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in Pancreatic Islets in the Nonobese Diabetic Mouse
Pharmacia & Upjohn, Lund Research Center, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is known to be produced by
islet-infiltrating mononuclear cells during insulitis and subsequent
ß cell destruction and has been implicated in the pathogenesis of
IDDM. Previously, T cells have been suggested as the main source of
TNF- in the islet infiltrate. However, on immunohistochemical
analysis of TNF- expression in islets, we are able to show that the
staining pattern of TNF- resembles that of dendritic cells (DC) and
macrophages (M
) rather than T cells and that TNF- is expressed in
islets at the very early stages of insulitis when no T cells are
detected. On double staining for TNF- and cell surface markers, we
can demonstrate that TNF- staining clearly correlates with DC and
M, whereas there is a poor correlation with T cells. This feature
was observed at both early and late stages of insulitis. TNF-
expression was also seen in NOD-SCID islets, in addition to a
peri-islet infiltration consisting of DC and M, indicating that T
cells are not required for the early DC and M infiltration and
TNF- expression in islets. In conclusion, our results show that DC
and M are the major, early source of TNF- in the NOD islet
infiltrate and that TNF- can be expressed independently of T cells,
indicating that the early DC and M infiltration and expression of
TNF- are crucial in initiation of diabetes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2, 3) as well as
CD4+ and CD8+ T cells (2, 4, 5) are required
for the development of the disease and cytokines such as IFN-
, IL-1,
and TNF- (6) are implicated in playing a role in disease
development.
TNF- is a pleiotropic cytokine known to be produced by
islet-infiltrating mononuclear cells during insulitis in the pancreas.
TNF- serves as a proinflammatory cytokine and affects several
aspects of the inflammatory process, including chemotaxis and
activation of leukocytes (7), up-regulation of adhesion molecules on
endothelial cells (8, 9, 10), and maturation and migration of dendritic
cells (DC) (11). In addition, TNF-, in combination with other
cytokines such as IL-1 and IFN-, has been shown to exert ß cell
cytotoxicity in vitro by inducing apoptosis (12, 13, 14). Thus TNF- may
act directly and indirectly in the autoimmune processes mediating ß
cell destruction. However, the role of TNF- in disease development
is still unclear, since treatment of NOD mice with rTNF- or blocking
anti-TNF- Abs may either prevent or exacerbate disease,
depending on the age of the mice. Thus, Yang et al. have shown that
treating newborn or 2-wk-old female NOD mice with rTNF- for 3 wk
leads to an earlier onset and a higher incidence of diabetes, whereas
treating them with anti-TNF- from birth prevents disease
development (15). In contrast, if TNF- treatment is initiated at 4
wk of age, onset is delayed (15). In addition to this, a dramatic
reduction in development of diabetes is seen in long term (4 mo)
treatment with TNF- when treatment is started at 10 wk of age (15, 16). Similar results have been obtained by Jacob and coworkers (17),
who were able to show that treatment of 8-wk-old NOD mice with rTNF-
for 4 or 8 wk reduced the incidence of insulitis, whereas treatment
with anti-TNF- for 8 wk resulted in an increased incidence. In
addition to these findings, the important role played by TNF- in
diabetes development is further demonstrated in p55 TNF-R transgenic
NOD mice, which constitutively express soluble neutralizing receptors.
In these mice, both insulitis and diabetes development are inhibited
(18). Furthermore, the expression of transgenic TNF- in the ß
cells of normal mice (19, 20) or diabetes-prone NOD mice (21) results
in a massive insulitis but does not lead to development of diabetes.
The protective effect of TNF- observed in TNF- transgenic NOD
mice appears to result from prevention of development of islet-specific
autoreactive T cells and may be explained by the fact that TNF-
fails to be expressed in young mice and is only expressed in adults
from 7 wk of age. In summary, TNF- appears to exacerbate disease in
young NOD mice, whereas it is protective in old mice.
