By
From the * Ontario Cancer Institute, Toronto M5G2M9, Canada; Institute of Experimental
Immunology, Department of Pathology, University of Zürich, CH-8091 Zürich, Switzerland; and
the § Department of Pathology, University of Zürich, CH-8091 Zürich, Switzerland
To investigate the role of T cell-mediated, perforin-dependent cytotoxicity in autoimmune diabetes, perforin-deficient mice were backcrossed with the nonobese diabetes mouse strain. It
was found that the incidence of spontaneous diabetes over a 1 yr period was reduced from 77%
in perforin +/+ control to 16% in perforin-deficient mice. Also, the disease onset was markedly delayed (median onset of 39.5 versus 19 wk) in the latter. Insulitis with infiltration of
CD4+ and CD8+ T cells occurred similarly in both groups of animals. Lower incidence and
delayed disease onset were also evident in perforin-deficient mice when diabetes was induced
by cyclophosphamide injection. Thus, perforin-dependent cytotoxicity is a crucial effector
mechanism for cell elimination by cytotoxic T cells in autoimmune diabetes. However, in
the absence of perforin chronic inflammation of the islets can lead to diabetogenic
cell loss by
less efficient secondary effector mechanisms.
Insulin-dependent diabetes mellitus (IDDM)1 is an autoimmune disease characterized by the loss of insulin-producing pancreatic In the past it has been attempted to address this last point
by defining the role of the CD4+ (helper) T cells versus the
CD8+ (cytotoxic) T cell subset. In these studies the nonobese diabetic (NOD) mouse strain has proved useful because it models the spontaneous initiation and the chronic
progressive course of the disease and the polygenic inheritance of susceptibility genes quite well (1). Several studies
have shown that CD4+ and CD8+ primary T cells are required to adoptively transfer diabetes (2, 3). However, cloned
islet cell-reactive NOD CD4+ T cells were able to induce
diabetes in NOD-SCID mice in the absence of CD8+ T
cells (4, 5). At the time, these findings were taken as evidence that both T cell subsets are required for the transfer of diabetes with polyclonal primary T cells but that cloned
CD4+ T cells are able to induce diabetes independently of
CD8+ T cells, given high numbers and specificity.
On the other hand, a cytofluorometric study of islet-infiltrating leukocytes has shown that CD8+ T cells infiltrated into the pancreas of young, prediabetic NOD mice
earlier than CD4+ T and B cells (6). Similarly, in a pancreas
from a human patient who had died only a month after diagnosis of diabetes the islet-infiltrating T cells consisted
mainly of the CD8+ subset (7). Several recent studies further supported the crucial role of CD8+ T cells in diabetes
of NOD mice: We report here that diabetes developed only with greatly
reduced incidence and delayed onset in perforin-deficient
NOD mice. This shows that perforin-dependent cytotoxicity is the main effector mechanism accounting for Breeding of Perforin-deficient NOD Mice.
Perforin-deficient mice
of the C57BL/6 strain have been previously described (14).
These mice were backcrossed for seven generations with NOD
mice (provided by Hans Acha-Orbea, Institute of Biochemistry,
University of Lausanne, Epalinges, Switzerland). At each backcross generation, heterozygous mice were identified by PCR
analysis for further breeding. In addition, the H-2g7 complex,
which contains the I-Ag7 locus and is strongly associated with diabetes susceptibility in the NOD strain, was enriched by screening
at the second backcross generation for the absence of the Kb allele
by flow cytofluorometry of blood cells with the antibody B8-24-3.
At the third generation, the second strongest susceptibility locus
Idd3 was enriched by PCR screening as previously described for
the microsatellite marker D3Nds1 (21). In brief, the microsatellite marker was amplified by PCR from genomic DNA with primers
5 Genotyping of the Perforin Allele.
The perforin genotype was
determined with PCR and two different primer pairs on DNA
prepared from tail biopsies. The first pair (5 Cytofluorometry.
T lymphocyte marker expression was analyzed by incubating spleen cell suspensions with PE-conjugated
CD8-specific and FITC-conjugated CD4-specific antibodies (PharMingen, San Diego, CA). After washing, the cell suspensions were
analyzed on a FACScan® flow cytometer (Becton Dickinson,
Mountain View, CA) using logarithmic scales. Viable lymphocytes were gated for by a combination of forward light scatter and
90° side scatter.
Measurement of Blood Glucose.
The glucose concentration in
blood obtained from a tail vein was measured using Haemo-Glucotest strips (Boehringer Mannheim, Mannheim, Germany).
Induction of Diabetes by Injection of Cyclophosphamide.
6 mg of
cyclophosphamide (Sigma Chemical Co., St. Louis, MO) was injected i.p. into 10-12-wk-old mice on day 0. If the first injection
failed to produce diabetes, 6 mg of cyclophosphamide was again
injected on day 14.
