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*
Roche Milano Ricerche, Milano, Italy;
The Jackson Laboratory, Bar Harbor, ME 04609; and
Department of Biotechnology, Hoffmann-La Roche, Nutley, NJ 07110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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80% in draining lymph nodes of these mice, and addition
of IL-12, but not IL-18, restored Th1 development in vitro, indicating
a nonredundant role of IL-12. Moreover, spontaneous Th1 responses to a
self Ag, the tyrosine phosphatase-like IA-2, were undetectable in
lymphoid organs from IL-12-deficient, in contrast to wild-type, NOD
mice. Nevertheless, wild-type and IL-12-deficient NOD mice developed
similar insulitis and IDDM. Both in wild-type and IL-12-deficient NOD
mice, 20% of pancreas-infiltrating CD4+ T cells
produced IFN-
, whereas very few produced IL-10 or IL-4, indicating
that IDDM was associated with a type 1 T cell infiltrate in the target
organ. T cell recruitment in the pancreas seemed favored in
IL-12-deficient NOD mice, as revealed by increased P-selectin ligand
expression on pancreas-infiltrating T cells, and this could, at least
in part, compensate for the defective Th1 cell pool recruitable from
peripheral lymphoid organs. Residual Th1 cells could also accumulate in
the pancreas of IL-12-deficient NOD mice because Th2 cells were not
induced, in contrast to wild-type NOD mice treated with an IL-12
antagonist. Thus, a regulatory pathway seems necessary to counteract
the pathogenic Th1 cells that develop in the absence of IL-12 in a
spontaneous chronic progressive autoimmune disease under polygenic
control, such as IDDM. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as well as IL-2 and TNF-ß, whereas Th2 cells are
characterized by secretion of IL-4, IL-5, and IL-10
(4). The important and nonredundant role of IL-12 in the induction of Th1 responses has been demonstrated in mice deficient for IL-12 (5), IL-12Rß1 (6), or Stat4 (7). IL-12-deficient mice fail to control mycobacterial infections due to a decreased ability to develop Th1-mediated protective immunity (8). Similarly, humans with genetic deficiency of IL-12 or the IL-12 receptor demonstrate systemic dissemination of otherwise poorly pathogenic bacteria (9). In contrast, IL-12 deficiency does not alter the control of viral infections, indicating that alternative pathways for the generation of type 1 responses may be induced (10, 11).
A pathogenic role of Th1 cells is documented in several autoimmune diseases, such as experimental allergic encephalomyelitis (EAE),4 collagen-induced arthritis (CIA), experimental autoimmune uveitis (EAU), and experimental autoimmune myasthenia gravis (EAMG) (reviewed in Refs. 12, 13). In all of these models, IL-12-deficient mice are protected from disease, either completely (14, 15) or partially (16, 17).
In contrast to induced autoimmune disease models, insulin-dependent diabetes mellitus (IDDM) develops spontaneously in nonobese diabetic (NOD) mice (18). The disease is characterized by a progressive mononuclear cell infiltration in the pancreatic islets of NOD mice, which starts at about 4 wk of age and leads to ß cell destruction and hyperglycemia (18, 19). Pancreas-infiltrating CD4+ and CD8+ T cells have a type 1 phenotype, as demonstrated by intracytoplasmic staining for cytokine production (13).
We have previously shown that daily administration of IL-12 accelerated IDDM, and this acceleration was associated with increased type 1 cytokine production by pancreas-infiltrating CD4+ and CD8+ T cells and selective elimination of islet ß cells (20). These data, consistent with a dominant role of Th1 cells in the pathogenesis of IDDM, did not determine whether IL-12 is obligatory for the development of diabetogenic Th1 cells. We addressed this question by targeting endogenous IL-12 in NOD mice with the IL-12 antagonist (p40)2 (21). Treatment with (p40)2 started at 3 wk of age, resulted in the deviation of pancreas-infiltrating CD4+ cells to the Th2 phenotype at the expense of Th1-type cells, as well as in delay and reduction of IDDM incidence. Reduction of IDDM incidence by IL-12 antagonist administration to NOD mice was also obtained by Rothe et al. (22). Nevertheless, it remained unclear whether Th2 cells exerted a direct protective role, inhibiting pathogenic Th1 cells, or whether the decrease in Th1 development could by itself account for IDDM protection. The role of Th2 cells in IDDM is still controversial. Th2 lymphocytes may be neutral (23, 24), may actively inhibit pathogenic Th1 activity (25), and, in immunodeficient hosts, they may even be pathogenic (26).
