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*
Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, London, Ontario, Canada;
Departments of Microbiology and Immunology, and Medicine, University of Western Ontario, London, Ontario, Canada;
Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, University of Michigan, Ann Arbor, MI 48109; and
§
Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1):MIP-1ß in the pancreas
correlated with destructive insulitis and progression to diabetes in
NOD mice, a decreased intrapancreatic MIP-1:MIP-1ß ratio was
observed in nonobese diabetes-resistant (NOR) mice. IL-4 treatment,
which prevents diabetes in NOD mice by polarizing intraislet Th2
responses, decreased CCR5 expression in islets and potentiated a high
ratio of MIP-1ß and monocyte chemotactic protein-1 (MCP-1): MIP-1
in the pancreas. Furthermore, NOD.MIP-1-/-
mice exhibited reduced destructive insulitis and were protected from
diabetes. Neutralization of MIP-1 with specific Abs following
transfer of diabetogenic T cells delayed the onset of diabetes in
NOD.Scid recipients. These studies illustrate that the
temporal expression of certain CC chemokines, particularly MIP-1,
and the CCR5 chemokine receptor in the pancreas is associated with the
development of insulitis and spontaneous type I
diabetes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1),5
MIP-1ß, and monocyte chemotactic protein-1 (MCP-1) are representative
of the CC family of chemokines that act primarily upon monocytes and T
cells (1, 3). A role for CC chemokines, particularly
MIP-1, has been noted in experimental autoimmune diseases, such as
experimental allergic encephalomyelitis (EAE) (4, 5, 6).
Certain chemokines may be associated with predominant Th1 or Th2 cell
responses, as evidenced by the linkage of MIP-1 to a Th1
inflammatory response and of MCP-1 to a Th2 response (4, 5, 7).
Chemokines act through specific receptors that belong to the
superfamily of serpentine G protein-coupled receptors. The expression
of chemokine receptors depends on the state of activation or
differentiation of a T cell. CXCR3 and CCR5 are expressed on activated
Th1 cells, whereas CCR3, CCR4, and CCR8 expression characterizes Th2
cells (8, 9, 10, 11). In addition, chemokine receptor expression
can be modulated by cytokines known to influence T cell polarization,
e.g., IFN-
, IL-4, and TGF-ß (12, 13).
Nonobese diabetic (NOD) mice spontaneously develop a form of type I
diabetes that shares many features of the human disease
(14). Mononuclear cell infiltration of pancreatic islets
and the progressive Th1 cell-mediated destruction of insulin-producing
ß cells herald the onset of autoimmune type I diabetes. Previously,
we found that the genetic control of NOD T cell proliferative
hyporesponsiveness to engagement of the TCR is linked to a central
region on chromosome 11 that includes the CC chemokine gene family and
the Idd4 diabetes susceptibility locus (15). We
also found that IL-4 treatment and CD28 costimulation prevent
Th1-mediated destructive insulitis and type I diabetes in NOD mice by
potentiation of regulatory Th2 cell function (16, 17, 18).
IL-4 (19) and CD28 (20) can each modulate CC
chemokine expression. Given the role of CC chemokines in inflammation
and autoimmunity, we analyzed the expression of certain of these
chemokines and their receptors in the pancreas of NOD mice at the
preinsulitis (0–5 wk), nondestructive insulitis (5–12 wk), and
destructive insulitis (>12 wk) inflammatory stages of onset of
diabetes. In particular, we determined whether the intrapancreatic
expression of selected CC chemokines and CCR receptors is associated
with the progression to diabetes onset. Our results demonstrate that
temporal expression of MIP-1 and its CCR5 receptor contributes to
the development of destructive insulitis and spontaneous type I
diabetes in NOD mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD/Del, nonobese diabetes-resistant (NOR), and severe combined immunodeficient NOD (NOD.Scid) female mice were bred in a specific pathogen-free barrier facility at The John P. Robarts Research Institute (London, Canada). Islet infiltration begins at 4–6 wk of age in our colony of female NOD mice, and progression to destructive insulitis and overt diabetes occurs by 4–6 mo of age. The incidence of diabetes in female NOD mice in our colony is 40–50% at 15 wk of age and 80–90% by 25 wk. C57BL/6J and 129/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME).
IL-4 treatment
Fifty nanograms (500 U) of recombinant murine IL-4 (Immunex,
Seattle, WA) in 0.2 ml of vehicle (PBS plus 1% serum from 
6-wk-old
prediabetic female NOD mice) was administered i.p. three times weekly
to female NOD mice from 2 wk (preinsulitis) to 10 wk of age as
previously described (18). Control mice received only
vehicle.