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 monoclonal antibodies YTS191.1 (anti-CD4) and
YTS169.4.2 (anti-CD8) (22). Alkaline phosphatase-labeled goat anti-
rat immunoglobulin antibodies, followed by alkaline phosphatase-
labeled donkey anti-goat immunoglobulin antibodies (Tago, Inc.,
Burlingame, CA) were used as secondary reagents. The substrate for
the red color reaction was naphtol AS-BI phosphate/New Fuchsin.
cells. In its early and clinically silent
phase T cells and other inflammatory cells infiltrate into the
islets causing a progressive loss of
cells. When a majority
of
cells has disappeared, the lack of insulin secretion leads
to a failure of blood glucose homeostasis and diabetes.
While there is a consensus that IDDM is caused by autoreactive T cells, many other aspects of the disease are still
poorly understood. These include the breakdown of tolerance against islet cell antigens, the failure of mechanisms
controlling self-reactive T cells, genetic and environmental
susceptibility factors, and the molecular effector mechanisms that are responsible for the elimination of
cells.
2-microglobulin-negative and hence CD8+
T cell-deficient NOD mice developed neither insulitis nor
diabetes (8). Also, depletion of CD8+ T cells by antibody treatment at 2-5-wk after birth prevents insulitis development and also abrogates the ability of CD4+ T cells to
induce insulitis (12). Finally, CD8+ T cell clones from NOD
mice that were generated by restimulation with transgenic
islet cells expressing the costimulatory molecule B7.1 were
able to transfer diabetes to irradiated NOD and NOD-SCID mice (13). These findings clearly demonstrated that CD8+
T cells are not only responsible for the lysis of
cells in the
late effector phase, but that they also may have a role in the
early induction phase by affecting the properties of autoreactive CD4+ T cells. Perforin-deficient mice lack a major
pathway of T cell-mediated cytotoxicity and NK cell-
mediated cytotoxicity (14). Since perforin-deficient
mice have no defect in activation and proliferation of T
cells and generate normal B cell responses (14), they are well suited to directly address the role of cytotoxicity in
vivo. We have previously crossed perforin-deficient mice
with transgenic mice expressing glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) in the pancreas.
Infection with LCMV triggers an acute virus-specific immune response which induces insulitis and diabetes in perforin-expressing transgenic mice by day 10 after infection
(19). In contrast, LCMV-GP transgenic perforin-deficient mice did not develop diabetes, although they developed
marked insulitis (20). These findings indicated that perforin-dependent cytotoxicity is not required for the initiation of insulitis but is crucial for the destruction of
cells in
the later effector phase. However, there was the possibility
that these findings were specific to this model system, since
LCMV induces a very strong cytotoxic immune response
and, unlike human diabetes, the diabetes develops very
acutely without chronic long-term insulitis. It was therefore of interest to test the role of perforin-dependent cytotoxicity in the NOD mouse model, where the spontaneous
onset and the chronic inflammation of the pancreatic islets
are more similar to the human disease. In addition, we surmised that the slower course of diabetes in the NOD
mouse may reveal additional perforin-independent effector
mechanisms, which may be masked during the very acute
progression of diabetes in the LCMV-GP transgenic model.
cell
loss in the NOD mouse but also indicates that one or several perforin-independent mechanisms, possibly involving
Fas, can cause diabetes with reduced efficiency.
-GGA TCT GGC ACC TCC AGG G-3
and 5
-TAT GTT
GCC TTG GCA AAT AGA TG-3
. The reaction product was
resolved on a 5% Nusieve agarose gel (fragment size: NOD>
C57BL/6; FMC BioProducts, Rockland, ME). Littermate controls were used in all experiments.
-TTT TTG AGA
CCC TGT AGA CCC A-3
, 5
-GCA TCG CCT TCT ATC
GCC TTC T-3
) yields a band of 665 bp for the mutated and is
negative for the wild-type allele. The second pair (5
-CCG GTC
CTG AAC TCC TGG CCA A-3
, 5
-CCC CTG CAC ACA
TTA CTG GAA G-3
) yields a 300-bp fragment for the wild-type and a 1,300-bp fragment for the mutated allele.
Measurement of the Volume Density of Islets in the Pancreas. Islets are more frequent in the head of the pancreas than in the tail. Thus, the head of the pancreas was fixed in formaldehyde and cut in sections at eight consecutive levels each 100 µm apart. The volume density was determined according to the Delesse principle by calculating the ratio of the sum of the islet areas to the sum of the section areas (23). Islet and section areas were determined by computer-assisted morphometry on a microscope equipped with a video camera. The results are given as the mean of the volume density from several individual mice together with the SEM.