We have generated IL-12-deficient NOD mice to clarify the requirement for IL-12 in the development of diabetogenic Th1 cells, as well as to provide further insight into the role of Th1/Th2 regulation in IDDM pathogenesis. Unexpectedly, our findings indicate that the development of diabetogenic type 1 T cells and their accumulation in pancreatic islets can occur via IL-12-independent mechanisms. In addition, they suggest that a regulatory pathway is required to counteract the pathogenic type 1 T cells that develop in the absence of IL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A functionally inactivated IL-12p40 allele originally generated on the 129/SvEv genetic background (5) was backcrossed to NOD/Lt mice obtained from The Jackson Laboratory (Bar Harbor, ME) for a total of eight generations. Progeny were screened for the presence of the IL-12p40 mutation by Southern blot analysis, as described previously (5). The IL-12p40 gene maps to a region of chromosome 11 close to the Idd4 locus. Thus, mice used for experimental analyses were all derived from a single seventh generation backcross female that was shown by the previously described PCR-typing protocols (27) to be homozygous for NOD allelic variants at the Idd4 microsatellite linkage markers D11 Mit115, D11 Mit140, and D11 Mit320. At the eighth backcross generation, IL-12p40+/- heterozygotes were then intercrossed to generate two types of mice on the NOD background: mice homozygous for the IL-12p40 mutation (IL-12-deficient NOD mice) and mice homozygous for the wild-type IL-12p40 allele (wild-type NOD mice). All mice were bred and maintained under specific pathogen-free conditions. A diagnosis of diabetes was made after two sequential measurements of blood glucose levels higher than 200 mg/dl.
Mouse IL-12p40 homodimer
Mouse IL-12(p40)2 [(p40)2] was produced by Chinese hamster ovary cells stably transfected with the mouse IL-12p40 cDNA and purified as previously described (28). The endotoxin contamination of the purified (p40)2 was <2 e.u./mg (p40)2, as assayed by the Limulus amebocyte assay. For in vivo administration, (p40)2 was diluted in Dulbecco’s PBS (Life Technologies, Grand Island, NY) containing 100 µg/ml mouse albumin (Sigma, St. Louis, MO). NOD/Lt mice were injected i.p. daily, from 3 to 10 wk of age, with 3 mg/kg recombinant mouse (p40)2 or with vehicle, as previously described (21).
LPS-induced IFN- production in vivo
Mice were injected i.p. with 400 µg of LPS from
Salmonella abortus equi (Sigma). The mice were
bled 3 and 6 h later, and serum IL-12p40 and IFN-,
respectively, were measured by ELISA.
Induced T cell responses to hen egg-white lysozyme (HEL) and protein-purified derivative (PPD)
NOD mice were immunized into the hind footpads with either CFA containing H37Ra mycobacteria (Difco, Detroit, MI) or with 3.5 nmoles/mouse HEL emulsified in IFA. Draining lymph nodes were removed 9 days after immunization, and 6 x 105 cells/well were cultured in 96-well culture plates with Ag in synthetic HL-1 medium (Ventrex Laboratories, Portland, ME) supplemented with 2 mM L-glutamine and 50 µg/ml gentamicin (Sigma). The recall Ag was either 30 µg/ml PPD (Statens Seruminstitut, Copenhagen, Denmark), for the mice immunized with CFA, or 10 µM HEL. In addition, cells were incubated with or without 0.5 ng/ml recombinant mouse IL-12 (kindly provided by Dr. M. K. Gately, Hoffmann-La Roche, Nutley, NJ) or 12 ng/ml IL-18 (Research Diagnostic, Flanders, NJ). After 72 h of culture, supernatants were collected for quantification of secreted cytokines by ELISA. The cells were harvested, washed, and recultured for an additional 72 h in RPMI 1640 medium (Life Technologies) supplemented with 50 µM 2-ME (Fluka Biochemica, Buchs, Switzerland), 2 mM L-glutamine, 50 µg/ml gentamicin, and 10% FCS (complete medium) for the detection of intracellular production of cytokines. Afterwards, the cells were stimulated by either PPD or PMA/ionomycin. In the first case, 2.5 x 105 cells/well were incubated in 96-well plates for 24 h at 37°C with PPD and 106 T cell-depleted mitomycin C-treated splenocytes from either IL-12-deficient or wild-type NOD mice in the presence of 10 µg/ml brefeldin A (BFA; Novartis, Basel, Switzerland). Cells were resuspended in PBS containing 10 µg/ml BFA before adding an equal volume of 4% paraformaldehyde. After fixing for 20 min, cells were stained for intracytoplasmic cytokines. Otherwise, 2.5 x 105 cells/well were stimulated with 50 ng/ml PMA and 750 ng/ml ionomycin (all from Sigma) for 4 h at 37°C, and 10 µg/ml BFA was added for the last 2 h. Cells were resuspended in PBS containing 10 µg/ml BFA, fixed as above, and stained for intracytoplasmic cytokines.