Assay of chemokine protein content in tissues
Pancreata and spleens from 1) age-matched nondiabetic NOD, NOR,
and NOD.Scid mice; 2) nondiabetic and diabetic female NOD
littermates; 3) nondiabetic IL-4-treated and control nondiabetic
vehicle-treated NOD females; and 4) age-matched nondiabetic and
diabetic female
NOD.MIP-1-/- and
NOD.MIP-1+/+ mice were snap-frozen in liquid
N2. Immediately before analysis, tissues were
homogenized and sonicated in an antiprotease buffer (Roche, Laval,
Canada) as previously described (21, 22, 23, 24). Homogenates were
centrifuged to remove debris and were then passed through 1.2-µm pore
size filters (Gelman Sciences, Ann Arbor, MI). Chemokine concentrations
were determined by an ELISA using polyclonal goat anti-murine Abs
(R&D Systems, Minneapolis, MN) with a sensitivity of 
5 pg/ml
(18). ELISA results were normalized relative to the total
protein (Bradford dye-binding protein assay, Bio-Rad, Mississauga,
Canada) derived from each tissue.
Cytokine/chemokine secretion by islet-infiltrated cells
Islet-infiltrated cells (106/ml) isolated
from purified islets (18) from 10-wk-old IL-4- or
control-treated NOD mice (n = 5) were pooled and
cultured for 48 h in complete RPMI 1640 medium (Life Technologies,
Grand Island, NY) and the plate-bound 145-2C11 anti-CD3
mAb
(1/500 dilution of ascites; Cedarlane, Hornby, Canada). Cytokine and
chemokine concentrations in culture supernatants were assayed by ELISA
(18). Cytokine standard curves were linear in the range of
20–2000 pg/ml.
Assay of chemokine receptor expression
Splenic T cells were purified on T cell purification columns
(R&D Systems) to a purity of 98%, as assayed by FACS analysis of CD3
cell surface expression. Islets were isolated as previously described
(18). Total RNA was purified from tissues by the
guanidinium isothiocyanate/silica gel-based membrane RNeasy method
(Qiagen, Valencia, CA) using the manufacturer’s recommended procedure
except that 0.14 M 2-ME was used during the isolation of total islet
RNA. Total RNA was digested with RNase-free DNase (Life Technologies,
Gaithersburg, MD) to remove contaminating genomic DNA,
phenol/chloroform extracted, and ethanol precipitated.
Reverse transcription of RNA (1 µg) was performed using Superscript II (Life Technologies) and oligo(dT) primers. Standardization of cDNA was determined by PCR amplification of GAPDH (sense primer, 5'-CCATGGAGAAGGCTGGGG-3'; antisense primer, 5'-CAAAGTTGTCATGGATGACC-3') in each sample. Equivalent amounts of cDNA (according to GAPDH standardization) were added for quantitation of CCR5 receptor levels. PCR amplification of cDNA was performed using platinum Taq polymerase (Life Technologies), CCR5-specific primers (sense, 5'-ATCATCCTCCTGACAATCGATAGGTACCTG-3'; antisense, 5'-AATCATGATGGTGAAGATAAGCCTCACAGC-3') and CCR2-specific primers (sense, 5'-GGTCATGATCCCTATGTGG-3'; antisense, 5'-CTGGGCACCTGATTTAAAGG-3').
Levels of CCR5 and CCR2 expression were determined by competitive RT-PCR using MIMIC template according to the manufacturer’s instructions (Clontech, Palo Alto, CA). Serial 2-fold (CCR5) or 10-fold (CCR2) dilutions of PCR MIMIC comprised of the primer sequences for CCR5 or CCR2 separated by nonhomologous DNA were amplified, and the products were separated on an agarose gel. Ethidium bromide-stained product band intensities of the MIMIC and cDNA targets were quantified by Gel Doc 1000 video gel documentation (Bio-Rad, Hercules, CA). The amount of target cDNA was determined by comparing the amount of MIMIC required to produce equimolar quantities of CCR5 gene or CCR2 product. The positions of migration of the MIMIC and cDNA targets were determined relative to DNA m.w. standards of 100-1500 bp (Life Technologies).
Adoptive T cell transfer
Female NOD.Scid mice (n = 6/group) at
8–10 wk of age were injected i.p. with splenic T cells (5 x
106) from newly diabetic female NOD mice.
Recipient mice received injections (100 µg/injection) of either a
neutralizing goat anti-mouse anti-MIP-1 or anti-MIP-1ß
polyclonal Ab (R&D Systems) or control normal goat IgG (Cedarlane)
three times a week for 3 wk. Beginning at 2 wk post-transfer, mice were
monitored twice weekly for the presence of hyperglycemia for up to 10
wk. A blood glucose level reading of 11.1 mmol/L glucose on two
consecutive occasions was indicative of the onset of diabetes.
