By
From the Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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Abstract |
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The islet-infiltrating and disease-causing leukocytes that are a hallmark of insulin-dependent diabetes mellitus produce and respond to a set of cytokine molecules. Of these, interleukin 1,
tumor necrosis factor (TNF)-
, and interferon (IFN)-
are perhaps the most important. However, as pleiotropic molecules, they can impact the path leading to
cell apoptosis and diabetes at multiple points. To understand how these cytokines influence both the formative and effector phases of insulitis, it is critical to determine their effects on the assorted cell types comprising the lesion: the effector T cells, antigen-presenting cells, vascular endothelium, and target
islet tissue. Here, we report using nonobese diabetic chimeric mice harboring islets deficient in
specific cytokine receptors or cytokine-induced effector molecules to assess how these compartmentalized loss-of-function mutations alter the events leading to diabetes. We found that
islets deficient in Fas, IFN-
receptor, or inducible nitric oxide synthase had normal diabetes
development; however, the specific lack of TNF-
receptor 1 (p55) afforded islets a profound
protection from disease by altering the ability of islet-reactive, CD4+ T cells to establish insulitis and subsequently destroy islet
cells. These results argue that islet cells play a TNF-
-dependent role in their own demise.
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Introduction |
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Insulin-dependent diabetes mellitus (IDDM)1 is an autoimmune disease caused by the T cell-mediated destruction of the insulin-producing cells of the pancreatic islets
of Langerhans (1, 2). The nonobese diabetic (NOD) mouse
spontaneously develops IDDM remarkably similar to that
seen in humans (2, 3). The disease process is characterized
by an initial, silent and nondestructive accumulation of a heterogeneous mixture of CD4+ and CD8+ T lymphocytes, B
lymphocytes, macrophages, and dendritic cells (4, 5) circumscribing the islet mass, termed peri-insulitis. An invasive
and malignant phase follows, termed insulitis, during which
the immune infiltrate invades the islet and induces the specific apoptotic destruction of
cells (6). The events leading to insulitis induction, its perpetuation, and the subsequent destruction of
cells are poorly understood.
Cytokines produced by the immune infiltrate itself are
clearly involved in the propagation of insulitis, but their
pluripotentiality has made it difficult to pinpoint their specific roles in diabetes. Nevertheless, TNF-, IFN-
, and
IL-1
have all been implicated as critical players in the disease process. As these are all potent proinflammatory molecules with the capability of affecting events critical to insulitis such as the expression of adhesion molecules (9), the
upregulation of MHC molecules (10), and the localized production of chemoattractants (11), it is not surprising to see a varied, and often conflicting, set of effects ascribed to them during the course of IDDM development.
TNF-, which is secreted principally by activated macrophages and CD4+ Th1 cells (for review see references
12, 13), can, for example, either retard or exacerbate the
development of IDDM in NOD mice depending on when
and where it acts (14). Thus, although these experiments reveal the potent ability of TNF-
to alter the development of autoimmune diabetes, the specific role TNF-
normally plays in diabetes development has not yet been
fully elucidated.
The effect of IFN- on IDDM is less clear. On the one
hand, a number of studies have revealed a critical role for
IFN-
in the induction of insulitis and subsequent development of diabetes (21, 22). On the other hand, an absolute deficiency of the IFN-
gene in NOD mice had little
effect on the overall development of diabetes (23).
To further complicate matters, many proinflammatory cytokines not only promote inflammation but may also facilitate the localized destruction of target tissue. For example,
IFN-, TNF-
, and IL-1
have all been implicated in
the cytolysis of a number of cell types, including pancreatic
cells (24, 25). TNF-
and IFN-
have been shown to
directly induce apoptosis (24), whereas IL-1
may act
indirectly through the induction of reactive nitric oxide (NO) intermediates to islet cell destruction (27). Here
again, the pluripotent nature of cytokines has made it difficult to dissect and ascribe precisely what role they play in
the actual destruction of
cells in vivo.
Similarly, understanding cell death has been hampered
by the inability to study individual apoptotic pathways in
isolation. This is perhaps best exemplified by recent studies
on Fas (CD95) as a potential inducer of
cell apoptosis
(30, 31). Although Fas-deficient NOD mice (NODlpr/lpr)
do not develop diabetes, the global loss of Fas expression in these mice affects not only the pancreatic
cell but the
critical T and B lymphocyte populations as well. Therefore,
it is difficult to assign the protection from diabetes seen in
these mice to the inability to kill Fas-deficient
cells or to
the gross abnormalities found in adaptive immunity.
How then does one root out the multiple effects cytokines have on a complex autoimmune disease such as
IDDM? Here, we report the use of chimeric NOD mice
carrying islets deficient in one of several defined cytokine
receptors or cytokine-induced effector molecules as a means
of specifically and physiologically isolating the target islet
tissue from the effects of locally produced cytokines. In this
way, we can examine the effect islet-specific deficiencies in
Fas, IFN-R, inducible NO synthase (iNOS), and TNFR
have on the development of IDDM in NOD mice when
confronted by a population of diabetogenic CD4+ T cells.
Contrary to previous reports, we found that Fas, IFN-
R, and iNOS deficiencies did not alter the development of
diabetes. However, the specific deficiency of the TNF-
receptor 1 (p55) rendered these islets profoundly impermeable to islet-reactive T cells. In fact, in the absence of
an islet response to locally produced TNF-
, infiltrating
T cells failed to proliferate, establish insulitis, and subsequently destroy islet
cells. Not only do these results argue
that TNF-
is a key player in the development of diabetes,
they argue that this molecule must act in part upon the target tissue. Therefore, the islet must play an active, and TNF-
-dependent, role in its own demise.
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Materials and Methods |
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Mice.