To date, the source of TNF- in the islet infiltrate has been
suggested to be the T cell (22, 23, 24), whereas expression of TNF- by
DC and M in the islet infiltrate has not been shown. In fact, it has
been suggested that M produce very little TNF- in the islet
infiltrate (22, 23). However, we are able to show that the TNF-
staining in the islets of Langerhans exhibits a dendritic morphology
and resembles the staining pattern observed for DC and M and that
TNF- is detectable in the islets in the very early stages of
insulitis, when DC and M, but no T cells are detected, suggesting
that DC/M may be a source of TNF- in the islet infiltrate. On
double staining for TNF- and cell surface markers, we demonstrate
that TNF- staining clearly correlates with the DC marker N418 as
well as the M markers F4/80 and Mac-1, whereas a poor correlation is
observed with the T cell markers CD4 and CD8. DC and M infiltration
and expression of TNF- occur independently of T cells, as these
processes can be observed in young NOD mice in the absence of
infiltrating T cells as well as NOD-SCID mice lacking T cells. In
conclusion, our results indicate that DC and M, rather than T cells,
are the major source of TNF- in the islet infiltrate; that DC and
M infiltration and TNF- expression may occur independently of T
cells; and that the early APC-associated TNF- may be important for
the initiation and/or development of diabetes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female NOD and NOD-SCID mice were purchased from Bommice (Bomhult Gård Breeding and Research Center, Ry, Denmark) and were used at the ages of 4 to 18 wk of age, as indicated. For pancreatic tissue sampling, mice were sacrificed by cervical dislocation, pancreata were removed and frozen in 2-methyl butane (KEBO Lab, Lund, Sweden), chilled on liquid nitrogen, and then stored at -70°C for sectioning.
Immunohistochemical and immunofluorescence staining
Levels of mononuclear cell infiltration in islets of Langerhans
were determined in NOD mice at various ages. Cryosections (8 µm
thick) were allowed to dry in air overnight and were subsequently fixed
with acetone (Histolab Products, Västra Frölunda, Sweden)
at -20°C for 10 min. Endogenous biotin and avidin binding activity
was blocked with an Avidin/Biotin Blocking Kit (Vector Laboratories,
Burlingame, CA) for 15 min. Sections were incubated with primary Abs
directed toward N418 (5 µg/ml, Serotec, Oxford, U.K.), F4/80 (1:10,
Serotec), Mac-1 (5 µg/ml, Boehringer Mannheim, Mannheim, Germany),
CD3 (5 µg/ml, PharMingen, San Diego, CA), CD4 (5 µg/ml,
PharMingen), and CD8 (5 µg/ml, PharMingen) for 1 h. Sections
were then incubated with the appropriate biotin-conjugated secondary
Abs, i.e., goat anti-Armenian hamster IgG (1:500, Jackson
ImmunoResearch Laboratories, West Grove, PA), for detection of N418 and
goat anti-rat IgG (1:400, Jackson ImmunoResearch Laboratories) for
detection of F4/80, Mac-1, CD4, or CD8 for 30 min, followed by
incubation with HRP-conjugated streptavidin (ABC-HRP, 1:110, Vector
Laboratories) for 30 min. Finally, the sections were incubated with 0.5
mg/ml diaminobenzamidine (DAB, Saveen Biotech, Malmö, Sweden)
diluted in Tris, pH 7.6, for 5 min, and counterstained with hematoxylin
(Apoteksbolaget, Malmö, Sweden) for 30 s. All incubations
were performed at room temperature, and Abs were diluted in PBS
supplemented with 10% FCS (HyClone Laboratories, Logan, UT). After
each incubation, sections were washed three times with PBS. For TNF-
staining, the sections were fixed for 20 min with 2% formaldehyde
(Sigma, St. Louis, MO) diluted in PBS, followed by incubation with 1%
H2O2 (ICN Pharmaceuticals, Costa Mesa, CA)
diluted in EBSS buffer (Life Technologies, Paisley, U.K.) supplemented
with 0.3 M sodium azide (BDH Laboratory Supplies, Poole, U.K.) and
0.1% saponin (Riedel-de Haen, Seelze, Germany) for 30 min. Then,
sections were blocked with avidin and biotin for 1 h and for 20
min, respectively. Sections were then incubated with primary Ab
directed toward TNF- (clone MP6-XT22, 1 µg/ml, PharMingen)
overnight, followed by incubation with biotin-labeled goat anti-rat
IgG for 1 h. Staining was visualized using Extravidin-HRP (1:3000,
Sigma) followed by incubation with DAB as described above. All
incubations were performed at room temperature, and reagents were
diluted in EBSS buffer, pH 7.4, supplemented with 10 mM HEPES buffer
(ICN Pharmaceuticals) and 0.1% saponin. Sections were washed three
times with the same buffer after each incubation.