Statistical Analysis. The time course of spontaneous diabetes onset in normal control, heterozygous, and perforin-deficient NOD mice was analyzed using Kaplan-Meier curves. The curves were tested for significance by log-rank tests. All statistical procedures were performed with the lifetest program of the SAS statistical software package (SAS Institute, Inc., Cary, NC).
We have previously shown that T cells from perforin-deficient C57BL/6 mice have no defect in maturation, activation, or proliferation (14). To exclude the possibility that backcrossing with the NOD strain revealed a defect in T lymphocyte maturation, we checked for the presence of CD4+ and CD8+ T cells in the spleens of 8-wk-old NOD mice. It was found that CD4- and CD8-expressing T lymphocytes were present at comparable percentages in normal control, heterozygous, and perforin-deficient NOD mice, indicating that the lack of perforin did not affect T lymphocyte development in the NOD strain (data not shown).
Similar Development of Insulitis in Control and Perforin-deficient NOD Mice.A hallmark of the NOD mouse model system for IDDM is the progressive infiltration of mononuclear cells into the islets. Inflammatory islet-infiltrating mononuclear cells start to appear at the age of 5 wk in most NOD mice. Histologic analysis in 8-wk-old heterozygous and perforin-deficient mice revealed varying degrees of insulitis but mostly periinsulitis in both types of mice with little islet cell damage detectable in heterozygous and perforin-deficient mice (see Fig. 2 A). At 55 wk of age, islets of perforin-expressing NOD mice which had not succumbed to diabetes were strongly infiltrated by mononuclear cells and often displayed little or no endocrine tissue. Since diabetes only develops when >90% of islet tissue is lost, the mice were still normoglycemic. In nondiabetic perforin-deficient mice severe insulitis had developed as well, but more frequently than in control NOD mice the infiltrates were associated with patches of endocrine tissue. Sections that were stained by immunohistochemistry for the lymphocyte surface markers CD4 and CD8 revealed that the mononuclear infiltrate in both groups of mice contained similar proportions of CD4+ and CD8+ T cells, with CD4+ outnumbering the CD8+ cells in both groups (see Fig. 2 A).
To quantitate the development of insulitis, 20-40 islets from individual 9-12-wk-old female NOD mice were assessed for the severity of insulitis. We found considerable variability in the degree of insulitis in individual mice with no significant difference between normal control, heterozygous, and perforin-deficient mice (Fig. 1 A). Thus, perforin-dependent cytotoxicity is not required for the breakdown of self tolerance nor ignorance in NOD mice, which causes the infiltration of T cells into the islets.
Lack of Perforin Leads to Reduced Incidence and Delayed Onset of Diabetes.
The role of perforin-dependent cytotoxicity in the diabetes disease process was evaluated by observing the incidence of spontaneous diabetes in female mice over a period of 55 wk (Fig. 1 B). In normal control NOD mice diabetes progressively developed between 15 and 30 wk (median: 19 wk) of age, and after that the incidence plateaued at 77% (n = 13). Heterozygous mice displayed a slightly delayed pattern of disease onset (median: 27 wk) with an incidence of diabetes at the end of the observation period of 67% (n = 27), possibly indicating a weak gene dosage effect (P = 4.75%). In contrast, in perforin-deficient mice, disease incidence was reduced to 16% (n = 21, P = 0.01%) and diabetes occurred only between 35 and 41 wk (median: 39.5 wk) of age. In all diabetic perforin-deficient mice, blood sugar rose to values comparable to values of diabetic control mice (28-44 mM) and did not spontaneously revert to lower values. Diabetes incidence in male perforin-expressing control mice was low (<20%). No diabetes was observed in perforin-deficient male NOD mice (data not shown).
To test whether the diabetes observed in perforin-deficient mice was insulin-dependent, spontaneously diabetic
NOD mice were injected with 1 IU of insulin (Fig. 1 C).
In perforin-expressing as well as perforin-deficient NOD
mice insulin administration drastically lowered blood sugar
levels 15 min after s.c. injection and normoglycemia was
reached within 60 min after injection. Thus, diabetes in
perforin-deficient mice is caused by a lack of insulin, either
due to the loss of cells or an inability of
cells to secrete
sufficient amounts of insulin.
