymphocytes were isolated by centrifugation over a Ficoll gradient (Pharmacia, Uppsala, Sweden) and washed thoroughly. The lymphocytes were stained with phycoerythrin-conjugated anti-CD8 and FITC-conjugated anti-CD4 Abs (both from PharMingen, San Diego, CA). Cytofluorometric analysis of surface marker expression on a FACScan flow cytometer with CellQuest software (Becton Dickinson, Mountain View, CA) included gating on live lymphocytes by forward and side scatter.
Induction of diabetes by injection of cyclophosphamide
Six milligrams of cyclophosphamide (Sigma, St. Louis, MO) was injected i.p. into 10- to 12-wk-old mice on day 0. If the first injection failed to produce diabetes, 6 mg of cyclophosphamide was again injected on day 14.
Diabetes induction by adoptive transfer of spleen cells from diabetic NOD mice
Nine- to 11-wk-old male recipient NOD mice were sublethally irradiated with a 137Cs source (800 rad) 24 h before transfer. Donor spleen cells from two freshly diagnosed diabetic control or perforin-deficient mice (20–40 wk old) were pooled, and 2 x 107 pooled spleen cells were i.v. injected into irradiated recipients.
Recombinant vaccinia virus
Recombinant vaccinia virus expressing the full length of the LCMV glycoprotein precursor molecule (vacc-GP) was a gift from Dr. D. H. L. Bishop, Institute of Virology, Oxford University (Oxford, U.K.) (38, 39). Recombinant vaccinia virus stocks were grown and quantitated on BSC 40 cells.
Diabetes induction by adoptive transfer of transgenic CD8+ T cells
Perforin-competent or perforin-deficient mice, both transgenic for a LCMV-GP-specific TCR, were i.v. infected with 2 x 106 pfu vacc-GP virus to activate LCMV-GP-reactive transgenic T cells. After 6 days, 5 x 106 spleen cells were adoptively transferred into nonirradiated transgenic mice expressing LCMV-GP in ß cells of the pancreas. To keep the adoptively transferred T cells in an activated state, the recipients were concomitantly infected with 2 x 106 pfu vacc-LCMV-GP i.v. Diabetes was monitored by measuring blood glucose levels.
Immunohistochemistry
Pancreata were immersed in HBSS and snap-frozen in liquid nitrogen. Cryostat sections (5 µm) of tissue were cut and fixed in cold acetone. Sections were incubated with rat anti-mouse mAbs YTS191.1 (anti-CD4), YTS169.4.2 (anti-CD8) (40), or M1-42 (anti-MHC class I). Since immunohistochemical staining with the mAb Jo-2 (41) could not be established, affinity-purified rabbit anti-Fas Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were used to stain for Fas expression. Alkaline phosphatase-conjugated goat anti-rat Ig Abs or goat anti-rabbit Ig Abs followed by alkaline phosphatase-labeled donkey anti-goat Ig Abs (Tago, Burlingame, CA) were used as secondary reagents. The substrate for the red color reaction was naphtol AS-BI phosphate/New Fuchsin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of genetic loci that are associated with the
susceptibility of NOD mice to diabetes have been identified (36, 37).
Idd6 is one of these susceptibility loci and is located on the same
chromosome (chromosome 6) as the TNFR1 gene (42). It was therefore
possible that Idd6 had cosegregated with the mutated TNFR1 gene during
backcrossing and that the presence of an Idd6 gene derived from the
SV129/Ola mouse strain interfered with the analysis of the role of
TNFR1 in diabetes. To test this possibility we tested two polymorphic
markers on chromosome 6 that have been shown to be linked to Idd6. PCR
analysis of D6 Mit14 and D6 Mit15 (36, 37) showed that the
129/Ola-derived forms of these markers were absent in heterozygous and
TNFR1 0/0 mice, indicating that in the course of backcrossing
TNFR1-deficient mice with the NOD strain, crossing-over between the
Idd6 and the TNFR1 locus had taken place (Fig. 1
).
Since we did not observe linkage between Idd6 and TNFR1, the
chromosomal region of Idd6 was derived from the NOD strain in all
animals tested. Thus, the possibility that Idd6 interfered with the
assessment of diabetes in TNFR1-deficient NOD mice could be excluded.