BDC2.5 TCR transgenic mice have been described previously (32). B6.lpr mice were obtained from Dr. John Russell (Washington University, St. Louis, MO), who originally obtained congenic breeding pairs from The Jackson Laboratory. These mice were backcrossed >12 generations to C57Bl/6 (B6) and were maintained by brother/sister mating. IFN-Flow Cytometry.
Flow cytometry was performed on a Becton Dickinson FACScan®. We purchased PE-conjugated anti-CD4 (Caltag Labs.), anti-B220 (PharMingen) and goat anti-mouse IgM (Jackson ImmunoResearch Labs.). The mAb to theDiabetes.
Diabetes was assessed by measurement of venous blood using a Bayer Glucometer Elite one-step blood glucose meter. Animals were considered diabetic after two consecutive measurementsStreptozotocin Treatment.
Streptozotocin was prepared fresh for each set of injections in sodium citrate-buffered saline. NOD.scid mice were weighed and streptozotocin was injected intravenously at a dose of 180 mg/kg, after which most mice became diabetic (>400 mg/dl) within 48-72 h. Diabetic mice were transplanted with islets within 48-72 h of the onset of diabetes.Islet Isolation and Culture.
Mouse islets were isolated by collagenase technique (38) and purified on Ficoll gradient. Individual clean islets were selected and cultured overnight at 24°C in 5% CO2 in DMEM supplemented with 5% FCS (Hyclone), 10 mM Hepes, 5 × 10Islet Transplantation.
250 islets from mutant or control mice (all H-2b on either a 129 or B6 background) were transplanted under the renal capsule of streptozotocin-induced diabetic NOD.scid mice as previously described (39). In brief, under anesthesia (87 mg/kg of sodium pentobarbital), islets were transplanted under the renal capsule by exposing the left kidney through a flank incision, pushing the kidney through the incision, and holding it in place with small clamp attached to fatty tissue; with the aid of a dissecting microscope the capsule was cut with a needle and islets were then delivered through the incision by a Hamilton syringe fitted with a polyethylene catheter. After the catheter was withdrawn and the capsule was sealed by a small, pen-size eyecautery, the kidney was returned to the abdomen and the incision was closed. Normoglycemia was reestablished within 24 h of successful transplantation. Mice were then rested for 10-14 d to allow for vascularization of the graft by host vascular endothelium before the introduction of diabetic T cells. In the mixed islet grafts, the number of islets was 300 (200 p55Nephrectomy.
Mice were anesthetized with 87 mg/kg of sodium pentobarbital and the engrafted kidney was exposed by a flank incision as above. The engrafted kidney was raised and freed from fatty tissue as before. The renal artery and vein along with the ureter were clamped off with a mosquito hemostat and were sutured distal to the kidney with 4-0 silk. The kidney was cut free with a scalpel. The clamp was released slowly and the suture was inspected for leaks and the incision closed.Immunohistochemistry.
Kidney graft sections were stained with antibodies against VT Cell Proliferation.
In vitro T cell proliferation to islet cell antigen was performed as previously described (32). In vivo T cell proliferation was performed using the cell surface dye, 5,6-carboxy-succinimidyl-fluorescein-ester (CSFE). Preparation and labeling of T cells with CSFE was performed as described in reference 43. CSFE-labeled BDC2.5 T cells (107 cells) were transferred intravenously in to NOD.scid mice carrying kidney grafts of either 250 p55-deficient or 250 p55-sufficient islets. T cells from the draining lymph nodes (peri-renal) were collected on the indicated day after transfer and analyzed by flow cytometer for evidence of cell division. ![]() |
Results and Discussion |
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The ability to target loss-of-function mutations to specific organ systems remains a major challenge. With the exception of certain mutations targeted to the immune system using the Rag-mutant complementation system (44) or the inducible knockouts (45), it has been difficult to study the effects of broadly expressed or broadly acting mutations on specific organ systems or disease models. This is particularly true of the ubiquitously expressed cytokine receptors and their pluripotent ligands, the cytokine molecules themselves.
Several experimental models have long suggested that cytokines influence the development of autoimmune diabetes (for review see reference 46). Although these models have been informative, it has remained difficult to attribute particular stage dependency to any given cytokine, especially under physiological conditions. Moreover, it has been difficult to determine what cellular conduit channels the action of each cytokine; is it through the effector lymphocyte, the APC, the vascular epithelium, or the islet tissue itself? Therefore, we undertook to develop a novel approach that compartmentalizes the action of cytokines and their receptors to specific cellular targets in an effort to establish the dependency of particular phases of diabetes development to the local effect of these mediators.
We did this by creating chimeric NOD mice. Unlike
prior models, these mice harbored wild-type NOD APCs
that express the disease-associated MHC class II, I-Ag7, and
wild-type NOD vascular endothelium. Moreover, they
carried a defined population of diabetogenic CD4+ T cells
(BDC2.5 TCR transgenic T cells) that respond to pancreatic cell antigen, presented uniquely by NOD APCs, and
are capable of mediating the autoimmune destruction of
pancreatic
cells (32, 47). What distinguishes our current approach was the prior replacement of the endogenous
cells with those derived from one of several mouse strains
deficient in key cytokine receptor or proapoptotic effector
molecules. In so doing, we created chimeric NOD mice
containing altered
cell target tissue. This allowed us to assess the potential impact of each genetic alteration on the
islet cell's ability to serve as a target for autoimmune-mediated destruction by altering the host effector lymphocyte,
APC, or vascular endothelium.
As shown schematically in Fig. 1, NOD.scid mice were
treated with streptozotocin, a cell toxin, to destroy endogenous
cells, producing a chemically-induced diabetes
(
400 mg/dl). Normoglycemia (<100 mg/dl) was rapidly
returned with the transplantation of ~250 islets under the
left kidney capsule. (It should be noted that the donor islets
can be from any strain of mouse as NOD.scid do not reject
allogeneic tissue. We routinely used islets of H-2b donors.)