Histological analysis of pancreatic sections from diabetic animals
revealed that in contrast to diabetic perforin-competent NOD mice, in which cells were not detected, hematoxylin and eosin-stained sections from diabetic perforin-deficient NOD mice showed endocrine tissue which contained
insulin-expressing
cells (Fig. 2 B). To investigate the
question whether these
cells represent a low or a high
percentage of the
cells present in healthy control mice,
i.e., whether perforin-deficient mice were able to eliminate
cells, we compared the amount of endocrine tissue between young NOD mice and diabetic perforin-deficient mice. Representative sections were prepared from the head
of the pancreas, where islets are more numerous than in the
tail, and the volume density of endocrine islet tissue was
calculated. The volume density of young 7-wk-old NOD
mice was 1.18 ± 0.10% (n = 2). In contrast, perforin-deficient diabetic mice had a drastically reduced volume density of 0.010 ± 0.002% (n = 3). Thus, diabetes in perforin-deficient mice was caused by a loss of
cells, indicating that alternative mechanism can cause
cell damage in the
absence of perforin-dependent cytotoxicity. This notion
was also supported by the observation of occasional cells in
islets from diabetic perforin-deficient mice with a prominent eosinophilic cytoplasm and condensed nuclei. The
prominent cytoplasms indicated that these cells were
cells
whereas eosinophilic staining and condensed nuclei are histological signs of cell death (Fig. 2 B).
Injection of cyclophosphamide induces diabetes in NOD mice (24). The underlying mechanism of this effect is not completely clear, but it has been found that a single injection of cyclophosphamide induces a temporary reduction of CD4+ and CD8+ T lymphocytes (25). After recovery of the T cell compartment diabetes develops in a high percentage of young NOD mice of both sexes, but not in other nondiabetes-prone strains (1). Diabetes induced by cyclophosphamide is dependent on T cells (26, 27) and is probably caused by a yet poorly understood deregulation of the T cell compartment.
Cyclophosphamide was injected into 8-12-wk-old control or perforin-deficient NOD mice, with the first injection on day 0 and the second on day 14. Some of the normal control and heterozygous mice developed diabetes on
days 12-14 before the second injection, but most became
diabetic only after the second injection of cyclophosphamide between days 25 and 30, with incidences of 80% for
normal control and 90% for heterozygous mice at 50 d after
the first injection (Fig. 3 A). However, in perforin-deficient mice diabetes occurred only between days 32 and 38 and the incidence was reduced to 18%. Whereas blood glucose values in all diabetic perforin-expressing mice quickly
rose to values of 44 mM, often followed by death from ketoacidosis, perforin-deficient mice displayed only temporary hyperglycemia with intermediate blood glucose values
of 17 or 28 mM and eventually returned to normoglycemia
(Fig. 3 B). Histological analysis of pancreas sections from
diabetic perforin-expressing mice showed pronounced insulitis and complete elimination of islet cells. In contrast, in
diabetic perforin-deficient mice secretory islet cells rich in
cytoplasm were still present despite a marked mononuclear
infiltrate. Antiinsulin immunohistochemical staining confirmed the absence of cells from pancreata of diabetic
perforin-expressing mice whereas diabetic perforin-deficient mice retained some insulin-containing
cells (Fig. 2
C). Thus the reduced incidence and later onset of diabetes
in perforin-deficient NOD mice in the presence of marked
insulitis confirm the finding with spontaneous diabetes and
indicate that perforin plays a crucial role in the development of diabetes.
The results of this study show that inactivation of perforin in NOD mice results in delayed onset and reduced incidence of diabetes. Perforin-dependent cytotoxicity is generally exerted by CD8+ T and NK cells (14, 18). In pancreatic islets of NOD mice, only a few NK cells have been found; perforin expression is thus confined to infiltrating CD8+ T cells (28). That perforin-dependent cytotoxicity mediated by CD8+ T cells is crucial in the late effector phase of diabetes in NOD mice is supported by the following evidence. First, the incidence of spontaneous and cyclophosphamide-induced diabetes is markedly reduced in perforin-deficient NOD mice. Second, the low percentage of perforin-deficient mice that developed diabetes showed a delayed disease process with significantly later onset. Third, in cyclophosphamide-induced diabetes, the blood sugar levels in perforin-deficient mice only temporarily reached diabetic levels. Finally, perforin-deficient NOD mice developed infiltration of CD4+ and CD8+ T cells into the islets comparable to those in control mice. This last result indicates that perforin-dependent cytotoxicity is not involved in the breakdown of tolerance and/or ignorance towards islet antigens in the CD4+ or in the CD8+ T cell subset which results in insulitis in the early stages of the disease. Therefore, the reduced incidence of diabetes in perforin-deficient mice is not explained by a correspondingly reduced presence of inflammatory cells in the islets, but rather reflects a defect at the level of the diabetogenic effector mechanism.