Generation of NOD.MIP-1-/-
mice
C57BL/6.MIP-1-/-
mice, previously shown to be resistant to virus-induced autoimmune
inflammation, were generated in 129 embryonic stem cells by
gene-targeted disruption (25). Mice were backcrossed six
generations onto the NOD background (>98% NOD-like;
Idd1–7, Idd10, Idd12,
Idd14, and Idd15 positive) and intercrossed
to generate mice with the
NOD.MIP-1-/- and
NOD.MIP-1+/+ genotypes. Genotyping was
performed using PCR primers specific for the
MIP-1-/- genotype (Neo
forward primer (5'-TAAAGCGCATGCTCCAGACT-3') and MIP-1 reverse primer
(5'-GAAGTGGAGAAGTCTACTCCA-3')) or the MIP-1+/+
genotype (MIP-1 542 forward primer (5'-CCTCTCTATGGTGTGGAAGG-3') and
MIP-1 reverse primer (5'-GAAGTGGAGAAGTCTACTCCA-3')).
Specific primers were also generated for the PCR genotyping of the D11 NdsI (43.7 cM), D11 Mit38 (49.0 cM), and D11 Mit325 (49.0 cM) polymorphic microsatellite markers in the Idd4 diabetes susceptibility locus of 129/J, C57BL/6, and NOD mice. The chromosomal positions of these markers (indicated in parentheses) were determined according to the most current map of mouse chromosome 11 (Mouse Genome Database, Mouse Genome Informatics, The Jackson Laboratory (http//www.informatics.jax.org)). These analyses narrowed the region of interest to a chromosomal distance of about 5–6 cM.
To address the possibility that the diabetes phenotype observed in
NOD.MIP-1-/- mice
may be due to a 129/J-derived (disease-resistant) linked gene(s), we
generated a control mouse strain, designated NOD.129/J, by backcrossing
129/J to NOD mice for six generations. Mice that expressed
129/J-derived alleles at the CC chemokine locus on mouse chromosome 11
were selected at each generation. Microsatellite markers used to
genotype this region of chromosome 11 were D11 Mit325, D11 Mit98, and
D11 Mit258.
Histopathology analysis
Pancreata were removed, fixed with 10% buffered formalin, embedded in paraffin, and sectioned at 5-µm intervals. The incidence and severity of insulitis were examined by hematoxylin and eosin staining as well as insulin immunostaining. A minimum of 30 islets from each mouse were observed, and the degree of mononuclear cell infiltration was scored independently and blindly by two observers using the following ranking: 0, normal; 1, peri-insulitis (mononuclear cells surrounding islets and ducts, but no infiltration of the islet architecture); 2, moderate insulitis (mononuclear cells infiltrating <50% of the islet architecture); and 3, severe insulitis (>50% of the islet tissue infiltrated by lymphocytes accompanied by a reduction in insulin staining). The immunohistochemical identification of insulin was performed using a porcine anti-insulin Ab and avidin-biotin peroxidase detection system (Dako, Carpinteria, CA).
Statistical analysis
Results were compared using Student’s t test for unpaired samples. p < 0.05 was chosen as the level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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:MIP-1ß ratio in NOD mice
To identify candidate CC chemokines that are associated with the
establishment and/or progression of insulitis, the intrapancreatic
levels of MIP-1, MIP-1ß, and MCP-1 in prediabetic female NOD, NOR,
and NOD.Scid mice (lack functional T and B cells, insulitis-
and diabetes-free) were quantified in whole tissue homogenates by
ELISA. NOD and NOR mice are MHC congenic and share 88% of their
genomes, but islet inflammation does not progress beyond a
nondestructive peri-insulitis in NOR mice (26).
Age-dependent analyses showed that relative to the pancreas of NOR and
NOD.Scid mice, high levels of MIP-1 (600 pg/mg tissue)
are detectable in pancreata of female NOD mice at 5 wk of age during
the onset of insulitis (Fig. 1
A). At 10 wk of age, the
expression of MIP-1 in NOR pancreata increased about 3-fold to the
level found in NOD pancreata. In contrast, a 3-fold higher
intrapancreatic level of MIP-1ß was observed in NOR (1200 pg/mg
tissue) than NOD (400 pg/mg tissue) mice at 5 wk of age, while a 4-fold
higher level (2300 vs 575 pg/mg tissue) was observed in 10-wk-old mice
(Fig. 1B). Equivalent levels of MCP-1 were found in the
pancreata of newborn to 10-wk-old NOD, NOR, and NOD.Scid
mice. MCP-1 content increased starting at 15 wk of age to about 450
pg/mg tissue in 20-wk-old NOR mice and 750 pg/mg tissue in 20-wk-old
NOD and NOD.Scid mice, respectively (Fig. 1D).