The rescued mice were then rested for 7-10 d, allowing for
host-derived vascularization of the graft. At this point, splenic T cells from diabetic BDC2.5/NOD.scid mice were
transferred into these chimeric NOD.scid mice, and the
mice were followed for onset of diabetes.
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These experiments are predicated on the following observations. First, as mentioned above, the recipient mice
are scid, hence they do not reject allogeneic islet grafts, as
confirmed by control experiments where each series of donor islets are engrafted under the kidney capsule and the
mice are left unmanipulated for >180 d. None of these mice
develop diabetes during this period. Moreover, the transplanted islets are functional, and are responsible for the
maintenance of normoglycemia, as removal of the engrafted kidney results in hyperglycemia (data not shown).
Second, the BDC2.5 T cells do not recognize the cells
directly but rather require the transfer of islet antigen to
NOD (H-2g7) APCs, which are supplied by the NOD.scid
recipient mice. Third, although the recognition of antigen
is MHC restricted, all strains of mice express the relevant
antigen in their pancreatic
cells (48). And finally, once
activated by antigen, BDC2.5/NOD.scid T cells can mediate the destruction of islet
cells in NOD.scid mice (6).
Pancreatic cell death during the course of T cell-mediated
diabetes is by apoptosis (6). One potential mediator of
cell apoptosis is Fas (CD95). The engagement of Fas, a proapoptotic member of the TNFR family, on the surface of
target cells by Fas ligand-expressing T lymphocytes leads to
the apoptotic destruction of the Fas-expressing target cell
(for review see reference 50). Treatment with IFN-
induces the expression of Fas on the surface of a variety of
cell types including
cells (24). Moreover, islet-infiltrating
T cells can induce Fas expression on
cells through localized production of IFN-
(31). Additionally, Fas-deficient, NODlpr/lpr mice do not develop diabetes (30, 31); however,
these mice had substantially altered T and B cell immunity
(30). These results notwithstanding, it has not been formally
demonstrated that Fas expression on
cells is required for
their autoimmune-mediated destruction.
To test whether Fas expression on cells is obligatory,
we used our chimeric NOD mice model to create mice
specifically lacking Fas expression on their islet cells. This
was done by either eliminating the islet's ability to respond
to IFN-
by replacing the existing islet mass with islets
lacking the IFN-
R or by using islets from B6.lpr/lpr mice
that lack functional Fas expression as islet donors. After
transplant, T cells from diabetic BDC2.5/NOD.scid mice
were transferred into these mice and diabetes onset was followed. We found that BDC2.5 T cells destroyed B6.lpr/lpr
islets as efficiently as control B6 islets, as shown in Fig. 2 a,
indicating that Fas does not play an obligatory role as the
critical inducer of
cell destruction, at least with respect to
disease transferred by diabetogenic CD4+ T cells. Similarly,
when NOD.scid mice were transplanted with islets deficient in IFN-
R, the chimeric mice developed diabetes at the same rate as control islet grafts (129/SvJ; Fig. 2 b). These results clearly demonstrate that islet cell Fas expression, either induced or constitutive, is not required for islet
destruction by diabetogenic CD4+ T cells. Moreover, this
would suggest that much of the protection seen in Fas-deficient NOD mice results from altered lymphoid development in the absence of Fas expression on T and B lymphocytes. Parenthetically, these results help to clarify the role
IFN-
may play in diabetes development. Wang et al., in
describing the introduction of systemic IFN-
receptor
deficiency onto the NOD background, found that both
NOD and BDC2.5/NOD mice lacking IFN-
R had severely retarded insulitis development (21). Our results
would suggest that this is probably due to an effect IFN-
has on the T cells, APCs, or host endothelium but not on
the islet mass itself.
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Previous studies have
indicated that IL-1 stimulates the production of nitric oxide
(NO) either by priming for Fas-mediated apoptosis or by
inducing the inducible form of the NO synthase gene
(iNOS or NOS2; references 51, 52). iNOS-mediated NO
production can lead to cell death in vitro (27). NO
can be produced by the islets themselves or by the infiltrating macrophage/dendritic population. We found that the
in vivo neutralization of IL-1
with a cocktail of antibodies
and soluble receptor did not prevent NOD mice from becoming diabetic (data not shown), leading us to question its
role in
cell death. However, to examine the effect that
the targeted iNOS deficiency in islets had on
cell destruction, we tested the ability of iNOS-deficient islets to resist T
cell-mediated destruction. As shown in Fig. 2 b, iNOS-deficient islets were destroyed with similar kinetics and magnitude as wild-type islets, indicating that iNOS gene expression is not critical for islet cell apoptosis. Although this result
does not rule out a role for NO production by infiltrating
macrophage/dendritic cells, it clearly demonstrates that islets themselves do not need to produce intracellular NO to
undergo T cell-mediated elimination.
TNF-, which is
secreted principally by activated macrophages and CD4+
Th1 cells (12, 13), can both retard and exacerbate the development of IDDM in NOD mice largely dependent on
the time of its administration (14). Thus, when TNF-
is given to NOD mice from birth to 3 wk of age, diabetes
is accelerated, and conversely the administration of neutralizing antibody to TNF-
during this period markedly reduces both insulitis and diabetes (16). Yet when administered to adult NOD mice with established insulitis, TNF-
attenuates the disease process, and its antibody neutralization exacerbates diabetes (16, 53). Moreover, the transgenic
expression of TNF-
in the islets of adult NOD mice leads
to insulitis without disease (17) and produces a state of
T cell tolerance to islet cell antigens (19, 20). Thus, although
these experiments reveal the potent ability of TNF-
to alter the development of autoimmune diabetes, the physiological role played by TNF-
has yet to be fully elucidated.