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Injection of newborn NOD mice for 3 wk with a neutralizing
anti-TNF Ab inhibits the development of insulitis in NOD mice (25).
To test whether this proinflammatory activity of TNF is mediated via
TNFR1, 20–40 islets from each wild-type, heterozygous, or
TNFR1-deficient female NOD mouse were semiquantitatively assessed for
the severity of insulitis. As shown in Fig. 2A, marked insulitis developed in all
three groups of mice at the age of 13 wk. As expected from the variable
onset of spontaneous diabetes in NOD mice, the severity of mononuclear
islet infiltration in individual mice from the same group displayed
considerable variation. Therefore, no significant differences in the
percentages of islets with strong, moderate, or weak insulitis between
TNFR1-deficient and heterozygous or wild-type NOD mice were found.
Thus, breakdown of tolerance toward ß cells and extravasation of
leukocytes into the islets were not measurably affected by the absence
of TNFR1. To test whether the absence of TNFR1 affected the relative
proportions of infiltrating T cell subsets, pancreatic lymphocytes were
isolated and analyzed by flow cytometry. The pancreatic infiltrate in
13-wk-old control mice consisted mainly of T cells with a predominance
of the CD4+ subset (Fig. 2B), as has been
described previously (43). In TNFR1-deficient mice, similar proportions
of CD4+ and CD8+ T cell subsets were present in
pancreatic infiltrates, showing that the absence of TNFR1 did not
measurably alter the composition of pancreas-infiltrating lymphocytes.
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To evaluate the role of TNRF1 in the spontaneous development of
diabetes, heterozygous and TNFR1-deficient female mice were observed
over a period of 55 wk (Fig. 3). In heterozygous
mice, diabetes developed in 61.5% of all animals, with the onset
mostly between 20–25 wk (median, 24 wk). This was comparable to the
incidence in nonlittermate TNFR1+/+ NOD mice in our
facility (data not shown). In TNFR1-deficient mice, in contrast,
diabetes did not develop in any of the 15 mice observed.
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In the NOD mouse, but not in other nondiabetes-prone mouse
strains, injection of cyclophosphamide induces diabetes in male and
female mice (44). It has been shown that this acceleration of diabetes
onset is a T cell-dependent process. Although the underlying pathogenic
mechanisms remain to be defined, the effector mechanisms involved in
ß cell destruction during spontaneous and cyclophosphamide-induced
diabetes may be similar. Cyclophosphamide injection on days 0 and 14
led to diabetes in all normal control mice by day 30, but in only one
of five TNFR1-deficient mice (Fig. 4). In contrast
to the development of spontaneous diabetes, only one in seven
heterozygous mice became diabetic. Because TNFR1+/0 mice
express TNFR1 at reduced levels (30), these data show that expression
of TNFR1 at wild-type levels is required in cyclophosphamide-induced
diabetes. The decreased expression level caused by inactivation of a
single allele or a complete ablation of TNFR1 expression similarly led
to a marked decrease in sensitivity to cyclophosphamide-induced
diabetes.
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TNFR1-deficient NOD mice may fail to develop diabetes because of
the absence of TNFR1 on immune cells, resulting in the lack of
coactivation and/or differences in autoantigen presentation.
Alternatively, the lack of TNFR1 on islet cells may render them
resistant to TNF-dependent effector functions. To differentiate between
these two possibilities, diabetes development was studied in
sublethally irradiated normal or TNFR1-deficient NOD recipient mice
after adoptive transfer of spleen cells from diabetic
TNFR1-expressing NOD donor mice. This allowed assessment of the
role of TNFR1 in a situation where the lymphocytes express TNFR1 but
pancreatic ß cells are deficient of TNFR1. Since
perforin-deficient NOD mice progress to diabetes only with delayed
onset and reduced incidence (10), it was interesting to test whether
diabetes caused by perforin-deficient effector cells is dependent on
TNFR1 expression of ß cells by also using diabetic perforin-deficient
NOD mice as donors. Transfer of spleen cells from diabetic normal
control NOD mice into TNFR1-expressing recipients induced diabetes in
all five recipient mice 3 wk after transfer (Table I). Transfer of these cells into TNFR1-deficient
recipients, however, significantly delayed diabetes, and hyperglycemia
developed only at 11 wk after transfer in all five recipients. An even
more pronounced delay of diabetes was observed in TNFR1-expressing
recipients after adoptive transfer of spleen cells from diabetic
perforin-deficient mice. In this case, diabetes developed in only four
of five recipients over the observation period of 11 wk. Finally,
transfer of perforin-deficient donor cells into TNFR1-deficient
recipients failed to induce diabetes.