Interestingly, C57BL/6J mice between 5 and 30 wk of age do not express
MIP-1, MIP-1ß, or MCP-1 in the pancreas (our unpublished
observations). This result demonstrates the importance of the NOD
genetic background in the control of the patterns of CC chemokine
expression observed in the pancreas of NOD, NOR, and
NOD.Scid mice. Thus, during the initial stages of islet
mononuclear cell infiltration (10 wk of age), the progression to
destructive insulitis in NOD mice vs a benign peri-insulitis in NOR
mice is associated with a higher intrapancreatic MIP-1:MIP-1ß plus
MCP-1 ratio in NOD mice despite the presence of similar pancreatic
levels of MCP-1 in NOD and NOR mice at this time (Fig. 1, C–E).
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:MIP-1ß
Next we investigated whether similar changes in the chemokine
profiles in the pancreas correlate with the onset of diabetes. To
minimize age, lineage, and/or environmental differences,
intrapancreatic chemokine concentrations were determined among female
NOD littermate pairs discordant for diabetes. A 5-fold higher level of
MIP-1 and a 3-fold lower amount of MIP-1ß was found in the
pancreata of 15- to 20-wk-old diabetic female NOD mice relative to that
in nondiabetic littermates (Fig. 2). No
differences in MCP-1 concentrations were observed in the pancreas of
recent onset diabetic and nondiabetic mice. These results indicate that
as destructive insulitis progresses in NOD mice, an additional increase
in the ratio of MIP-1:MIP-1ß in the pancreas is associated with
the onset of overt diabetes.
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Since we have shown that IL-4 treatment protects female NOD mice
from diabetes by stabilizing a Th2-like cytokine environment in the
pancreas (18), we determined whether this IL-4-mediated
protection is accompanied by changes in the levels of intrapancreatic
expression of chemokines. At 5 wk of age, MIP-1 was reduced about
3-fold in IL-4-treated compared with control NOD mice (Fig. 3A), and MIP-1ß was
increased about 6-fold in 10-wk-old IL-4-treated female NOD mice (Fig. 3B). With the exception that a 2-fold increase in the
intrapancreatic content of MCP-1 was detected in 10- and 20-wk-old NOD
mice treated with IL-4 (Fig. 3D), this chemokine profile was
comparable to that found in NOR mice (Fig. 1
, A and
B). At 10 wk of age, MIP-1 content was reduced about
2-fold in the spleen of IL-4-treated female NOD mice relative to that
in control mice, while MIP-1ß and MCP-1 levels were similar in
IL-4-treated and control mice (data not shown). Thus, a reduced ratio
of MIP-1:MIP-1ß plus MCP-1 and a late increase in MCP-1 expression
in the pancreas following IL-4 treatment is associated with protection
from diabetes (Fig. 3, C–E).
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Islet-infiltrated T cells from 10-wk-old IL-4-treated NOD mice
secreted higher levels of IL-4 (5-fold increase) and MIP-1ß
(
50-fold) compared with islet-infiltrated T cells from age-matched
control mice (Fig. 4). IFN- secretion
by islet-infiltrated T cells was detectable (5000 pg/ml) only in
10-wk-old control mice. Less MIP-1 was secreted by islet-infiltrated
T cells of IL-4-treated mice than in control mice; however, the
observed differences were generally less significant (20–25%
decrease). The levels of secretion of MCP-1 (Fig. 4) as well as the
magnitude of proliferative responses by islet-infiltrated T cells from
IL-4-treated and control NOD mice did not differ significantly (our
unpublished observations). Interestingly, basal T cell proliferative
responses in IL-4-treated and control NOD mice were generally higher
than those of splenic T cells in parallel cultures (data not shown).
The latter result probably reflects the increased activity of T cells
isolated from a site of inflammation and may explain the high basal
chemokine production observed in Fig. 4. Thus, the results shown in
Fig. 4 are compatible with those obtained for the CC chemokine levels
(Fig. 3) and cytokine content (18) in the pancreas of
IL-4-treated NOD mice of similar age and support the idea that a higher
IL-4 plus MIP-1ß:IFN- plus MIP-1 ratio is associated with
protection from diabetes.
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In addition to cytokine and chemokine expression, the abilities of
different T cell subsets to migrate to sites of inflammation depend on
their expression of certain chemokine receptors (1, 12).
To further investigate how IL-4 treatment affects lymphocyte
recruitment to the pancreas and prevents diabetes, we compared the
expression of CC chemokine receptor mRNA in splenic T cells and islets
from IL-4-treated and control female NOD mice by semiquantitative
RT-PCR. While no reproducible differences in the expression of CCR1,
CCR2, CCR3, CCR4, CXCR3, and CXCR4 were observed using this method, a
consistent reduction in CCR5 mRNA expression was detected in splenic T
cells and islets of IL-4-treated mice (our unpublished observations).