To investigate the role that localized production of TNF-
can have on the development of diabetes, we transplanted
streptozotocin-treated NOD.scid mice with islets rendered
doubly deficient for both TNF-
Rs (TNF-
R1, p55;
TNF-
R2, p75). As before, the transfer of diabetogenic T
cells led to the rapid destruction of wild-type islet grafts ( 7 out
of 8); however, doubly deficient islets (p55
/
p75
/
) remained functional (Fig. 2 c). Mice engrafted with p55
/
p75
/
islets remained normoglycemic for up to 52 d after
the transfer of T cells. To confirm that the introduced islets were responsible for the maintenance of blood glucose,
normoglycemic p55
/
p75
/
islet recipients were hemi-nephrectomized at day 28 to remove the engrafted kidney.
As shown in Fig. 2 c, these mice became hyperglycemic
within 24 h of nephrectomy, proving that the transplanted p55
/
p75
/
islets were indeed responsible for the maintenance normoglycemia. Interestingly, the mice carrying
p55
/
p75
/
islets contained BDC2.5 T cells as measured
by flow cytometric analysis of spleen and lymph node. In
addition, these T cells were phenotypically normal in that
they could still transfer disease to unmanipulated NOD.scid
mice (data not shown).
To assess which receptor conferred the protection, we
produced chimeric mice carrying islets defective in either
the p55 receptor or the p75 receptor. Fig. 2 c shows that
p55/
islets were protected from T cell-mediated destruction, whereas the p75
/
islets were efficiently destroyed.
All the p75
/
transplanted mice (11 out of 11) became
diabetic by day 12, whereas the p55
/
transplanted mice
(9 out of 9) remained normoglycemic until end of the assay
(
28 d). We therefore concluded that the engagement of
the p55 receptor by locally produced TNF-
was critical in the subsequent destruction of
cells.
One explanation for the lack of islet
destruction of the p55/
grafts is that the p55
/
islets are
non- or poorly antigenic. To test this, BDC2.5 T cells were
cocultured with NOD APCs in the presence of dispersed
p55
/
and p75
/
islet cells for 72 h under standard conditions, and T cell proliferation was measured. As shown in
Fig. 3, BDC2.5 T cells proliferated equally well to both
receptor-deficient islet cells, indicating that islet cell antigenicity does not depend on TNF-
R expression. We performed a similar assay with intact islets in vitro in the presence and absence of exogenous recombinant TNF-
and
were unable to detect a difference in the induced proliferation of BDC2.5 T cells (data not shown). We therefore
concluded that at least in vitro, there is no difference in the
antigenicity of p55
/
and p75
/
islet cells. This is somewhat discordant with recent results by Green et al., who
reported that the localized production of TNF-
in
cells
of transgenic NOD mice enhanced autoantigen presentation to BDC2.5 T cells in vitro (54).
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An alternative explanation for the p55-deficient islets' resistance to T cell-mediated destruction resides with a fundamental modification in
the cellular constituency of the infiltrate. To evaluate this
possibility, we performed an immunohistochemical analysis
of both p55/
and wild-type (or p75
/
) islet grafts after T
cell transfer. Engrafted kidneys were sampled at day 5, 7, and 9 after T cell transfer as well as at the time of diabetes
or in the case of normoglycemic mice at day 28. As seen in
Fig. 4, there was no infiltration in either p55-deficient or
wild-type islet grafts at day 5. At day 7, however, the wild-type graft showed distinct signs of peri-islet accumulation
of leukocytes, with some grafts showing evidence of intra-islet infiltration and destruction. The same was not true for
the p55-deficient islet grafts, which showed only modest
peri-islet infiltration and no intra-islet infiltration. As seen
in Fig. 4, the most dramatic difference was revealed at day
9 when the wild-type islet grafts were completely infiltrated. These islets showed discrete foci of apoptotic
cells
as revealed by TUNEL analysis (data not shown). In contrast, the p55-deficient islet grafts were only mildly infiltrated at day 9 (Fig. 4) and showed no signs of apoptosis
(data not shown). Moreover, by day 13, the wild-type islet
grafts were destroyed and the mice were overtly diabetic.
Surprisingly, the mild infiltration of the p55
/
grafts failed
to progress, and in fact resolved, so that by day 28 they
were nearly indistinguishable from those seen on or before day 5. We therefore concluded that the p55
/
islet grafts
could not sustain the infiltrating CD4+ T cells and that
the propagation of destructive insulitis requires a TNF-
-
dependent response on part of the islets.
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We then asked if the composition of the infiltrate was
modified between the p55/
and wild-type lesions. We
compared the cellular components of the transient infiltration of p55-deficient islet grafts at day 9 with those of the
wild-type grafts. In general, the composition of the infiltrate was similar to that seen in the pancreata of NOD.scid
recipients of T cells from diabetic BDC2.5 mice (42). Moreover, we saw no difference between the p55
/
and the
wild-type grafts in the activation state of the high endothelial venule (HEV) as revealed by staining for madCAM
(MECA 367) and peripheral node addressin (MECA 79, data not shown). We were also able to identify the presence of roughly equal numbers of BDC2.5 T cells in both
infiltrates as revealed by V
4 (KT4) and anti-CD4 staining.
Both lesions contained similar subsets of macrophage (F4/80,
MOMA 1, MOMA 2) and dendritic cells (NLDC-145) as
well. Despite these similarities, there was one striking difference between the p55
/
and wild-type grafts: the complete absence of
cell apoptosis in the p55-deficient grafts
(data not shown). We therefore concluded that apart from
the lack of continued progression of the lesions and the
lack of
cell apoptosis, there was little difference in the nature of the infiltrates and the vascular endothelium between
wild-type and p55-deficient islet grafts.