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) with a similar degree of lymphocytic
infiltration into the islets, but the inflammatory infiltrate in
TNFR1-deficient mice was often localized peripheral to the endocrine
islet tissue, and little tissue damage was detectable, whereas in
normal control recipients the inflammatory infiltrate often invaded the
islet tissue, and some endocrine cells displayed signs of cell death.
Immunohistochemical staining of serial sections with CD4, CD8, MHC
class I, and Fas-specific Abs revealed that the islet infiltrates of
normal control and TNFR1-deficient NOD recipients contained
CD4+ and CD8+ at comparable frequencies (Fig. 5). CD4+ outnumbered CD8+ T cells in both types
of recipients. Also, MHC class I Ag up-regulation on the endocrine
cells of infiltrated islets developed equally in both types of
recipients. Fas expression was found mainly on infiltrating
lymphocytes, with no specific staining on endocrine islet cells from
normal and TNFR1-deficient mice. Thus, the absence of TNFR1 in the
recipient mice did not reduce the capacity of autoreactive donor cells
to extravasate and infiltrate into the islets, did not result in
changes in T cell subsets present in this infiltrate, and did not
prevent MHC class I up-regulation and Fas expression in the infiltrated
islets.
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Adoptive transfer of diabetogenic CD8+ transgenic T cells
We have shown previously that diabetes induction by adoptive
transfer of LCMV-GP-specific TCR-transgenic CD8+ T cells
into recipients expressing a transgenic LCMV-GP in pancreatic ß cells
(RIP-GP mice) is mediated by perforin-dependent cytotoxicity (11, 34).
This experimental system was used to test whether TNFR1 expression in
the recipient is required for diabetes induction by adoptively
transferred CD8+ T cells. Spleen cells (5 x
106) containing activated TCR-transgenic CD8+ T
cells from control and perforin-deficient mice were adoptively
transferred into control or TNFR1-deficient RIP-GP transgenic recipient
mice. To further stimulate the transgenic T cells, the recipients were
i.v. infected with 2 x 106 pfu LCMV
glycoprotein-recombinant vaccinia virus on the day of transfer. It was
found that diabetes was induced similarly in control and
TNFR1-deficient recipients on day 9 after adoptive transfer (Table II). As described earlier, spleen cells from
perforin-deficient TCR transgenic mice did not induce diabetes (11).
Thus, the absence of functional TNFR1 in the recipients did not delay
diabetes induction by adoptively transferred CD8+ T cells,
paralleling the previous finding that diabetes induction after LCMV
infection in RIP-GP mice was independent of TNFR1 (35).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown before that TNF can strongly modulate insulitis in
NOD mice. Treatment with a TNF-neutralizing Ab (25) prevents insulitis,
and transgenic expression of TNF or LT in ß cells (28, 45, 46)
induces insulitis. Also, transgenic expression of TNF in ß cells
prevented the development of islet-specific T cells in NOD mice (47)
and reduced a potential autoimmune T cell response against a transgenic
Ag coexpressed in the pancreas (29). Thus, expression of TNF in the
islet is probably involved in chemotactically attracting islet-invading
leukocytes and may modulate the process of tolerance induction against
ß cell Ags. Since neither spontaneous insulitis nor the very severe
islet infiltration following transfer of spleen cells from diabetic
donor mice was detectably reduced in TNFR1-deficient mice, development
and chemotaxis of autoreactive lymphocytes are functional in the
absence of TNFR1, possibly because activating signals transmitted by
TNFR2 can compensate for the lack of TNFR1. The development of normal
insulitis with similar proportions of infiltrating CD4 and
CD8+ T cells in TNFR1-deficient NOD mice also shows that
the absence of TNFR1 on vascular endothelial cells in the pancreas did
not inhibit extravasation by, e.g., preventing the up-regulation of
adhesion molecules. This confirms the reported normal extravasation of
lymphocytes in the absence of TNFR1 upon LCMV infection of transgenic
mice expressing LCMV-GP in pancreatic ß cells, upon LCMV inoculation
into the foot (31), and upon Leishmania major infection
(48). Also, infiltration of neutrophils and macrophages after
Listeria monocytogenes infection (49) was not affected in
TNFR1-deficient mice.