We therefore used a MIMIC RT-PCR technique to determine more
quantitatively the difference in CCR5 mRNA levels noted between splenic
T cells and islets of IL-4-treated and control NOD mice. The level of
CCR5 mRNA was reduced about 4-fold in islets from 10-wk-old
IL-4-treated NOD mice compared with that expressed by islets from
control mice (Fig. 5A,
lane 2 vs lane 11). No significant changes in
mRNA expression of the control CCR2 were detected in islets from
IL-4-treated mice (Fig. 5B). Similarly, a 4-fold reduction
in the expression of CCR5 mRNA (Fig. 5C, lane 3
vs lane 12), but not CCR2 mRNA (Fig. 5D), was
detected in splenic T cells from 10-wk-old IL-4-treated vs control NOD
mice. Thus, the mRNA expression of CCR5, a CC chemokine receptor
associated with Th1-type immune responses (9, 10, 11), is
selectively down-regulated in splenic T cells and islets of NOD mice
treated with IL-4.
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plays a role in the pathogenesis of type I diabetes
We adopted two approaches to investigate whether a
cause-and-effect relationship exists between the level of expression of
MIP-1 in pancreatic islets and the development of type I diabetes.
First, NOD.Scid recipients of splenic T cells (5 x
106) from diabetic NOD mice received injections
(100 µg/injection) of either a neutralizing polyclonal goat
anti-mouse anti-MIP-1 Ab or control normal goat IgG three
times weekly for 3 wk. At 4 wk after the transfer of diabetogenic T
cells, the incidence of diabetes was 100% in control IgG-treated mice
(Fig. 6). Whereas the incidence of
diabetes was significantly reduced from 100 to 50% in
anti-MIP-1-treated mice at 8 wk post-cell transfer,
anti-MIP-1ß treatment provided only minimal protection, yielding
an incidence of 85% diabetes at this time.
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-/- mice.
Histological sections of pancreas from
NOD.MIP-1-/- and
NOD.MIP-1+/+ mice were scored for insulitis at
10 wk of age. While 60% of the islets displayed either normal
histology or peri-insulitis in the pancreas of 10-wk-old
NOD.MIP-1-/- mice, a
normal histology or peri-insulitis was evident in only 30% of islets
from NOD.MIP-1+/+ mice (Fig. 7A). Conversely, the
percentage (40%) of islets displaying moderate or severe insulitis in
10-wk-old NOD.MIP-1-/-
mice was also lower than that (70%) observed in
NOD.MIP-1+/+ mice. To test the statistical
significance of these data, the severity of islet inflammation was also
scored as nondestructive (combined scores of 0 and 1) and destructive
(combined scores of 2 and 3) infiltration (Fig. 7B). Note
that 10-wk-old NOD.MIP-1-/- mice exhibit a
significantly higher proportion of islets of a nondestructive phenotype
(p < 0.01) and a significantly lower
proportion of islets of a destructive phenotype
(p < 0.01) relative to
NOD.MIP-1+/+ mice. Thus, deficient MIP-1
expression in NOD mice reduces the amount of destructive insulitis in
the pancreas.
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-/- female
mice compared with NOD.MIP-1+/+ mice (Fig. 7C). We determined that
NOD.MIP-1-/- mice
express the Idd1, Idd2, Idd3,
Idd4, Idd5, Idd6, Idd7,
Idd10, Idd12, Idd14, and
Idd15 diabetes susceptibility loci. Despite the observed
lower incidence of 60% diabetes in
NOD.MIP-1+/+ mice compared with the 80–90%
incidence noted in wild-type NOD female mice, we found that the
incidence of diabetes approached 90% in three of the seven
NOD.MIP-1+/+ litters. The overall lower
incidence of diabetes in NOD.MIP-1+/+ mice may
result from the incomplete fixation of other Idd loci in
these mice. Clinical determinations of these mice following the onset
of overt diabetes, including blood glucose measurements, physical
assessments, and response to insulin therapy, have not revealed any
differences in the severity of disease between
NOD.MIP-1-/- and
NOD.MIP-1+/+ mice.
The Idd4 locus located on mouse chromosome 11 encompasses
the CC chemokine gene family. This raises the possibility that 129- or
C57BL/6-derived Idd4 genes cosegregated with the mutated
MIP-1 gene during the backcrossing of this gene onto the NOD genetic
background. To examine whether such 129/J- or C57BL/6-derived
cosegregating genes influence the incidence of diabetes in our
NOD.MIP-1+/+ or
NOD.MIP-1-/- mice, we
analyzed the genotype in chromosome 11 regions centromeric and
telomeric to the CC chemokine locus. We found that there is probably
minimal influence of these cosegregating genes on the incidence of
diabetes, since regions flanking this chemokine locus were genotyped as
being NOD in origin. Thus, the Idd4 locus in
NOD.MIP-1-/- mice was
derived from the NOD strain and could not account for the phenotype of
NOD.MIP-1-/- mice.