Backcrossing perforin-deficient mice with the NOD strain and screening for the disrupted, C57BL/6-derived perforin allele results in coselection of C57BL/6-derived chromosomal regions linked to the perforin gene on chromosome 10 (29). These regions may carry a diabetes susceptibility gene in the NOD mouse. Theoretically, the reduced incidence of diabetes in perforin-deficient mice may be explained by the absence of a diabetes susceptibility gene linked to the perforin locus. However, since none of the known 13 susceptibility markers has been mapped to chromosome 10 (30) and since in a backcross analysis no association with diabetes was found for a marker on chromosome 10 (21), this possibility is unlikely. A weak and only barely significant (P = 4.75%) gene dosage effect between perforin +/+ and +/0 mice was observed in diabetes incidence. During the functional analysis of perforin-deficient mice, gene dosage effects were observed in other experiments as well, e.g., in the cytotoxic activity of peritoneal exudate lymphocytes against allogeneic fibroblast target cells and in the control of injected syngeneic fibrosarcoma tumor cells (14). This, together with the low abundance of perforin mRNA in vivo (31), indicates that the amount of perforin in CTLs may be a limiting factor for certain effector functions.
Recently, absence of insulitis has been reported in 2-microglobulin-deficient NOD mice (9) and in NOD
mice that were treated during an age window of 2-5 wk
after birth with CD8+ T cell-depleting antibodies (12).
Transfer experiments indicated that CD8+ T cells are required during that period to induce the ability to infiltrate
the pancreatic islets in CD4+ T cells. It has been suggested
that
cell antigens released by CD8+ T cell-mediated lysis
are required to trigger a CD4+ T cell response (32). However, in perforin-deficient mice CD4+ T cells infiltrate the
pancreas to a similar degree as in control mice. Thus induction of insulitis by CD4+ T cells does not require the lysis
of
cells by perforin-dependent cytotoxicity. This also indicates that perforin-dependent cytotoxicity is not involved
in the early initiation phase but rather in the later effector
phase, accounting for the lysis of
cells. Taken together,
the current evidence favors a model where disease progresses in three phases. The first phase, which is required for the
development of islet-reactive CD4+ T cells, is dependent
on CD8+ T cells but does not require perforin. In the second phase, CD4+ T cell-dependent insulitis develops. In
the third phase, diabetes is induced as CD8+ T cells lyse
cells via perforin-dependent cytotoxicity.
The observation that cell loss and diabetes develop in
perforin-deficient NOD mice argues for the existence of
one or several perforin-independent effector mechanisms.
However, since the increase of blood sugar in cyclophosphamide-induced diabetes was only temporary and since
spontaneous diabetes developed only with delayed and reduced incidence in the absence of perforin, these alternative mechanisms appear to be of lower efficiency. They may
act more slowly and therefore are able to induce diabetes only
in chronic situations, in which diabetes develops from insulitis over the course of several weeks. We recently reported
the complete absence of diabetes in perforin-deficient
transgenic mice expressing LCMV-GP in
cells of the
pancreas (20) whereas diabetes develops in control mice 10 d
after LCMV infection. This suggests that in a more acute
situation, perforin-independent mechanisms are not able to
induce diabetes, as observed in the LCMV-GP transgenic model, or induce only temporary diabetes, as in the cyclophosphamide-induced model. However, the more chronic
inflammatory process which occurs spontaneously in the islets of NOD mice may allow sufficient time for alternative
mechanisms with low efficiency to cause diabetogenic
cell damage.
We can only speculate on the molecular nature of the
mechanisms causing diabetes in perforin-deficient mice. A
possible secondary mechanism for elimination of cells is
the elimination of
cells via the perforin-independent Fas
pathway of T cell-mediated cytotoxicity, which would require the expression of Fas by
cells. Fas expression associated with chronic inflammation has been reported in a
number of other glandular tissues including the salivary
gland and the prostate (33). Islets that are free of inflammatory infiltrates do not express Fas (33, 34); however, incubation with IL-1
induced Fas expression on cultured human
cells (34). Moreover, a recent publication has shown
that
cells express Fas after transfer of a
cell-specific
CD8+ T cell clone (35). From the failure of Fas-deficient
NOD-lpr mice to develop diabetes, and the accelerated diabetes in transgenic mice expressing Fas ligand on
cells,
this group postulated that Fas-mediated death of
cells
may be the main pathogenic mechanism in autoimmune
diabetes. However, since the lpr mutation leads to multiple
manifestations in the immune system including lymphadenopathy, accumulation of CD4
, CD8
, B220+ T cells,
and constitutive upregulation of Fas ligand on lymphocytes, the absence of diabetes in NOD-lpr mice could be
explained by these factors and not by the lack of Fas expression on
cells. Nevertheless, it is conceivable that
cell elimination in perforin-deficient NOD mice is caused
by upregulation of the Fas molecule on
cells in response
to inflammatory cytokines produced by the infiltrate. Cognate interaction with Fas ligand-expressing
cell-specific CTLs could then induce
cell death. Noncognate interaction with any other Fas ligand-expressing cell population
in the islets also is conceivable, as it has been suggested for
the elimination of thyrocytes in Hashimoto's thyroiditis
(36).
Alternatively, soluble factors secreted by T cells and
other infiltrating leukocytes could account for cell lysis.