To be able to correlate TNF- staining with particular cell types,
double immunofluorescence staining was performed as follows.
Cryosections (8 µm thick) were fixed with 2% formaldehyde and
treated with 1% H2O2 as described above. All
primary and secondary Abs were diluted in EBSS buffer, pH 7.4,
supplemented with 10 mM HEPES buffer and 0.1% saponin, which was also
used to wash sections after each incubation. To double stain for N418
and TNF-, sections were incubated with N418 (hamster anti-mouse)
for 1 h, blocked for endogenous biotin and avidin binding activity
(for 1 h and for 20 min, respectively) and then incubated with
biotin-labeled goat anti-hamster secondary Ab for 30 min, followed
by streptavidin-Cy3 (5 µg/ml, Amersham Life Science, Arlington
Heights, IL) for 30 min. Sections were then incubated with
anti-TNF- (rat IgG1) overnight followed by FITC-labeled mouse
anti-rat IgG1 (5 µg/ml, PharMingen) for 1 h. For double
staining of TNF- and the other cell surface markers, sections were
incubated with primary Abs directed toward F4/80 (rat IgG2b), Mac-1
(rat IgG2b), CD4 (rat IgG2a), or CD8 (rat IgG2a), diluted as described
above, for 1 h. Sections were then incubated with Cy3-labeled goat
anti-rat IgG (10 µg/ml, Amersham Life Science) for 30 min,
followed by 1% normal rat serum for 30 min, and then anti-TNF-
overnight. Finally, sections were incubated with FITC-labeled mouse
anti-rat IgG1 for 1 h. Slides were mounted with Dako
Fluorescence Mounting Medium (Dako, Glostrup, Denmark).
Image analysis
DAB staining was examined with a Leitz Aristoplan light
microscope (Leica, Göteborg, Sweden) equipped with a PC-based
Quantimet Q 500 image analysis program (Leica). Quantification of
levels of infiltration by cells expressing the N418, F4/80, Mac-1, CD4,
or CD8 cell surface markers, as well as expression of TNF- in the
islets, was achieved by setting an interval of brown staining (DAB),
which was detected as positive by the computer, whereas the blue
hematoxylin counterstaining was ignored. The percentage of stained area
within each islet was determined for 
40 different islets in five
animals per age group. To analyze the degree TNF- staining
correlating with the different cell surface markers in double
immunofluorescence-stained sections, a Leica DMRX fluorescence
microscope with a red-green filter equipped with a PC-based modified
Quantimet Q 600 program was used. The single-stained Cy3-labeled cell
surface markers N418, F4/80, Mac-1, CD4, and CD8 were seen as red,
single-stained FITC-labeled TNF- was seen as green, and therefore
double-stained areas were seen as yellow. The Quantimet Q 600 program
was used to discriminate between the three different colors to
determine the relative percentage of single- and double-stained areas
within the islets. At least 40 different islets were examined in five
animals per group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the NOD islet
infiltrate resembles that seen for DC and M
On immunohistochemical analysis of pancreata from NOD mice,
TNF- is detectable during the very early stages of insulitis, i.e.,
at 4 wk of age. At this age, DC and M are detectable in the islets,
whereas T cells are undetectable in the majority of islets (Fig. 1
, Table I). In 3/5 four week old animals, the
islets lacked detectable T cell infiltration, whereas in two animals
CD4+ T cells could be detected in 3/7 and 3/13 islets
respectively, and CD8+ T cells could be detected in 2/6 and
3/12 islets respectively. TNF- is continuously expressed in the
islets throughout the course of insulitis development from at least 4
wk of age and the level of expression increases with increasing
mononuclear cell infiltration (Fig. 1). Thus, the kinetics of TNF-
expression in the islet infiltrate appears to correlate more closely
with the infiltration kinetics of DC and M rather than T cells.