To verify that the resistance to diabetes resided with the
p55-deficient islets rather than with the host endothelium
or APC populations, reciprocal transplants were performed
in which wild-type islets (from 129 mice) were transplanted
under the kidney capsule of streptozotocin-treated p55-deficient NOD.scid mice (N7 generation). Under these conditions, both the host vasculature and APC population lacked
p55 receptor expression, whereas the engrafted islet mass retained full p55 functionality. When purified diabetogenic
CD4+ T cells were transferred into these mice, diabetes developed with similar kinetics in both these mice and control recipients (Fig. 5 a). This indicated that functional expression of the p55 receptor on the islet mass alone was
sufficient to drive cell destruction regardless of the p55
receptor expression status of the host APCs and the vascular
endothelium.
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Although the p55-deficient islets were no less
antigenic than the p55-sufficient islets and were equally capable of attracting similar subsets of infiltrating leukocytes,
they were clearly incapable of providing a microenvironment that supported the maturation of the immune response
to a point were cell death could occur. This could be for
one of two reasons. First, the propagation of insulitis may
require TNF-
-mediated
cell death. In this case, the
TNF-
produced locally by the infiltrating T cells and
macrophages would fail to kill the p55-deficient
cells and
insulitis would subside due to a lack of
cell damage. This
would be consistent with our failure to observe
cell apoptosis in p55-deficient islets during the early phase of infiltration, yet it also seemed unlikely as ectopic expression of
super-physiologic levels of TNF-
by the islets of transgenic mice did not lead to
cell death or diabetes (17,
55). Alternatively, the evolution of insulitis from a benign
accumulation of leukocytes to a destructive infiltrate may
require a TNF-
-dependent change in the islet mass
either the release of an islet cell-produced chemoattractant or activation factor or an alteration in the secretion or production
of antigen (something we are unable to mimic in vitro, but
which has been observed by others, see reference 54). Either way, the net result would be the full activation of the
infiltrating BDC2.5 T cell population such that it can now
act to target
cells for destruction in a TNF-
-independent fashion.
To distinguish between these two possibilities, we designed and produced chimeric NOD.scid mice that carried
mixed grafts containing both varying amounts of p55-deficient and p55-sufficient islets. If TNF- acted strictly as
a cytolytic agent, only the p55-sufficient islets would be
destroyed upon transfer of diabetogenic T cells, while the
p55-deficient islets would be spared and normoglycemia would be maintained, provided that adequate amounts of
p55-deficient islets were included in the mixed graft. On
the other hand, if TNF-
acted to alter the local environment in favor of T cell activation, the presence of even a
modicum of TNF-
-responsive islets would result in T cell
activation and the destruction of both the p55-deficient
and -sufficient islets and diabetes.
We first ascertained the minimum number of islets required in our grafts to maintain a persistent state of normoglycemia in our Streptozotocin-treated NOD.scid mice.
We found that as few as 100 islets could maintain blood
glucose levels at 100 mg/dl (data not shown). Therefore,
for our initial experiments we chose to mix
200 p55-deficient islets with ~100 wild-type islets per graft. In this way,
the "protected" p55-deficient islets were in sufficient excess to assure normoglycemia if all of the wild-type islets
were destroyed. As before, mixed islet recipients received diabetogenic T cells 7-10 d after islet transplantation. As
depicted in Fig. 5 b, both the mixed islet recipients and the
control mice engrafted with 300 wild-type islets developed
diabetes with comparable kinetics (between days 16 and 18 after transfer). In subsequent experiments, the numbers of
wild-type islets were reduced until as few as 10 wild-type
islets were mixed with ~300 p55-deficient islets, yet the
results (islet graft destruction and diabetes) were the same
(data not shown). Therefore, we concluded from these experiments that the stimulation of islet cells through their p55 receptor altered the local environmental conditions favoring the development of a productive BDC2.5 T cell infiltrate.
Having determined that a
small number of islet cells can, in response to locally produced TNF-, support the transition from benign to destructive insulitis, it was now critical to determine if this
was a purely localized effect. To address this, we performed kidney grafts on streptozotocin-treated NOD.scid mice in
which wild-type islets (100 islets) were engrafted under the
right kidney capsule and p55-deficient islets (250 islets)
were engrafted under the left kidney capsule of the same
animal. By physically separating the grafts we sought to
minimize the effects between the wild-type and p55-deficient graft. If, upon transfer of diabetogenic T cells, the
p55-deficient graft survived in these mice, despite the destruction of the wild-type islet grafts, then the original destruction of the p55-deficient islets in the mixed islet grafts
described above (Fig. 5 b) resulted merely by virtue of their
intimate proximity to the wild-type islets. On the other hand,
if the distant p55-deficient grafts were likewise destroyed, it is
more likely that the transferred T cells were altered by an
encounter with wild-type islet cells.
We found that the twin-kidney engrafted NOD.scid
recipient mice did, in fact, become diabetic 12-16 d after
receiving BDC2.5 T cells, at a rate coincidental with the
development of diabetes in mice harboring dual wild-type
grafts (Fig. 6 a). As before, those mice engrafted with only
p55-deficient islets did not develop disease. Additionally,
histological analysis of the p55-deficient bilateral graft
showed signs of cell apoptosis within 1 d of the onset of
destructive infiltration of the p55-sufficient graft (~day 5-7
after transfer, Fig. 6 b). The ability of wild-type grafts to influence the outcome of the contralateral p55-deficient grafts indicated that TNF-
responsiveness on the part of
the wild-type islets led to the activation of the transferred
BDC2.5 T cells such that they were now capable of homing to and destroying the p55-deficient graft on the opposite kidney.