Since Idd4 may mask the effects of cosegregating 129/J genes
on chromosome 11, we reasoned that the
NOD.MIP-1+/+ strain is an appropriate genetic
control for
NOD.MIP-1-/- mice. A
similar approach was previously used to rule out possible effects of
the Idd6 locus cosegregating with a mutated TNFR1
gene on mouse chromosome 6 in TNFR1-deficient mice
(27). The possibility that undefined resistant
Idd alleles were linked to a mutated CIITA gene
on mouse chromosome 16 in CIITA-deficient mice was also
eliminated using this approach (28).
We also observed that G6F1 NOD.129/J backcross control female mice were
completely protected from diabetes (12 of 12) at 25 wk of age (data not
shown), a time at which the incidence of diabetes is >80% in female
NOD mice in our colony. This result was expected due to the
colocalization of the Idd4 locus and the MIP-1 gene on
chromosome 11. Since a large region of 129/J-derived genes on
chromosome 11 probably confers resistance to diabetes, the NOD.129/J
strain may be a less appropriate control for the
NOD.MIP-1-/- mice than
the NOD.MIP-1+/+ strain. Analyses of
NOD.MIP-1+/+ mice enabled us to examine the
possibility that Idd4 derived from the NOD strain interfered
with the assessment of diabetes in
NOD.MIP-1-/- mice.
These results provide further evidence that MIP-1 plays a unique
effector role in the pathogenesis of type I diabetes in NOD
mice.
To examine the changes in chemokine profiles in the pancreas of female
NOD.MIP-1-/- and
NOD.MIP-1+/+ mice and their correlation to the
onset of diabetes, intrapancreatic chemokine concentrations were
determined in whole tissue homogenates by ELISA. MIP-1 was not
detected in 15- to 20-wk-old nondiabetic and diabetic
NOD.MIP-1-/- mice
(Fig. 7D). While an average of about 400 pg/mg tissue of
MIP-1ß was found in the pancreata of nondiabetic
NOD.MIP-1-/- mice,
MIP-1ß was not detected in diabetic
NOD.MIP-1-/- mice
(Fig. 7D). No significant differences in MCP-1
concentrations were observed in the pancreas of recent onset diabetic
and nondiabetic
NOD.MIP-1-/- mice.
Levels of intrapancreatic chemokines in 15- to 20-wk-old nondiabetic
and diabetic NOD.MIP-1+/+ mice (data not
shown) were similar to results obtained from 15- to 20-wk-old
nondiabetic and diabetic wild-type NOD mice (Fig. 2). Thus, while the
absence of MIP-1 reduces insulitis and diabetes incidence in
NOD.MIP-1-/- mice,
impaired expression of MIP-1ß in the pancreas is characteristic of
NOD.MIP-1-/- mice that
progress to overt diabetes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-1ß, and MCP-1 in the pancreas are
associated with either the progression or the prevention of insulitis
and spontaneous type I diabetes. We found that early pancreas
inflammation (10 wk of age) correlates with a high relative
expression of MIP-1 in NOD mice. During this stage, NOD islets are
infiltrated by both APCs and T cells, and this infiltration is
accompanied by the subsequent expression of proinflammatory Th1
cytokines associated with an invasive insulitis (26). By
comparison, NOR islet infiltrates are composed primarily of APCs and
relatively few, if any, T cells at this time. Our findings that
pancreatic MIP-1 levels are high and MIP-1ß levels are low during
early islet infiltration in NOD mice may partially explain the
differences in composition of the NOD and NOR islet cellular
infiltrates.
The transgenic expression of MCP-1 in islet ß cells yields an
insulitis characterized by a nondestructive peri-islet monocyte
infiltrate (29). During the early stages of islet
infiltration in NOD and NOR mice, MCP-1 expression by pancreas-resident
cells may contribute to the recruitment of a first wave of mononuclear
cells composed mainly of APCs with a consequent attraction of
lymphocytes. The outcome of this inflammatory response may depend on
the presence of other chemokines, such as MIP-1 and MIP-1ß. In
this manner early intrapancreatic expression of CC chemokines may
determine the quality of the islet infiltrate as destructive insulitis
develops in female NOD mice. Differences in the severity of insulitis
between NOD and NOR mice may also be regulated by genetically
determined factors, such as the ability of resident APCs to interact
with autoreactive T cells and modulate the profiles of intraislet
cytokines and chemokines. Indeed, NOD.Scid mice, which do
not have functional T or B cells, express detectable levels of MCP-1
but neither MIP-1 nor MIP-1ß in the pancreas, suggesting that the
intrapancreatic expression of MIP-1 and MIP-1ß depends on the
presence of infiltrated mononuclear cells.