Spontaneous T cell responses to IA-2
Total spleen cells (106 cells/well) from
10-wk-old nonimmunized NOD mice were cultured in 96-well plates in
complete medium containing 2.5% horse serum (Life Technologies) with
or without 0.3 µM recombinant mouse IA-2. After 48 h of
incubation at 37°C, IFN- and IL-10 secretion were determined in
culture supernatants by ELISA. Alternatively, splenic or mesenteric
lymph node CD4+ cells were sorted by positive
selection on MiniMACS (Miltenyi Biotec, Auburn, CA). These
CD4+ cells (2 x
105/well) were cultured with T cell-depleted and
mitomycin C-treated spleen cells (106 cells/well)
in 96-well plates with the indicated concentrations of IA-2. After
72 h, IFN- and IL-10 were determined in culture supernatants.
Purified recombinant mouse IA-2 protein (protein tyrosine
phosphatase-like, PTP 35) (29) was a kind gift of Dr.
Antonella Isacchi (Pharmacia-Upjohn, Milan, Italy).
Quantification of secreted cytokines
Cytokines were quantified by two-sites ELISA. To detect IFN-,
polyvinyl microtiter plates (Falcon 3012) were coated with 100 µl of
AN-18.17.24 mAb (30) in carbonate buffer. After blocking,
samples (50 µl/well) diluted in test solution (PBS containing 5% FCS
and 1 g/L phenol) were incubated together with 50 µl
peroxidase-conjugated XMG1.2 mAb (31). After overnight
incubation at room temperature, bound peroxidase was detected by
3,3',5,5'-tetramethylbenzidine (Fluka Chemical, Ronkonkoma, NY), and
adsorbance read at 450 nm with an automated microplate ELISA reader
(MR5000; Dynatech Laboratories, Chantilly, VA). To detect IL-12p40, the
mAb used for capture was 10F6 anti-IL-12p40 followed by
biotin-conjugated goat anti-IL-12, as described (32).
Anti-IL-12 Abs were kindly provided by Dr. M. K. Gately
(Hoffmann-LaRoche). IL-4 and IL-10 were determined using paired mAb
from PharMingen (San Diego, CA). For capture, the mAb were BVD4-1D11 or
11B11 (anti-IL-4) and JESS-2A5 (anti-IL-10). Samples were
titrated in test solution and incubated overnight at 4°C. To detect
bound cytokines, plates were then incubated with the biotinylated mAb
BVD6-24G2 (anti-IL-4) or SXC-1 (anti-IL-10) in PBS containing
0.1% Tween 20 and 1% BSA (PBSA-Tw). After washing, the bound
biotinylated Abs were revealed by an additional 30-min incubation with
alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch
Laboratories, Avondale, PA) diluted 1/5000. The plates were washed
again and incubated with the developing substrate
p-nitrophenylphosphate disodium (Sigma) in diethanolamine
buffer (pH 9.6; 100 µl/well). The reaction was stopped by adding 50
µl/well NaOH 3N, and absorbance was read at 405 nm. Cytokines were
quantified from two to three titration points using standard curves
generated by purified recombinant mouse cytokines and results expressed
as cytokine concentration in ng or pg/ml. Detection limits were 15
pg/ml for all cytokines, unless otherwise stated.