It has been shown in vitro that IL-1 induces the formation
of nitric oxide selectively in
cells but not in
cells (37,
38). Thus the production of IL-1 by monocytes and macrophages recruited into the islets by T cell-derived chemoattractants may account for
cell elimination in perforin-
deficient mice. The islet toxicity of IL-1 may be potentiated further by IFN-
and TNF-
, which synergistically
inhibit glucose-induced insulin release and cause islet cell
disintegration in vitro (39, 40). Neutralization of TNF-
precludes the development of islet-reactive T cells in NOD
mice, indicating that TNF-
has a critical role in the early development of the islet-specific autoimmune response
(41). On the other hand, IFN-
was shown to be essential
for upregulation of class I and class II MHC molecules in
the islets of LCMV-GP transgenic mice. Absence of IFN-
completely prevented insulitis and diabetes in this model
system for diabetes (42). However, in IFN-
-deficient
NOD mice absence of IFN-
had less dramatic consequences and merely led to reduced incidence and delayed
onset of diabetes, but had no consequence on insulitis (43).
These findings show that TNF-
and IFN-
seem to be
involved in the modulation of the autoreactive T cell response and it remains to be seen whether these molecules
also mediate
cell elimination.
In two systems, CD4+ T cells were found to induce diabetes in the absence of CD8+ T cells. Cloned islet-reactive
CD4+ T cells were able to transfer diabetes to NOD-SCID
recipients (4). Also, transgenic mice expressing a TCR
from the same CD4+ T cell clone developed insulitis and
diabetes even when they were rendered incapable of rearranging the endogenous TCR- locus and therefore lacked
CD8+ T cells (5). In both of these systems, the requirement
for CD8+ T cells may be overcome by the high number of
autoreactive T cells. Similar effector mechanisms may underlie both the induction of
cell loss in the absence of
perforin and diabetes induced by cloned or TCR transgenic CD4+ T cells.
In conclusion, we have confirmed the important role of
perforin-dependent cytotoxicity in the destruction of cells leading to diabetes. However, our findings also show
that this role is not exclusive and that perforin-independent
mechanisms acting in situations of chronic inflammation
can cause diabetes with a slower time course and lower incidence.
Address correspondence to David Kägi, c/o Professor T.W. Mak, Ontario Cancer Institute, RM 8-622, 610 University Avenue, Toronto, Ontario M5G2M9, Canada. Phone: 416-204-5310; FAX: 416-204-5300; E-mail: dkagi{at}amgen.com
Received for publication 11 March 1997 and in revised form 7 July 1997.
1 Abbreviations used in this paper: GP, glycoprotein; IDDM, insulin-dependent diabetes mellitus; LCMV, lymphocytic choriomeningitis virus; NOD, nonobese diabetic.We thank Hans Acha-Orbea for helpful discussions and providing NOD mice, Kajsa Karlsson and Jolanda Bretscher for excellent technical assistance with genotyping mutant mice, Christine Quarrington and Rudolf Jörg for maintaining the colony of specific pathogen-free NOD mice, James Ho for immunohistological analysis of insulin expression, and Lisa Martin for help with the statistical analysis. We appreciate the scientific editorial assistance of Mary Saunders.
This work was supported by the Swiss National Science Foundation.
1. | Makino, S., K. Kunimoto, Y. Muraoka, Y. Mizushima, K. Katagiri, and Y. Tochino. 1980. Breeding of a non-obese diabetic strain of mice. Exp. Anim. (Tokyo). 29: 1-13 . |
2. | Bendelac, A., C. Carnaud, C. Boitard, and J.F. Bach. 1987. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates: requirement for both L3T4+ and Lyt-2+ T cells. J. Exp. Med. 166: 823-832 [Abstract]. |
3. |
Miller, B.J.,
M.C. Appel,
J.J. O'Neil, and
L.S. Wicker.
1988.
Both the Lyt-2+ and L3T4+ T cell subsets are required for
the transfer of diabetes in nonobese diabetic mice.