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staining in the islet infiltrate (Fig. 2a) strongly resembles
a DC/M staining pattern, with thin elongated processes or dendrites
and irregularly shaped cell bodies, a staining pattern which is very
similar to the pattern observed when staining with antibodies toward
the DC marker N418 and the M markers F4/80 and Mac-1 (Fig. 2, b–d, respectively). A similar TNF- staining pattern is
observed in NOD islets from mice at all ages, ranging from 4 to 18 wk
of age, the only difference being that the level of TNF- expression
increases with age. Similarly, TNF- associated with T lymphocytes
would be expected to stain with a discrete staining pattern as can be
seen when staining for CD4 or CD8 (Fig. 2, e and
f, respectively), but TNF- staining with this morphology
cannot be observed. The early presence of TNF-, at 4 wk of age, as
well as its dendritic appearance in staining suggests that DC and M
rather than T cells could be a main source of TNF- in the NOD islet
infiltrate.
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staining with DC and M markers
To investigate whether DC and M, rather than T cells, were the
main source of TNF- in the NOD islet infiltrate, we performed double
immunofluorescence staining where staining for TNF- with
FITC-labeled Abs was combined with staining for the cell surface Ags
N418, F4/80, Mac-1, CD4, and CD8 with Cy3-labeled Abs. In Figure 3, parallel sections of representative
islets from 4- and 14-wk-old animals stained for TNF- and the
various cell surface markers are shown. Figure 3, a and g, shows
that the TNF- staining in islets from 4- and 14-wk-old mice,
respectively, have a similar dendritic staining pattern, although
levels of TNF- expression are increased in 14-wk-old mice compared
with 4-wk-old mice. It can be seen that TNF- expression correlates
very well with the DC marker N418 (Fig. 3, b and
h) as well as with the M markers F4/80 (Fig. 3, c and i) and Mac-1 (Fig. 3, d
and j) in both young and old NOD mice. In contrast to
this, there is a poor correlation of TNF- with CD4+
(Fig. 3, e and k) and CD8+ T
cells (Fig. 3, f and l). Note that the
localization as well as the morphology of the TNF- staining is
similar to that observed for DC and M, but differs from that seen
for T cell staining. Islets are normally surrounded by
TNF--expressing cells, and in addition to this, a few
TNF--expressing cells with dendritic morphology can be seen in
intraislet positions (Fig. 3a). A similar
distribution is seen for M and DC. M are usually found in
peri-islet locations, whereas DC are found in peri-islet as well as in
intraislet locations, close to the front of ß cell destruction and at
the site of T cell infiltration. T cells are mainly intra-islet, often
with TNF- positive dendritic structures in close proximity. The
correlation of TNF- with DC and M was seen at both 4 and 14 wk of
age, the staining pattern being similar except that T cells were only
rarely detected in islets of 4-wk-old mice. The percentage of TNF-
associated with the different cell surface markers was quantified by
image analysis (Fig. 4). It was found
that most of the TNF- expressed in the islet infiltrate correlated
with the cell surface markers N418, F4/80, or Mac-1. Thus, in 4-wk-old
mice, 37% of the TNF- expressed in the islet infiltrate correlated
with the N418 marker, 50% with F4/80, and 16% with Mac-1. In
14-wk-old mice, the corresponding figures were 41% for N418, 46% for
F4/80, and 26% for Mac-1. A certain degree of correlation between
TNF- and the CD4 and CD8 cell surface Ags could also be observed
(1.6% for CD4 and 0.2% for CD8 in 4-wk-old mice and 20% for CD4 and
17% for CD8 in 14- wk-old mice). However, most of this correlation
appeared to be due to TNF--expressing DC/M overlapping with
CD4+ or CD8+ T cells in the islet infiltrate,
especially in highly infiltrated islets. In Figure 3, k and
l, dendritic-like structures, which appear double stained,
can be seen in very close proximity to T cells. As judged by the
staining morphology, these TNF--expressing cells are probably
DC/M rather than T cells, and therefore the true correlation between
TNF- and T cells may be overestimated by image analysis. In
addition, a certain proportion of these cells may be DC that are
expressing CD4 or CD8 (25, 26). When primary Abs were substituted for
their relevant isotype control Abs in the double immunofluorescence
staining method described in Materials and Methods, no
staining could be detected (data not shown). Substitution of
anti-TNF- Ab by rat IgG1 on sections, after staining for the
cell surface Ags, resulted in staining for the cell surface markers
alone (data not shown); and when preincubating the anti-TNF- Ab
with rTNF- before adding it to the sections, staining was prevented
(data not shown).