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These results led us to assess the in vivo proliferative status of BDC2.5 T cells after transfer. We reasoned that the
lack of progression in the p55-deficient islet engrafted mice
may be due to the inability of the p55/
islets to fully activate the transferred BDC2.5 T cells. In the mixed and
twin-kidney graft experiments, the wild-type islets would
provide an environment capable of furnishing this activation and therefore of leading to the subsequent destruction
of the p55
/
islets in a TNF-
-independent fashion. If this
were true, T cell proliferation in vivo might differ between
mice harboring only p55
/
islets and those carrying only
wild-type islets. This was tested by monitoring the degree
of in vivo proliferation of the BDC2.5 T cells in the efferent lymph of mice harboring one or the other islet grafts
under the left kidney using the decay of the integral membrane dye, CSFE, as an indicator of cell division (43, 56, 57). As depicted in Fig. 7, draining lymph nodes from mice
engrafted with p55
/
islets were devoid of reactivated
BDC2.5 T cells, whereas the renal lymph nodes from mice
engrafted with wild-type islets contained T cells that clearly
had undergone several rounds of replication. Therefore, we
concluded that the most likely explanation for the infiltration and subsequent destruction of p55
/
islets in both the
mixed and twin-kidney grafts was due in part to the activation of the BDC2.5 T cells in response to wild-type islets
either proximal or distal to the p55
/
islets. This process
required a TNF-
response on the part of the target islet
cells but the subsequent destruction of the islet tissue was
TNF-
-independent. The nature of the TNF-
response on the part of islets remains unknown, but could be an increase in antigen delivery either in direct response to TNF-
or as a result of islet cell death. In either case, this leads to
the subsequent activation of our infiltrating islet-reactive
BDC2.5 T cells, which then act to mediate the destruction
of islet
cells in a process that does not require TNF-
.
|
Using the same BDC2.5 TCR transgenic model, André
et al. have proposed two checkpoints in the progression of
diabetes, the first the formation of a benign infiltrate and
the second the transition to destructive insulitis (58). We
would propose that the transition through checkpoint two
is dependent on the active response of islets to TNF-.
Moreover, our results are consistent with the hypothesis
that early and local production of TNF-
in the islet acts to
enhance the islet's antigenicity and the subsequent activation of disease-causing lymphocytes (16, 17, 53).
In conclusion, these data, taken together, demonstrate
that Fas, IFN-, and iNOS do not play an obligatory role
in the apoptotic destruction of pancreatic
cells induced
by a diabetogenic CD4+ T cell population, but that TNF-
plays a critical role in the transformation of islet-reactive
CD4+ T cells from a benign state of
cell indifference to
an activated state of
cell reactivity. Moreover, these results suggest, for the first time, that the islet cells themselves
play an active and TNF-dependent role in facilitating their
own death by providing an environment capable of perpetuating T cell-mediated insulitis.
![]() |
Footnotes |
---|
Address correspondence to J.D. Katz, Center for Immunology and Department of Pathology, Washington University School of Medicine, Campus Box 8118, 660 South Euclid Ave., St. Louis, MO 63110. Phone: 314-747-1221; Fax: 314-747-0728; E-mail: jkatz{at}immunology.wustl.edu
Received for publication 19 November 1998 and in revised form 19 January 1999.
We particularly wish to thank Dr. Charles Kilo and his Kilo Diabetes and Vascular Research Foundation for their generous financial support. In addition, this work was supported by grants to J.D. Katz from the Juvenile Diabetes Foundation International (JDFI; No. 197030), the Washington University Diabetes Research and Training Center, the National Institutes of Health (R01 AI44416), and a joint NIH/JDFI program project grant (P01 AI39676/995012; Dr. E.R. Unanue, program director). J.D. Katz is a recipient of an American Diabetes Association Career Development Award.We would like to thank Ms. Olga Strots and Mr. Larry McClendon for their excellent technical assistance and animal care. We would also like to thank Drs. Robert D. Schreiber, John H. Russell, Osami Kanagawa, John Mudgett, Werner Lesslauer, and Mark Moore for gifts of reagents, mice, and critical technical advice, and Dr. Stacey Smith for use of her cryostat. We would also like to thank Dr. Paul E. Lacy for his advice on transplantation experiments and critical review of the manuscript.
Abbreviations used in this paper CFSE, 5,6-carboxy-succinimidyl-fluorescein-ester; IDDM, insulin-dependent diabetes mellitus; iNOS, inducible NO synthase; NO, nitric oxide; NOD, nonobese diabetic.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bach, J.F.. 1994. Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr. Rev. 15: 516-542 [Abstract]. |
2. |
Atkinson, M.A., and
N.K. MacLaren.
1994.
The pathogenesis of insulin-dependent diabetes mellitus.
N. Engl. J. Med.
331:
1428-1436
|
3. | Tisch, R., and H. McDevitt. 1996. Insulin-dependent diabetes mellitus. Cell. 85: 291-297 [Medline]. |
4. |
Charlton, B.,
A. Bacelj, and
T.E. Mandel.
1988.
Administration of silica particles or anti-Lyt2 antibody prevents ![]() |
5. |
Miller, B.J.,
M.C. Appel,
J. O'Neil, and
L.S. Wicker.
1988.
Both the LYT-2+ and L3T4+ T cell subsets are required for
transfer of diabetes in nonobese diabetic mice.
J. Immunol.
140:
52-58
|
6. |
Kurrer, M.O.,
S.V. Pakala,
H.L. Hanson, and
J.D. Katz.
1997.
Beta cell apoptosis in T cell-mediated autoimmune diabetes.
Proc. Natl. Acad. Sci. USA.
94:
213-218
|
7. | O'Brien, B.A., B.V. Harmon, D.P. Cameron, and D.J. Allan. 1997. Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes 46: 750-757 [Abstract]. |
8. | O'Brien, B.A., B.V. Harmon, D.P. Cameron, and D.J. Allan. 1996. Beta-cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model. J. Pathol. 178: 176-181 [Medline]. |
9. |
Pober, J.S., and
R.S. Cotran.