J. Immunol.
140:
52-58
|
4. | Bradley, B.J., K. Haskins, F.G. La, Rosa, and K.J. Lafferty. 1992. CD8 T cells are not required for islet destruction induced by a CD4-positive islet-specific T cell clone. Diabetes. 41: 1603-1608 [Abstract]. |
5. | Katz, J.D., and C. Benoist. 1995. T helper cell subsets in insulin-dependent diabetes. Science (Wash. DC). 268: 1185-1188 [Medline]. |
6. | Jarpe, A.J., M.R. Hickman, J.T. Anderson, W.E. Winter, and A.B. Peck. 1991. Flow cytometric enumeration of mononuclear cell populations infiltrating the islets of Langerhans in prediabetic NOD mice: development of a model of autoimmune insulitis for type I diabetes. Reg. Immunol. 3: 305-317 . |
7. | Bottazzo, G.F., B.M. Dean, J.M. McNally, E.H. MacKay, P.G.F. Swift, and D.R. Gamble. 1985. In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N. Engl. J. Med. 313: 353-360 [Abstract]. |
8. | Katz, J., C. Benoist, and D. Mathis. 1993. Major histocompatibility complex class I molecules are required for the development of insulitis in non-obese diabetic mice. Eur. J. Immunol. 23: 3358-3360 [Medline]. |
9. |
Wicker, L.S.,
E.H. Leiter,
J.A. Todd,
R.J. Renjilian,
E. Peterson,
P.A. Fischer,
P.L. Podolin,
M. Zijlstra,
R. Jaenisch, and
L.B. Peterson.
1994.
![]() |
10. |
Sumida, T.,
M. Furukawa,
A. Sakamoto,
T. Namekawa,
T. Maeda,
M. Zijlstra,
I. Iwamoto,
T. Koike,
S. Yoshida,
H. Tomioka, and
M. Taniguchi.
1994.
Prevention of insulitis
and diabetes in ![]() |
11. | Serreze, D.V., E. Leiter, J. Christianson, D. Greiner, and D.C. Roopenian. 1994. Major histocompatibility complex class I-deficient NOD-B2mnull mice are diabetes and insulitis resistant. Diabetes. 43: 505-509 [Abstract]. |
12. | Wang, B., A. Gonzalez, C. Benoist, and D. Mathis. 1996. The role of CD8+ T cells in the initiation of insulin-dependent diabetes mellitus. Eur. J. Immunol. 26: 1762-1769 [Medline]. |
13. | Wong, F.S., I. Visintin, L. Wen, R.A. Flavell, and C.A. Janeway. 1996. CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells. J. Exp. Med. 183: 67-76 [Abstract]. |
14. | Kägi, D., B. Ledermann, K. Bürki, P. Seiler, B. Odermatt, K.J. Olsen, E. Podack, R.M. Zinkernagel, and H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature (Lond.). 369: 31-37 [Medline]. |
15. | Kägi, D., F. Vignaux, B. Ledermann, K. Bürki, V. Depraetere, S. Nagata, H. Hengartner, and P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science (Wash. DC). 265: 528-530 [Medline]. |
16. | Kojima, H., N. Shinohara, S. Hanaoka, Y. Someya-Shirota, Y. Takagaki, H. Ohno, T. Saito, T. Katayama, H. Yagita, K. Okumura, et al . 1994. Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. Immunity. 1: 357-364 [Medline]. |
17. |
Lowin, B.,
F. Beermann,
A. Schmidt, and
J. Tschopp.
1994.
A null mutation in the perforin gene impairs cytolytic T lymphocyte- and natural killer cell-mediated cytotoxicity.
Proc.
Natl. Acad. Sci. USA.
91:
11571-11575
|
18. |
Walsh, C.M.,
M. Matloubian,
C.-C. Liu,
R. Ueda,
C.G. Kurahara,
J.L. Christensen,
M.T.F. Huang,
J.D.-E. Young,
R. Ahmed, and
W.R. Clark.
1994.
Immune function in
mice lacking the perforin gene.
Proc. Natl. Acad. Sci. USA.
91:
10854-10858
|
19. | Ohashi, P.S., S. Oehen, K. Bürki, H.P. Pircher, C.T. Ohashi, B. Odermatt, B. Malissen, R. Zinkernagel, and H. Hengartner. 1991. Ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice. Cell. 65: 305-317 [Medline]. |
20. | Kägi, D., B. Odermatt, P.S. Ohashi, R.M. Zinkernagel, and H. Hengartner. 1996. Development of insulitis without diabetes in transgenic mice lacking perforin-dependent cytotoxicity. J. Exp. Med. 183: 2143-2152 [Abstract]. |
21. | Todd, J.A., T.J. Aitman, R.J. Cornall, S. Ghosh, J.R. Hall, C.M. Hearne, A.M. Knight, J.M. Love, M.A. McAleer, and J.B. Prins. 1991. Genetic analysis of autoimmune type 1 diabetes mellitus in mice. Nature (Lond.). 351: 542-547 [Medline]. |
22. | Cobbold, S.P., A. Jayasuriya, A. Nash, T.D. Prospero, and H. Waldmann. 1984. Therapy with monoclonal antibodies by elimination of T cell subsets in vivo. Nature (Lond.). 312: 548-551 [Medline]. |
23. | Weibel, E.R. 1973. Stereological techniques for electron microscopy. In Principles and Techniques of Electron Microscopy. Biological Applications. Vol.3. M.A. Hayat, editor. Van Nostrand Reinhold Company, New York. 237-296. |
24. | Harada, M., and S. Makino. 1984. Promotion of spontaneous diabetes in non-obese diabetic-prone mice by cyclophosphamide. Diabetologia. 37: 604-606 . |
25. | Zhang, Z.L., H.M. Georgiou, and T.E. Mandel. 1993. The effect of cyclophosphamide treatment on lymphocyte subsets in the nonobese diabetic mouse: a comparison of various lymphoid organs. Autoimmunity. 15: 1-10 [Medline]. |
26. |
Charlton, B., and
T.E. Mandel.