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surface markers used. Thus, we investigated whether the major
part of the TNF- staining observed in the islet infiltrate was
associated with the total DC/M population or whether other cell
types could also account for a proportion of the TNF- staining
observed. To this end, pancreatic sections from 4- and 14-wk-old
animals were double stained for TNF- (FITC) and a cocktail of Abs
directed to the DC/M surface markers N418, F4/80, and Mac-1 (Cy-3).
The vast majority of the TNF- expressed in the islet infiltrate is
contained within the DC/M population, as indicated by the double
staining shown in yellow (Fig. 5). On
image analysis of the sections, it was found that 70 ± 2.9% and
78 ± 2.2% (mean ± SEM) of the TNF- expressed in the
islet infiltrate correlated with the DC/M markers in 4- and
14-wk-old NOD mice, respectively. The TNF- that did not correlate
with these cell surface markers had the same dendritic morphology,
suggesting that it may be produced by DC/M, which are not detectable
by the markers used in this study.
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infiltration and TNF- expression is
independent of T cells
To confirm that DC/M infiltration in the islets of Langerhans
is a feature specific for diabetes development, we stained pancreatic
sections of control 8-wk-old C57BL/6 mice for the DC marker N418 and
the M marker F4/80. Low numbers of DC/M could be observed in the
exocrine pancreas, whereas islets lacked DC/M infiltration (Fig. 6, a and b),
suggesting that DC/M infiltration in the endocrine pancreas is a
feature of diabetes development and occurs only in mice of a diabetes
prone background.
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infiltration and TNF- expression in pancreatic islets. We have been
able to show that expression of TNF- in early stages (undetectable T
cell infiltration in most islets) as well as late stages (profound T
cell infiltration) of insulitis correlates well with the DC/M
population, suggesting that these cells could be important producers of
TNF- in the islet infiltrate. However, T cells have been shown to
produce TNF- in the late stages of insulitis and ß cell
destruction (22, 23, 24), and although we find poor correlation between
TNF- and T cell markers, it could be argued that the TNF- found
to correlate with DC/M could be exogenously produced, for example by
T cells, and then taken up by DC/M expressing TNF-R, DC/M are
detectable histochemically in islets before T cells, and it has been
suggested that DC/M are the first cells to infiltrate the islets and
therefore initiate the autoreactive process (27, 28, 29). However, PCR
analysis of TCR Vß elements has shown that T cell clones are present
in the islets of Langerhans as early as 2 wk of age (30), suggesting
that ß cell-specific T cells initiate the disease.
To investigate whether DC/M are able to infiltrate islets and
express TNF- in the absence of T cells, pancreatic sections from 8-
wk-old NOD-SCID mice, which lack T cells, were stained for a panel of
cell surface Ags and TNF-. Indeed, infiltration of N418- and
F4/80-positive cells, as well as TNF- expression, was detected in
islets from NOD-SCID mice (Fig. 7,
a–c), demonstrating that in the early
stages of insulitis, DC/M infiltration occurs independent of T
cells. However, it was noted that levels of DC/M infiltration and
TNF- expression were lower in NOD-SCID mice than in age-matched NOD
mice (Fig. 7, a–f), suggesting that the presence of
T cells accelerates the development of the DC/M infiltration. Thus,
our results strongly suggest that whereas T cells are required for the
development of a massive insulitis and subsequent ß cell destruction,
initiation of insulitis can occur in the absence of T cells. No T cell
infiltration, as determined by CD3+, CD4+, and
CD8+ lymphocytes, could be detected in islets of either
NOD-SCID mice or C57BL/6 mice, whereas it was detectable in NOD islets
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the NOD islet infiltrate at both the early and
late stages of insulitis correlates well with DC and M markers,
whereas a poor correlation with T cells is observed, suggesting that
DC/M are an important source of TNF- in the islet infiltrate. In
previous publications, Held et al. (22) and Toyoda et al. (23) studied
TNF- mRNA expression by islet-infiltrating cells by in situ
hybridization and RT-PCR, respectively, and concluded that
CD4+ T cells and not Mac-1+ macrophages
are the source of TNF- in islets. These results are in contrast to
our own, but the contradictions may be explained by several differences
in the experimental settings. Whereas we have studied the expression of
TNF- protein by immunohistochemistry and immunofluorescence in
islets, previous investigators have studied the expression of TNF-
mRNA by in situ hybridization or RT-PCR. In addition, we studied the
expression of TNF- protein over a broad time period, from 4 wk to 18
wk of age, whereas previous investigators studied TNF- mRNA
expression in older animals, aged 12–18 wk. It could be argued that
the TNF- protein found to correlate with the DC/M population
could be produced by other cell types, for example T cells, and then
taken up by DC/M via TNF-R. However, as TNF- protein expression
can be detected in islets of 4-wk-old NOD mice lacking a detectable T
cell infiltration, as well as in NOD-SCID islets, it is likely that, at
least in young animals, TNF- is produced by islet-infiltrating
DC/M. Another possibility is that production of other cytokines,
such as IL-1, by DC or M may induce ß cells to produce TNF-,
which would then be taken up by islet-infiltrating DC and M.