An increase in the intrapancreatic MIP-1:MIP-1ß ratio was also
noted in recent onset diabetic NOD female mice relative to their
nondiabetic littermates. This pattern of chemokine content correlates
with the presence of a Th1-enriched environment in the pancreas at
diabetes onset (30, 31, 32), consistent with the presence of a
higher IFN-:IL-4 concentration ratio in the pancreata of diabetic
female NOD mice than in nondiabetic NOD mice (31, 32).
Collectively, these findings implicate a temporal relationship between
a higher intrapancreatic MIP-1:MIP-1ß ratio and the development of
destructive insulitis and overt diabetes in NOD mice. Thus, although
MIP-1 and MIP-1ß are highly related proteins, they may have
opposing functions during autoimmune inflammation
(33, 34, 35).
Our previous finding that IL-4 protects NOD mice from diabetes
(18) was informative about the biological relevance of the
chemokine profiles observed in unmanipulated NOD pancreata. Most
importantly, we found that the MIP-1:MIP-1ß plus MCP-1 ratio in
the pancreas was reduced in IL-4-treated compared with control NOD
mice. MIP-1 can stimulate macrophage secretion of IL-1, IL-6, and
TNF-, which may promote NO production by macrophages and induce
Fas-mediated apoptosis of islet ß cells (36, 37).
MIP-1 also preferentially attracts CD8+ T
cells, which are required for the development of diabetes and are a
source of MIP-1 in vivo (34, 38, 39). Thus, inhibition
of MIP-1 by IL-4 may help protect islet ß cells from attack by
effector CD8+ T cells. In addition, since IL-4
can suppress MIP-1 secretion by stimulated monocytes
(19), IL-4 treatment of NOD mice may inhibit the
production of MIP-1 by monocytes/macrophages and/or islet-derived
endothelial cells, thereby reducing early islet infiltration. Finally,
the results obtained with IL-4 treatment suggest a mechanism of Th2
cell-mediated protection of NOD mice from diabetes (18).
Thus, the level of MIP-1 expression is down-regulated in the spleen
and pancreas of IL-4-treated mice consistent with its association with
Th1 responses (4, 5, 6, 7).
Previous studies have shown that in vivo neutralization of the activity
of MIP-1 prevented EAE, while the neutralization of MIP-1ß
significantly exacerbated the disease (5, 33). In
contrast, we found that anti-MIP-1ß treatment not only did not
accelerate the development of diabetes in NOD.Scid
recipients of transferred diabetogenic T cells but, rather, provided a
small amount of protection of these recipients. This may have resulted
from the high autoreactivity of the T cells from diabetic mice and
their ability to rapidly transfer disease in this acute model. Indeed,
increased MIP-1ß levels in the pancreas are associated with
IL-4-mediated protection from diabetes only at 10 wk of age. The latter
finding is consistent with our recent report that the transfer of
regulatory T cells induced by oral insulin treatment of NOD mice
elicits a nondestructive insulitis characterized by a MIP-1ß-enriched
environment in the pancreas of NOD.Scid recipients
(40). Inasmuch as an increased expression of MIP-1ß
occurs only in the pancreata of IL-4-treated NOD mice, MIP-1ß may
mediate the down-regulation of Th1 cell responses in the pancreas, but
not secondary lymphoid organs (e.g., spleen). This idea is also
reflected by our finding that the relative amount of MIP-1ß is high
in the pancreas of diabetes-free
NOD.MIP-1-/- compared
with that in diabetic
NOD.MIP-1-/- mice. Our
results support the ideas that a higher ratio of IL-4 plus
MIP-1ß:IFN- plus MIP-1 in the pancreas affords protection from
diabetes and that islet-infiltrated T cells are probably the primary
source of MIP-1 and MIP-1ß in the pancreas.
The intrapancreatic content of MCP-1 increased in 10- and 20-wk-old NOD
mice treated with IL-4. This is an interesting finding, since our
analyses of NOD.Scid mice demonstrated that MCP-1 expression
does not depend on pancreas inflammation or the presence of B and T
cells. C57BL/6 mice (diabetes-resistant) between 5 and 30 wk of age do
not express MIP-1, MIP-1ß, or MCP-1 in the pancreas, demonstrating
the importance of the NOD/NOR genetic background in pancreas chemokine
expression (our unpublished observations). MCP-1 and IL-4 are expressed
coordinately during several immune responses, and MCP-1 blocks the
adoptive transfer of EAE (4, 7). An increase in mucosal
MCP-1 elicited by an oral autoantigen regulates tolerance induction by
down-regulating Th1 responses and increasing IL-4 in EAE
(41). IL-4 may induce a Th2 environment and the production
of MCP-1 in pancreatic islets, particularly by endothelial cells
(42). Indeed, our detection of differences in MCP-1
production in whole pancreata but not islet T cell infiltrates is
compatible with the idea that the source of increased MCP-1 following
IL-4 treatment may be islet-resident cells.