Induction of diabetes with cyclophosphamide confirmed the crucial role of TNFR1; the incidence of cyclophosphamide-induced diabetes in TNFR1-deficient mice was markedly reduced compared with the incidence in normal control mice. In contrast to the findings in spontaneous diabetes, TNFR1+/0 mice showed a similarly drastic reduction of diabetes incidence as TNFR10/0 mice. It has been shown previously that TNFR1+/0 mice express TNFR1 at about half the density of normal control mice (30). Thus, our results indicate that cyclophosphamide-induced diabetes proceeds with full efficiency only if TNFR1 is expressed at high density resulting from the presence of two functional TNFR1 alleles. The different thresholds of TNFR1 expression for cyclophosphamide-induced vs spontaneous diabetes may reflect the more acute character of the former vs the chronic progressive character of the latter autoimmune process.
TNF has a variety of effects on the development of immune responses. It has been shown to promote cell proliferation (50), to be required for the formation of B cell follicles and germinal centers (51), and to play a role in homeostatic death induction of peripheral T cells (52, 53, 54, 55). Thus, the failure of TNFR1-deficient mice to spontaneously progress from insulitis to diabetes may not necessarily indicate the resistance of ß cells to TNF-mediated effector mechanisms, but could alternatively reflect a modulation of the autoimmune inflammatory process due to the absence of TNFR1 on lymphohemopoietic cells. This view is supported by the induction of diabetes in TNFR1-deficient recipient mice upon adoptive transfer of spleen cells from diabetic TNFR1-expressing NOD mice and by the susceptibility, albeit low, of TNFR1-deficient mice to cyclophosphamide-induced diabetes.
Although insulitis appeared normal in TNFR1-deficient NOD mice, more
subtle differences in the affinity, specificity, kinetics, and type
(e.g., Th1 vs Th2) of the autoimmune response may have developed. Any
of these parameters could have been affected by the lack of TNFR1
without necessarily causing conspicuous differences in insulitis. To
circumvent the complications caused by the complex immunomodulatory
effect of TNF on immune cells, diabetes development after adoptive
transfer of spleen cells from diabetic NOD mice was investigated. This
allowed us to study the effect of TNFR1-expressing lymphocytes, which
are not expected to develop these potential subtle differences, on a
TNFR1-deficient pancreas. By studying diabetes in TNFR1-deficient
recipients upon transfer of TNFR1-expressing donor cells, the
pleiotropic effects of TNF and LT on lymphocytes and macrophages
were separated from the consequences of TNFR1 expression on islet cells
at the level of effector mechanisms. In addition, transfer of spleen
cells from perforin-deficient diabetic mice allowed us to address
whether diabetes induced by perforin-deficient effector cells was
dependent on the expression of TNFR1 on ß cells. These experiments
confirmed the previously identified major role of perforin-dependent
cytotoxicity (10) and, more importantly, revealed the involvement of an
additional diabetogenic pathway that is dependent on the expression of
TNFR1 by ß cells. Since TNFR1 is the main receptor that mediates the
cytotoxicity of TNF and LT, this could suggest that TNF and/or LT
secreted by infiltrating T cells and macrophages is directly toxic to
ß cells. However, transgenic mice expressing TNF in pancreatic ß
cells developed insulitis, but did not progress to diabetes (28),
indicating that either the constant exposure of ß cells to TNF in
that model led to desensitization of ß cells or that TNF is only
toxic to ß cells in synergy with other cytokines. The latter idea is
supported by in vitro experiments with islet cells showing potent ß
cell-toxic synergies of TNF- and LT with IL-1 and IFN- (19, 20, 56). Thus, it is likely that the TNFR1-dependent effector pathway
of ß cell elimination proposed here is activated by a combination of
TNF/LT with one or several other synergistic cytokines that are
selectively toxic to ß cells in a paracrine fashion not requiring
direct cell-to-cell contact. This idea also accommodates the finding
that CD4+ T cells can induce diabetes despite the apparent
absence of MHC class II expression on ß cells (reviewed in 16).