IL-1-induced TNF- production by the ß cell line ßTC1, as well as
by mouse ß cells, has been demonstrated in vitro (31, 32).
Furthermore, on phenotypic analysis of TNF--expressing cells, we
have investigated the correlation of TNF- and mononuclear cell
markers in situ on pancreatic sections from NOD mice, whereas previous
studies were performed on mononuclear cells purified from pancreata by
collagenase digestion and subsequently sorted by FACS. When purifying
islet-infiltrating cells in this manner, it is probable that mainly
intra-islet-infiltrating cells are obtained, whereas there is the risk
of loosing cells at peri-islet locations, which is where most of the
F4/80+ and Mac-1+ M are situated. In
addition, DC/M are more sensitive to handling than lymphocytes, and
therefore intra-islet DC/M may be underrepresented. This may explain
the low numbers of TNF- mRNA-expressing M observed in these
investigations. Finally, we have been using three different markers to
detect DC/M, whereas previous investigators used only Mac-1, which
is the DC/M marker found to have the lowest correlation with TNF-
in this study.
The aim of this study was to investigate the source of TNF- in the
islet infiltrate. With the methods used in this study, it is difficult
to define the role of TNF- in the development of insulitis and
diabetes. However, the pathogenic role of TNF- at the early stages
of diabetes development has been clearly demonstrated. Treatment of
newborn or 2-wk-old NOD mice with rTNF- leads to an earlier onset
and a higher incidence of diabetes, whereas treatment with
anti-TNF- Abs from birth prevents disease (15), thus supporting
our claim that early production of TNF- is crucial to disease
development. Furthermore, in p55 TNF-R transgenic NOD mice that
constitutively express soluble neutralizing p55 TNF receptors, diabetes
development is inhibited (18). The precise role of TNF- in the
islets at these early time points is not known, but TNF-, in
combination with other cytokines such as IL-1, has been shown to be
toxic to ß cells (12, 13, 14); thus, early expression of TNF- and
other cytokines in islets may induce a partial ß cell dysfunction or
destruction facilitating the uptake of ß cell Ags by APC. TNF- is
also known to be an important maturation factor for DC, inducing the
up-regulation of MHC class I and II, CD40, CD80, CD86, and ICAM-1 on
the cell surface, down-regulating Ag uptake and processing, and
inducing migration into draining lymph nodes, resulting in subsequent
presentation of processed Ag to T lymphocytes (11, 33, 34, 35). These
effects of TNF- on DC may be of major importance for the development
of IDDM. In this report, we are able to show a clear correlation of
TNF- with N418+ DC in young NOD mice. It is possible
that islet-infiltrating DC that have taken up and processed ß cell
Ags are activated by locally produced TNF- in an autocrine or
paracrine fashion. The TNF- signal would then induce the DC to
mature and migrate to the draining lymph nodes where ß cell peptides
would be presented to potentially autoreactive T cells. In addition to
this, TNF- has been shown to induce the up-regulation of endothelial
cell adhesion molecules such as ICAM-1, VCAM-1, and MadCAM-1,
participating in the recruitment of mononuclear cells into the islets
(8, 9, 18). Thus, the correlation of TNF- with APC demonstrated in
this study may be important for the initiation of insulitis leading to
the massive T cell-mediated ß cell destruction and subsequently
resulting in overt diabetes.