Th1 and Th2 cells differentially express chemokine receptors and
migrate in response to different chemokines. The selective
down-regulation of splenic T cell and intrapancreatic CCR5 mRNA may be
significant due to its association with Th1 immune responses
(9, 10, 11). Accordingly, reduced CCR5 mRNA in splenic T cells
and islet-infiltrated cells after IL-4 treatment may reflect diminished
Th1 activity. It is also possible that reduced CCR5 message in islets
merely reflects the lower levels of T cell infiltration in IL-4-treated
mice. We believe that the reduction in CCR5 mRNA expression is not
solely due to a general reduction in insulitis afforded by IL-4
treatment, since the levels of expression of several other chemokine
receptors, i.e., CCR1, CCR2, CCR3, CCR4, CXCR3, and CXCR4, remain
unaltered, and an identical reduction in the level of CCR5 mRNA was
observed in splenic T cells from IL-4-treated mice. In addition, we
observed similar selective decreases in CCR5 expression in the
infiltrated islets of 10-wk-old NOR mice (our unpublished
observations), which, as discussed above, exhibit a intrapancreatic
profile of MIP-1, MIP-1ß, and MCP-1 expression similar to that in
IL-4-treated NOD mice.
Our findings that neutralization of MIP-1 in
NOD.Scid mice upon adoptive transfer of diabetogenic T cells
and that deficient expression of MIP-1 in NOD mice protects them
from destructive insulitis and diabetes onset suggest that MIP-1
profoundly influences the pathogenesis of type I diabetes in NOD mice.
Since MIP-1 may both influence the quality of the islet infiltrate
and act as a costimulatory factor (Refs. 2, 39 , and
43 and this report), a deficiency in MIP-1 may
establish a higher threshold for the activation and expansion of
autoreactive T cells and other effector cells, such as macrophages. In
this regard it is noteworthy that increases in the level of MIP-1 as
well as the number of T cells expressing the CCR5 receptor occur in the
CNS of patients with active multiple sclerosis (44). Thus,
interactions between MIP-1 and CCR5 may contribute to the
pathogenesis of inflammatory lesions in several T cell-mediated
autoimmune diseases.
In conclusion, our findings provide in vivo evidence for a role
of certain CC chemokines and CC chemokine receptors in the development
and pathogenesis of type I diabetes. MIP-1 and MCP-1 play an early
role in the recruitment of mononuclear cells to the islet and the
establishment of insulitis in NOD mice. Young NOR mice exhibit a
decreased intrapancreatic MIP-1:MIP-1ß plus MCP-1 ratio consistent
with their diabetes resistance. A close correlation exists between Th2
cytokine responses, a high MIP-1ß plus MCP-1:MIP-1 chemokine
ratio, and reduced CCR5 expression elicited following IL-4 treatment in
the pancreas of NOD mice protected from diabetes. Disruption of
MIP-1 expression in NOD mice prevents and delays the onset of overt
disease, an observation that can be explained in part by a reduction in
destructive insulitis in these mice. There is growing evidence that the
interrelationship of cytokines, chemokines, chemokine receptors, and
adhesion molecules determines the extent of leukocyte migration to and
the nature of inflammation at different target sites (1
and 44, 45, 46 and this report). Further experimentation is
required to determine the chemokine and chemokine receptor interactions
as well as the phenotypes of islet infiltrated cells in the pancreas of
NOD.MIP-1-/- mice.
Temporal modulation of CC chemokines and their interactions with CC
chemokine receptors may represent a novel therapeutic approach in the
prevention of the development of insulitis and spontaneous type I
diabetes.
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Acknowledgments |
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Footnotes |
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2 M.J.C. and G.A.A. contributed equally to this work.
3 Current address: Department of Immunology, Schering Plough Research Institute, Kenilworth, NJ 07033.
4 Address correspondence and reprint requests to Dr. Terry L. Delovitch, Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, 1400 Western Road, London, Ontario, Canada N6G 2V4.
5 Abbreviations used in this paper: MIP, macrophage inflammatory protein; EAE, experimental allergic encephalomyelitis; NOD, nonobese diabetic; NOR, nonobese diabetes resistant; NOD.Scid, severe combined immunodeficient NOD; MCP, monocyte chemotactic protein.
Received for publication October 14, 1999. Accepted for publication April 25, 2000.
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