1990.
Cytokines and endothelial cell biology.
Physiol. Rev.
70:
427-451
|
10. | Pujol-Borrell, R., I. Todd, M. Doshi, G.F. Bottazzo, R. Sutton, D. Gray, G. Adolf, and M. Feldmann. 1987. HLA class II induction on human islets by interferon-gamma plus tumor necrosis factor or lymphotoxin. Nature. 326: 304-306 [Medline]. |
11. | Feldmann, M., F.M. Brennan, and R. Maini. 1996. Role of cytokines in rheumatoid arthritis. Annu. Rev. Immunol. 14: 397-440 [Medline]. |
12. | Old, L.J.. 1985. Tumor necrosis factor (TNF). Science. 230: 630-632 [Medline]. |
13. | Abbas, A.K., K.M. Murphy, and A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature. 383: 787-793 [Medline]. |
14. |
Satoh, J.,
H. Seino,
T. Abo,
S. Tanaka,
S. Shintani,
S. Ohta,
K. Tamura,
T. Sawai,
T. Nobunaga,
T. Oteki, et al
.
1989.
Recombinant human tumor necrosis factor ![]() |
15. |
Jacob, C.O.,
S. Aiso,
S.A. Michie,
H.O. McDevitt, and
H. Acha-Orbea.
1990.
Prevention of diabetes in nonobese diabetic mice by tumor necrosis factor (TNF): similarities between TNF-![]() |
16. |
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 ![]() |
17. |
Picarella, D.E.,
A. Kratz,
C.B. Li,
N.H. Ruddle, and
R.A. Flavell.
1993.
Transgenic tumor necrosis factor (TNF)-alpha
production in pancreatic islets leads to insulitis, not diabetes.
Distinct patterns of inflammation in TNF-alpha and TNF-beta transgenic mice.
J. Immunol.
150:
4136-4150
|
18. |
Higuchi, Y.,
P. Herrera,
P. Muniesa,
J. Huarte,
D. Belin,
P. Ohashi,
P. Aichele,
L. Orci,
J.D. Vassalli, and
P. Vassalli.
1992.
Expression of a tumor necrosis factor alpha transgene
in murine pancreatic ![]() |
19. |
Grewal, I.S.,
K.D. Grewal,
F.S. Wong,
D.E. Picarella,
C.A. Janeway Jr., and
R.A. Flavell.
1996.
Local expression of
transgene encoded TNF-![]() |
20. |
Cope, A.P.,
R.S. Liblau,
X.D. Yang,
M. Congia,
C. Laudanna,
R.D. Schreiber,
L. Probert,
G. Kollias, and
H.O. McDevitt.
1997.
Chronic tumor necrosis factor alters T cell
responses by attenuating T cell receptor signaling.
J. Exp.
Med.
185:
1573-1584
|
21. |
Wang, B.,
I. Andre,
A. Gonzalez,
J.D. Katz,
M. Aguet,
C. Benoist, and
D. Mathis.
1997.
Interferon gamma impacts at
multiple points during the progression of autoimmune diabetes.
Proc. Natl. Acad. Sci. USA.
94:
13844-13849
|
22. |
von Herrath, M.G., and
M.B. Oldstone.
1997.
Interferon ![]() ![]() |
23. | Hultgren, B., X. Huang, N. Dybdal, and T.A. Stewart. 1996. Genetic absence of gamma-interferon delays but does not prevent diabetes in NOD mice. Diabetes. 45: 812-817 [Abstract]. |
24. | Boehm, U., T. Klamp, M. Groot, and J. Howard. 1997. Cellular responses to interferon-gamma. Annu. Rev. Immunol. 15: 749-795 [Medline]. |
25. |
Deiss, L.,
H. Galinka,
H. Berissi,
O. Cohen, and
A. Kimichi.
1996.
Cathepsin-D protease mediates programmed cell death
induced by interferon-gamma, Fas/APO-1 and TNF-![]() |
26. |
Chaudhary, P.,
M. Eby,
A. Jasmin,
A. Bookwalter,
J. Murray, and
L. Hood.
1997.
Death receptor 5, a new member of
the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-![]() |
27. | Ankarcrona, M., J.M. Dypbukt, B. Brune, and P. Nicotera. 1994. Interleukin-1 beta-induced nitric oxide production activates apoptosis in pancreatic RINm5F cells. Exp. Cell Res. 213: 172-177 [Medline]. |
28. |
Cailleau, C.,
A. Diu-Hercend,
E. Ruuth,
R. Westwood, and
C. Carnaud.
1997.
Treatment with neutralizing antibodies
specific for IL-1![]() |
29. |
Corbett, J.A.,
J.L. Wang,
M.A. Sweetland,
J.R. Lancaster Jr., and
M.L. McDaniel.
1992.
Interleukin 1![]() ![]() ![]() |
30. |
Itoh, N.,
A. Imagawa,
T. Hanafusa,
M. Waguri,
K. Yamamoto,
H. Iwahashi,
M. Moriwaki,
H. Nakajima,
J. Miyagawa,
M. Namba, et al
.
1997.
Requirement of Fas for the
development of autoimmune diabetes in nonobese diabetic
mice.