The data from the adoptive transfer experiment allow the following additional conclusions. First, diabetes did not develop upon transfer of perforin-deficient NOD spleen cells into TNFR1-deficient recipients. This argues against the involvement of additional, perforin- and TNFR1-independent pathways, such as, e.g., Fas-mediated ß cell death, in this system.
Secondly, each of the two pathways was able to induce delayed diabetes in the absence of the other, confirming the spontaneous development of diabetes in a low percentage of perforin-deficient NOD mice (10). The data argue for a higher efficiency of perforin-dependent compared with TNFR1-dependent ß cell depletion, since transferring perforin-deficient spleen cells into normal control recipients resulted in a longer delay of diabetes onset than transferring normal control donor cells into TNFR1-deficient recipients.
Thirdly, the finding that in both of the latter experiments diabetes was significantly delayed compared with the transfer of perforin-expressing donor cells into TNFR1-expressing recipients shows that both mechanisms together operate more efficiently than either one alone.
It has been proposed that ß cells up-regulate Fas during diabetes
development and die by Fas-mediated apoptosis (14). In such a scenario,
activation of TNFR1 on ß cells could be necessary to induce Fas
expression in vivo, despite the observation that IL-1, but not TNF,
induced the up-regulation of surface Fas on isolated ß cells in vitro
(12, 13). However, immunohistochemical staining with Fas-specific Abs
failed to demonstrate the up-regulation of Fas expression on ß cells
in TNFR-expressing mice suffering from severe insulitis after adoptive
transfer of diabetogenic spleen cells (Fig. 5
). Up-regulation of Fas on
ß cells in vivo has also been questioned by the absence of Fas
expression on ß cells in mice expressing a transgenic MHC class
II-restricted diabetogenic TCR (57). It is, of course, possible that
Fas expression on ß cells is below the detection limit of
immunohistochemistry or takes place only in those ß cells that are in
direct contact with diabetogenic T cells. Nevertheless, these
observations render it unlikely that the resistance of TNFR1-deficient
recipients to diabetes induced by the transfer of perforin-deficient
spleen cells is explained by a failure of TNFR1-deficient ß cells to
up-regulate Fas.
The experiments involving the adoptive transfer of TCR transgenic T
cells showed that CD8+ T cells induce diabetes
independently of the expression of TNFR1 by the recipient (Table II).
While it is established that perforin is expressed mainly by
CD8+ T cells in islets of NOD mice (9), it is less obvious
to identify the effector cell population responsible for the
TNFR1-dependent diabetogenic pathway. Both TNF and LT are ligands
for TNFR1. Unfortunately, no data are available about the expression of
LT in islets of diabetic or prediabetic mice. The expression pattern
of TNF- in infiltrated islets is controversial. Earlier data
obtained by in situ hybridization on sorted pancreatic lymphocytes
favored a preferential expression of TNF- by CD4+ T
cells in infiltrated islets of NOD mice (23). This was challenged,
however, by a recent immunohistochemical study of pancreatic islets
from NOD mice that found a correlation of TNF- staining with
macrophage and dendritic cell markers and no correlation with CD4 or
CD8 (24). Both expression patterns would suggest that the two
diabetogenic effector mechanisms identified by our experiments would be
delivered by distinct subsets of effector cells: CD8+ T
cells lyse ß cells via contact-dependent perforin-mediated
cytotoxicity, whereas CD4+ T cells and/or macrophages
secrete proinflammatory cytokines that contribute to diabetes
development by TNFR1-dependent ß cell toxicity.
In the islet infiltrate from prediabetic NOD mice, CD4+ outnumber CD8+ T cells (5). Unfortunately, only limited information about the presence of T cell subtypes present in islet infiltrates from human diabetes patients is available. The data available from a young patient who died only 1 mo after diabetes onset (58) and from a number of patients who received pancreas grafts from HLA-identical siblings (59) all point to a markedly higher proportion of CD8+ vs CD4+ T cells in human islet infiltrates. Thus, CD8+ T cell-mediated cytotoxicity may play a more prominent role in human diabetes than in the NOD mouse.