Isolation and staining of pancreas-infiltrating T cells
After removal of all visible pancreatic lymph nodes, pancreata were individually digested in 3 ml HBSS containing 1 mg/ml collagenase IV (Sigma), by shaking (200 rpm) at 37°C for 15 min. Single cell suspensions were collected after diluting the enzyme with ice-cold HBSS containing 5% FCS and removal of the aggregates by settling for 2 min on ice. Aggregates were further digested with collagenase IV at 0.5 mg/ml for 10 min, and at 0.25 mg/ml for 6 min. Single cell suspensions were washed three times, and CD4+ and CD8+ cells were sorted by positive selection on MiniMACS. Pancreas-infiltrating CD4+ and CD8+ cells were double stained by incubating them with optimal concentrations of PE-labeled rat anti-mouse CD4 (RM4-4) or CD8 (53-6.7) in the presence of rat anti-mouse FcR (2.4G2) (all from PharMingen) and P-selectin-IgG chimeric protein (kind gift of Dr. D. Vestweber, University of Muenster, Muenster, Germany) followed by FITC-labeled rabbit F(ab')2 anti-human IgG. Alternatively, pancreas-infiltrating CD4+ and CD8+ cells were directly stimulated with PMA and ionomycin in complete medium, fixed, and stained the next day for intracellular production of cytokines.
Intracellular staining for cytokine production
Cells were stained for IFN-, IL-4, and IL-10 using the method
described by Openshaw (33) and mAbs obtained from
PharMingen. Reagents for intracytoplasmic staining contained 1% FCS,
0.5% saponin (Sigma), and 0.1% sodium azide, and all incubations were
performed at room temperature. Cells were washed, preincubated for 10
min with PBS/FCS/saponin, and then incubated with FITC-labeled rat
anti-mouse IFN- (XMG1.2) and PE-labeled rat anti-mouse IL-4
(11B11), or PE-labeled rat anti-mouse IL-10 (JES5-16E3). Isotype
controls were FITC- and PE-labeled rat IgG1 (R3-34). After 30 min,
cells were washed twice with PBS/FCS/saponin and then with PBS
containing 5% of FCS without saponin to allow membrane closure. The
cell surface was then stained with CyChrome-labeled anti-CD4 (L3T4)
for 15 min at room temperature. Analysis was performed with a FACScan
flow cytometer (Becton Dickinson, Mountain View, CA) equipped with
CellQuest software, and 10,000 events were acquired.
Immunohistology
Pancreata were snap-frozen in Tissue Tek (Miles Laboratories, Elkhart, IN), and 5-µm-thick sections stained with hematoxilin and eosin. Insulitis score was quantified as follows: 0, no insulitis; 1, peri-insulitis; 2, insulitis in <50% of the islet; 3, insulitis in >50% of the islet. A mean score was calculated from 40–50 islets per individual pancreas. In addition, pancreas cryostat sections were stained with biotinylated mAb directed against CD4, CD8, B220, CD11b (all purchased from PharMingen), or CD11c (N418; American Type Culture Collection, Manassas, VA), followed by streptavidin-peroxidase conjugate. 3-amino-9-ethylcarbazole (Dako, Carpenteria, CA) was used as chromogen and hematoxylin as a counterstain.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production
LPS-induced serum IFN- levels in IL-12-deficient B6 or BALB/c
mice are reduced by 80%, as compared with wild-type littermates
(5). We analyzed LPS-induced serum levels of IL-12p40 and
IFN- in IL-12-deficient and wild-type NOD mice. Results shown in
Fig. 1
confirmed the absence of IL-12p40
in IL-12-deficient NOD mice and the reduction of serum IFN- levels
by 80%. Therefore, IFN- production in response to endotoxin is
strongly, although not completely, inhibited in IL-12-deficient NOD
mice, consistent with results obtained in other mouse strains.
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To test the role of endogenous IL-12 in the development of Th1
responses in NOD mice, we examined cytokine production in response to
two different Ags, HEL and PPD. PPD was chosen for its capacity to
preferentially induce IL-12-dependent Th1 responses (34).
IL-12-deficient and wild-type NOD mice were immunized with either HEL
emulsified in IFA or with CFA alone. Nine days later, draining lymph
node cells were restimulated in vitro with HEL or PPD, respectively.
The immune cells from NOD mice secreted high levels of IFN-, 15 and
23 ng/ml, respectively (Fig. 2,
A and B). In contrast, the amount of IL-4
secreted by HEL or PPD-specific cells was 120 pg/ml and <15 pg/ml,
respectively. As compared with wild-type mice, IL-12-deficient NOD mice
showed near abrogation of Ag-specific IFN- secretion, whereas IL-4
was increased and IL-10 secretion was slightly enhanced (Fig. 2, A and B). The phenotype of PPD-specific
CD4+ was characterized at the single cell level
by intracytoplasmic staining for IFN- and IL-4 production. Immune
lymph node cells, after culture for 72 h with PPD, were rested for
an additional 72 h without Ag and restimulated with either PPD or
PMA/ionomycin before staining for intracytoplasmic cytokines and
surface CD4 expression. With both stimuli, IL-4-producing
CD4+ cells were undetectable either in wild-type
or IL-12-deficient NOD mice (Fig. 2C). In contrast, 10% and
25% of CD4+ lymph node cells from wild-type NOD
mice produced IFN- when restimulated with PPD or PMA/ionomycin,
respectively (Fig. 2C). These proportions were reduced by
80% and 64%, respectively, in CD4+ cells from
IL-12-deficient NOD mice. Thus, a qualitatively similar pattern of
intracytoplasmic cytokine production was induced by restimulation with
PPD or PMA/ionomycin. Altogether, the results show that Th1 development
is impaired in IL-12-deficient NOD mice, without an appreciable
induction of Th2 cells.