J. Exp. Med.
186:
613-618
|
31. | Chervonsky, A.V., Y. Wang, F.S. Wong, I. Visintin, R.A. Flavell, C.A. Janeway Jr., and L.A. Matis. 1997. The role of Fas in autoimmune diabetes. Cell. 89: 17-24 [Medline]. |
32. | Katz, J.D., B. Wang, K. Haskins, C. Benoist, and D. Mathis. 1993. Following a diabetogenic T cell from genesis through pathogenesis. Cell. 74: 1089-1100 [Medline]. |
33. | Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, J. Vilcek, R.M. Zinkernagel, and M. Aguet. 1993. Immune response in mice that lack the interferon-gamma receptor. Science. 259: 1742-1745 [Medline]. |
34. | Rothe, J., W. Lesslauer, H. Lotscher, Y. Lang, P. Koebel, F. Kontgen, A. Althage, R. Zinkernagel, M. Steinmetz, and H. Bluethmann. 1993. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature. 364: 798-802 [Medline]. |
35. | Erickson, S.L., F.J. de Sauvage, K. Kikly, K. Carver-Moore, S. Pitts-Meek, N. Gillett, K.C. Sheehan, R.D. Schreiber, D.V. Goeddel, and M.W. Moore. 1994. Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature. 372: 560-563 [Medline]. |
36. | MacMicking, J.D., C. Nathan, G. Hom, N. Chartrain, D.S. Fletcher, M. Trumbauer, K. Stevens, Q.W. Xie, K. Sokol, N. Hutchinson, et al. 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell. 81:641-650. (See published erratum 81: following 1170.) |
37. |
Tomonari, K.,
E. Lovering, and
S. Spence.
1990.
Correlation
between the V![]() |
38. | Lacy, P.E., and M. Kostianovsky. 1967. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes. 16: 35-39 [Medline]. |
39. | Sullivan, F.P., C. Ricordi, V. Hauptfeld, and P.E. Lacy. 1987. Effect of low temperature culture and site of transplantation on hamster islet xenograft survival (hamster to mouse). Transplantation. 44: 465-468 [Medline]. |
40. | Leenen, P.J., M.F. de Bruijn, J.S. Voerman, P.A. Campbell, and W. van Ewijk. 1994. Markers of mouse macrophage development detected by monoclonal antibodies. J. Immunol. Methods. 174: 5-19 [Medline]. |
41. | Streeter, P.R., B.T. Rouse, and E.C. Butcher. 1988. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes. J. Cell Biol. 107: 1853-1862 [Abstract]. |
42. |
Pakala, S.V.,
M.O. Kurrer, and
J.D. Katz.
1997.
T helper 2 (Th2) T cells induce acute pancreatitis and diabetes in immune-compromised nonobese diabetic (NOD) mice.
J. Exp.
Med.
186:
299-306
|
43. | Lyons, A.B., and C.R. Parish. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods. 171: 131-137 [Medline]. |
44. | Shinkai, Y., S. Koyasu, K. Nakayama, K.M. Murphy, D.Y. Loh, E.L. Reinherz, and F.W. Alt. 1993. Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes. Science. 259: 822-825 [Medline]. |
45. | Kuhn, R., F. Schwenk, M. Aguet, and K. Rajewsky. 1995. Inducible gene targeting in mice. Science. 269: 1427-1429 [Medline]. |
46. |
Gianani, R., and
N. Sarvetnick.
1996.
Viruses, cytokines,
antigens, and autoimmunity.
Proc. Natl. Acad. Sci. USA.
93:
2257-2259
|
47. | Haskins, K., and M. McDuffie. 1990. Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone. Science. 249: 1433-1436 [Medline]. |
48. | Haskins, K., M. Portas, B. Bradley, D. Wedmann, and K.J. Lafferty. 1988. T-lymphocyte clone specific for pancreatic islet antigen. Diabetes. 37: 1444-1448 [Abstract]. |
49. | Haskins, K., M. Portas, B. Bergman, K. Lafferty, and B. Bradley. 1989. Pancreatic islet-specific T-cell clones from nonobese diabetic mice. Proc. Natl. Acad. Sci. USA. 86: 8000-8004 [Abstract]. |
50. | Nagata, S., and P. Golstein. 1995. The Fas death factor. Science. 267: 1449-1456 [Medline]. |
51. |
Stassi, G.,
R.D. Maria,
G. Trucco,
W. Rudert,
R. Testi,
A. Galluzzo,
C. Giordano, and
M. Trucco.
1997.
Nitric oxide
primes pancreatic ![]() |
52. | McDaniel, M.L., G. Kwon, J.R. Hill, C.A. Marshall, and J.A. Corbett. 1996. Cytokines and nitric oxide in islet inflammation and diabetes. Proc. Soc. Exp. Biol. Med. 211: 24-32 [Abstract]. |
53. | Jacob, C.O., S. Aiso, R.D. Schreiber, and H.O. McDevitt. 1992. Monoclonal anti-tumor necrosis factor antibody renders non-obese diabetic mice hypersensitive to irradiation and enhances insulitis development. Int. Immunol. 4: 611-614 [Abstract]. |
54. |
Green, E.A.,
E.E. Eynon, and
R.A. Flavell.
1998.
Local
expression of TNF-![]() |
55. | Picarella, D.E., A. Kratz, C.B. Li, N.H. Ruddle, and R.A. Flavell. 1992. Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas. Proc. Natl. Acad. Sci. USA. 89: 10036-10040 [Abstract]. |
56. | Kurts, C., W.R. Heath, F.R. Carbone, J. Allison, J.F. Miller, and H. Kosaka. 1996. Constitutive class I-restricted exogenous presentation of self antigens in vivo. J. Exp. Med. 184: 923-930 [Abstract]. |
57. |
Kurts, C.,
H. Kosaka,
F.R. Carbone,
J.F. Miller, and
W.R. Heath.
1997.
Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8+ T
cells.
J. Exp. Med.
186:
239-245
|
58. |
André, I.,
A. Gonzalez,
B. Wang,
J. Katz,
C. Benoist, and
D. Mathis.
1996.
Checkpoints in the progression of autoimmune disease: lessons from diabetes models.
Proc. Natl. Acad.
Sci. USA.
93:
2260-2263
|