Moreover, we are able to show DC/M infiltration in the islets of
NOD-SCID mice. The SCID mutation renders mice immunodeficient by
preventing the normal rearrangement of TCR and Ig genes, resulting in a
lack of mature T and B lymphocytes (36). NOD-SCID mice were generated
by an initial CB17-SCID x NOD Lt cross followed by several
backcrosses of +/SCID heterozygotes (37); NOD-SCID mice remain free
from insulitis and IDDM throughout life (37, 38, 39, 40). To investigate
whether the early DC/M infiltration in islets is T cell dependent,
we stained pancreatic sections from 8-wk-old NOD-SCID mice for a panel
of cell surface markers and TNF- and found peri-islet infiltration
of cells expressing F4/80, N418, and TNF-. These results showed that
the early DC/M infiltration into the islets can occur independently
of T cells but requires the NOD autoimmune background, as C57BL/6
islets lack DC/M infiltration, suggesting that DC/M and not T
cells initiate the autoreactive inflammation and that the TNF-
expressed in islets of young mice is not T cell derived or T cell
dependent.
Although we were unable to observe any T cell infiltration in the
islets of the NOD-SCID mice used in this study, the leakiness that can
be observed in some SCID mice must be considered. It has been shown,
especially in older CB17-SCID mice, that despite the SCID mutation,
clones of mature B and T lymphocytes may develop (41, 42, 43, 44) and that the
frequency of these cells has been shown to increase with age (42, 44).
Thus, in a report by Bosma et al., evidence for mature B cells could be
found in 2.5 to 5.4% of 3- to 4-mo-old CB17-SCID mice, and in 14- to
15-mo-old mice this figure had increased to 32.5% (44). In a later
report by Nonoyama et al., it was demonstrated that varying levels of
leakiness could be observed in 79% of 2- to 3-mo-old CB17-SCID mice
(43). However, the leakiness of SCID mice has been shown to be strain
dependent, and it is much lower in C3H-SCID (43) and in NOD-SCID mice
(40). Indeed, a very low level of T and B cell development has been
demonstrated in NOD-SCID mice, with only 2 of 30 mice, age 100 to 200
days, having detectable serum Ig, whereas Ig could be detected in the
serum from 21 of 29 CB17-SCID mice at the same age (40). Furthermore,
no CD3+ cells could be detected in the spleen of these
mice, and islets remained free from lymphocyte infiltration throughout
life (40). Therefore, we conclude that the DC/M infiltration and
expression of TNF- observed in the islets of NOD-SCID mice is T cell
independent.
What mediates DC/M recruitment and activation in islets is not
clear. However, intrinsic features of pancreatic islets may contribute
to this early recruitment and activation of APC. ß Cell dysfunctions
such as increased insulin responses to glucose (45, 46), increased
basal levels of plasma glucagon (47), some degree of insulin resistance
(46), and an increased number of large size islets (48) have been
observed early in the disease process of NOD mice. In addition, primary
destructive events due to a viral infection of the pancreas have been
suggested (49, 50); any of these features may contribute to the early
DC/M inflammation in NOD islets. Although these results suggest that
initial DC/M infiltration into the islets of Langerhans can occur
independently of T cells, it could clearly be seen that age-matched NOD
mice exhibited a higher level of DC/M infiltration than NOD-SCID
mice, arguing for a critical role of T cells in the development of a
pronounced DC/M infiltration in islets as well as in subsequent ß
cell destruction. We conclude that the early DC/M infiltration and
expression of TNF- in NOD islets can occur independently of T cells
and thus initiate insulitis development, but that a subsequent T cell
infiltration accelerates the process of insulitis and is required for
extensive ß cell destruction and overt diabetes development.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; M, macrophage; HRP, horseradish peroxidase; DAB, diaminobenzamidine.
Received for publication October 6, 1997. Accepted for publication December 2, 1997.
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. J. Exp. Med. 179:1109.This article has been cited by other articles:
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