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, increasing the frequency of Th1 cells by
2- to 3-fold (Fig. 2
C), and the amount of secreted IFN-
increased accordingly (Fig. 2B). Addition of IL-12, together
with PPD, to lymph node cells from IL-12-deficient NOD mice yielded a
similar proportion of IFN--producing CD4+
cells and similar levels of IFN- secretion, as in controls. In
addition, it inhibited the limited secretion of IL-4 (Fig. 2B).
Since IL-18 shares some biological functions with IL-12
(35), we also tested the capacity of IL-18 to induce
differentiation of Th1 cells. Addition of IL-18 to the cell culture
from wild-type NOD mice did not modify the proportion of cells
secreting IFN-, in contrast to the results obtained with exogenous
IL-12 (Fig. 2C). However, the total amount of IFN- in the
culture supernatant increased substantially, indicating that IL-18
stimulated individual Th1 cells to produce more IFN-. This effect
was IL-12-dependent, since it was abrogated in cells from
IL-12-deficient NOD mice (Fig. 2B). Addition of IL-18 to the
cell culture from IL-12-deficient NOD mice induced a slight increase,
2-fold, in the number of IFN--producing
CD4+ cells (Fig. 2C) and,
consequently, in the level of secreted IFN- (Fig. 2B).
This suggests that IL-18 may have a minor role in the development of
Th1 cells in IL-12-deficient NOD mice.
In conclusion, IL-12-deficient NOD mice have greatly reduced Th1 responses to exogenous Ags, confirming the important role of IL-12 for Th1 development in NOD mice. However, as in other mouse strains, a residual low level of IL-12-independent Th1 development does occur, which could be mediated in part by IL-18.
Insulitis and IDDM develop equally well in IL-12-deficient and wild-type NOD mice
A defect in peripheral Th1 responses was expected to reduce the
progression to IDDM in IL-12-deficient NOD mice. Strikingly, the onset
and cumulative incidence of diabetes were similar in wild-type and
IL-12-deficient NOD mice. About 70% female and 60% male wild-type or
IL-12-deficient NOD mice developed spontaneous IDDM by 45 wk of age
(Fig. 3). No statistically significant
difference in the insulitis score could be detected between either male
or female wild-type and IL-12-deficient NOD mice (Fig. 4). Histologic examination of the
pancreata from 12-wk-old wild-type and IL-12-deficient NOD mice
revealed in both a severe insulitis composed of
CD4+ and CD8+ T cells, B
cells, macrophages, and dendritic cells, as determined by CD4, CD8,
B220, Mac-1, and CD11c expression (Fig. 4).
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The lack of difference in IDDM progression between wild-type and
IL-12-deficient NOD mice was rather unexpected, since administration of
the IL-12 antagonist (p40)2 to NOD mice could
inhibit disease development (21). In an attempt to
determine the reason for this discrepancy, we analyzed the
CD4+ T cell response to a candidate autoantigen
in IDDM, the tyrosine phosphatase-like IA-2. Like glutamic acid
decarboxylase (GAD)65, another major autoantigen in IDDM, IA-2 induces
a spontaneous cytokine response in spleen cells from unprimed NOD, but
not other mouse strains (S. Trembleau et al., manuscript in
preparation). Spleen cells from NOD mice treated with the
IL-12-antagonist (p40)2 from 3 wk of age for 50
consecutive days, IL-12-deficient, and control NOD mice were stimulated
in vitro with recombinant mouse IA-2. IFN- and IL-10 secretion were
measured after 48 h of culture. The IFN- response to IA-2 was
inhibited by 50% in spleen cells from
(p40)2-treated NOD mice and was abrogated in
spleen cells from IL-12-deficient NOD mice (Fig. 5A). In contrast,
IA-2-specific IL-10 secretion was similar in spleen cells from IL-12
antagonist-treated, IL-12-deficient, and control NOD mice (Fig. 5A). To determine whether these cytokines were secreted by
IA-2-specific CD4+ T cells,
CD4+ cells were purified from peripheral lymphoid
organs and stimulated with IA-2 presented by T cell-depleted mitomycin
C-treated NOD spleen cells. Splenic and mesenteric lymph node
CD4+ T cells from control NOD mice secreted
dose-dependent high levels of IFN- in response to IA-2, whereas
CD4+ cells from IL-12-deficient NOD mice did not
secrete any detectable IFN- (Fig. 5B). IA-2-induced IL-10
secretion by CD4+ T cells was similar in both
groups (Fig. 5B). Thus, Th1 responses to IA-2 are decreased
in IL-12 antagonist-treated NOD mice and undetectable in
IL-12-deficient NOD mice.