1988.
Progression from insulitis to ![]() |
27. |
Charlton, B.,
A. Bacelj, and
T.E. Mandel.
1988.
Administration of silica particles or anti-Lyt2 antibody prevents ![]() |
28. |
Young, L.H.,
L.B. Peterson,
L.S. Wicker,
P.M. Persechini, and
J.D.-E. Young.
1989.
In vivo expression of perforin by
CD8+ lymphocytes in autoimmune disease. Studies on spontaneous and adoptively transferred diabetes in nonobese diabetic mice.
J. Immunol.
143:
3994-3999
|
29. | Trapani, J.A., B.Y. Kwon, C.A. Kozak, C. Chintamaneni, J.D.-E. Young, and B. Dupont. 1989. Genomic organization of the mouse pore-forming protein (perforin) gene and localization to chromosome 10: similarities to and differences from C9. J. Exp. Med. 171: 545-557 [Abstract]. |
30. | Wicker, L.S., J.A. Todd, and L.B. Peterson. 1995. Genetic control of autoimmune diabetes in the NOD mouse. Annu. Rev. Immunol. 13: 179-200 [Medline]. |
31. | Müller, C., D. Kägi, T. Aebischer, B. Odermatt, W. Held, E.R. Podack, R.M. Zinkernagel, and H. Hengartner. 1989. Detection of perforin and granzyme A mRNAs in infiltrating cells during LCMV infection of mice. Eur. J. Immunol. 19: 1253-1259 [Medline]. |
32. | Shehadeh, N.N., and K.J. Lafferty. 1993. The role of T-cells in the development of autoimmune diabetes. Diabetes Rev. 1: 141-151 . |
33. | Leithäuser, F., J. Dhein, G. Mechtersheimer, K. Koretz, S. Brüderlein, C. Henne, A. Schmidt, K.-M. Debatin, P.H. Krammer, and P. Möller. 1993. Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab. Invest. 69: 415-429 [Medline]. |
34. |
Stassi, G.,
M. Todaro,
P. Richiusa,
M. Giordano,
A. Mattina,
M.S. Sbriglia,
A. Lo,
Monte,
G. Buscemi,
A. Galluzo, and
C. Giordano.
1995.
Expression of apoptosis-inducing CD95
(Fas/Apo-1) on human ![]() |
35. | Chervonsky, A.V., Y. Wang, S. Wong, I. Visintin, R. Flavell, C.A. Janeway, and L.A. Matis. 1997. The role of Fas in autoimmune diabetes. Cell. 89: 17-24 [Medline]. |
36. |
Giordano, C.,
G. Stassi,
R. De Maria,
M. Todaro,
P. Richiusa,
G. Papoff,
G. Ruberti,
M. Bagnasco,
R. Testi, and
A. Galluzzo.
1997.
Potential involvement of Fas and its ligand in
the pathogenesis of Hashimoto's thyroiditis.
Science (Wash.
DC).
275:
960-963
|
37. | Bendtzen, K., T. Mandrup-Poulsen, J. Nerup, J.H. Nielsen, C.A. Dinarello, and M. Svenson. 1986. Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans. Science (Wash. DC). 232: 1545-1547 [Medline]. |
38. |
Corbett, J.A., and
M.L. McDaniel.
1992.
Perspectives in diabetes: does nitric oxide mediate autoimmune destruction of
![]() |
39. |
Campbell, I.L.,
A. Iscaro, and
L.C. Harrison.
1988.
IFN-![]() ![]() |
40. |
Mandrup-Poulsen, T.,
K. Bendtzen,
C.A. Dinarello, and
J. Nerup.
1987.
Human necrosis factor potentiates human interleukin-1 mediated rat pancreatic ![]() |
41. |
Yang, X.-D.,
R. Tisch,
S.M. Singer,
Z.A. Cao,
R.S. Liblau,
R.D. Schreiber, and
H.O. McDevitt.
1994.
Effect of tumor
necrosis factor ![]() |
42. |
von Herrath, M.G., and
M.B.A. Oldstone.
1996.
Interferon
![]() ![]() |
43. |
Hultgren, B.,
X. Huang,
N. Dybal, and
T.A. Stewart.
1996.
Genetic absence of ![]() |