In conclusion, the analysis of TNFR1-deficient mice, especially by using these mice as recipients in adoptive transfer studies, has yielded evidence that autoimmune diabetes is caused by two independent synergistic mechanisms. According to this model, ß cells are eliminated by a cooperation between CD8+ T cell-mediated perforin-dependent cytotoxicity and a TNFR1-dependent mechanism that may be mainly delivered by CD4+ T cells, macrophages, and dendritic cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Kägi, Ontario Cancer Institute, Room 8-622, 610 University Ave., Toronto, Ontario, Canada M5G2M9. E-mail address:
3 Abbreviations used in this paper: NOD, nonobese diabetic; LCMV, lymphocytic choriomeningitis virus; LT, lymphotoxin ; LCMV-GP, glycoprotein of lymphocytic choriomeningitis virus; vacc-GP, vaccinia virus expressing the full length of the lymphocytic choriomeningitis virus glycoprotein precursor molecule; pfu, plaque-forming unit; RIP, rat insulin promoter.
Received for publication November 30, 1998. Accepted for publication January 28, 1999.
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  C. Pfleger, H. B. Mortensen, L. Hansen, C. Herder, B. O. Roep, H. Hoey, H.-J. Aanstoot, M. Kocova, N. C. Schloot, and on behalf of the Hvidore Study Group on Childhood Association of IL-1ra and Adiponectin With C-Peptide and Remission in Patients With Type 1 Diabetes Diabetes, April 1, 2008; 57(4): 929 - 937. [Abstract] [Full Text] [PDF]
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 S. Kim, I. Millet, H. S. Kim, J. Y. Kim, M. S. Han, M.-K. Lee, K.-W. Kim, R. S. Sherwin, M. Karin, and M.-S. Lee NF-{kappa}B prevents beta cell death and autoimmune diabetes in NOD mice PNAS, February 6, 2007; 104(6): 1913 - 1918. [Abstract] [Full Text] [PDF]
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S. S. Smith, T. Patterson, and M. E. Pauza Transgenic Ly-49A Inhibits Antigen-Driven T Cell Activation and Delays Diabetes J. Immunol., April 1, 2005; 174(7): 3897 - 3905. [Abstract] [Full Text] [PDF]
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 S. T. Ildstad, P. M. Chilton, H. Xu, M. A. Domenick, and M. B. Ray Preconditioning of NOD mice with anti-CD8 mAb and costimulatory blockade enhances chimerism and tolerance and prevents diabetes, while depletion of {alpha}{beta}-TCR+ and CD4+ cells negates the effect Blood, March 15, 2005; 105(6): 2577 - 2584. [Abstract] [Full Text] [PDF]
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P. M. Chilton, F. Rezzoug, I. Fugier-Vivier, L. A. Weeter, H. Xu, Y. Huang, M. B. Ray, and S. T. Ildstad Flt3-Ligand Treatment Prevents Diabetes in NOD Mice Diabetes, August 1, 2004; 53(8): 1995 - 2002. [Abstract] [Full Text] [PDF]
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L. Xu, H. Yoon, M. Q. Zhao, J. Liu, C. V. Ramana, and R. I. Enelow Cutting Edge: Pulmonary Immunopathology Mediated by Antigen-Specific Expression of TNF-{alpha} by Antiviral CD8+ T Cells J. Immunol., July 15, 2004; 173(2): 721 - 725. [Abstract] [Full Text] [PDF]
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I. Chang, N. Cho, S. Kim, J. Y. Kim, E. Kim, J.-E. Woo, J. H. Nam, S. J. Kim, and M.-S. Lee Role of Calcium in Pancreatic Islet Cell Death by IFN-{gamma}/TNF-{alpha} J. Immunol., June 1, 2004; 172(11): 7008 - 7014. [Abstract] [Full Text] [PDF]
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D. W. Draper, V. G. Harris, C. A. Culver, and S. M. Laster Calcium and Its Role in the Nuclear Translocation and Activation of Cytosolic Phospholipase A2 in Cells Rendered Sensitive to TNF-Induced Apoptosis by Cycloheximide J. Immunol., February 15, 2004; 172(4): 2416 - 2423. [Abstract] [Full Text] [PDF]
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