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Th1-mediated autoimmune diseases can develop, in particular
conditions, in the context of a deviant Th2-like effector response
(26, 36, 37). To test whether this was the case, we
characterized the phenotype of pancreas-infiltrating T cells in
IL-12-deficient and IL-12 antagonist-treated NOD mice. Pancreatic
CD4+ and CD8+ T cells were
isolated, stimulated with PMA/ionomycin, and stained for
intracytoplasmic IFN- and IL-4, as well as IFN- and IL-10
production. As shown in Fig. 2, a short restimulation with
PMA/ionomycin is qualitatively comparable to Ag in revealing the
cytokine profile of previously activated cells. Surprisingly, both in
wild-type and IL-12-deficient NOD mice, 20% of pancreatic
CD4+ cells produced IFN- only, 6% IFN- and
IL-10, and 3% IL-10 only (Fig. 5C). No IL-4-producing cells
were detected (data not shown). Therefore, a similar percentage of
Th1-type pancreas-infiltrating CD4+ cells was
present both in wild-type and IL-12-deficient NOD mice. In contrast,
the proportion of IL-10-producing CD4+ cells
increased by 3-fold in NOD mice treated with the IL-12 antagonist
(p40)2 (Fig. 5C), consistent with
previous results (21). The proportion of IFN--producing
CD4+ cells remained similar in vehicle and
IL-12(p40)2-treated NOD mice (Fig. 5C). However, when IL-12(p40)2-treated
mice were tested individually at a later age and stratified in diabetic
and still normoglycemic, the increase of pancreas-infiltrating Th2-type
cells was found associated with decreased Th1 and with protection
from IDDM (21). Pancreas-infiltrating
CD8+ cells were also tested for their cytokine
profile. CD8+ cells from IL-12 antagonist-treated
NOD, IL-12-deficient NOD, and control NOD mice produced IFN-, but
not IL-4 nor IL-10, with a similar percentage of IFN--producing
cells in all groups (data not shown). These results are in agreement
with the lack of deviation to the type 2 phenotype in
pancreas-infiltrating CD8+ cells from
IL-12(p40)2-treated NOD mice
(21).
In conclusion, in IL-12-deficient NOD mice, the development of Th1 cells was strongly impaired in peripheral lymphoid organs and nevertheless they did accumulate and/or expand in the pancreas leading to IDDM development. Only Th1 and not Th2 cells were found in the pancreas of IL-12-deficient NOD mice. In contrast, administration of the IL-12 antagonist (p40)2 induced Th2 cells in the pancreas of NOD mice. This immune deviation was found to be associated with protection from IDDM (21). Thus, the lack of Th2 cells may leave the pathogenic Th1 cells that develop in the absence of IL-12 unchecked.
Increased frequency of P-selectin-binding T cells in the pancreatic infiltrate of IL-12-deficient NOD mice
Leukocyte extravasation into inflamed tissues requires P- and
E-selectin expression on endothelial cells, and the ligand for
P-selectin is expressed by chronically stimulated, but not resting,
CD4+ T lymphocytes (38) and by Th1
but not Th2 cells (39, 40). High numbers of
P-selectin-binding CD4+ T cells were found at
inflammatory sites in delayed-type hypersensitivity and autoimmune
colitis, two Th1-mediated conditions (41, 42). Therefore,
it was of interest to compare the expression of P-selectin ligand on
pancreas-infiltrating T cells from IL-12-deficient and wild-type NOD
mice. Unexpectedly, a larger fraction of ex vivo labeled
CD4+ and CD8+
pancreas-infiltrating cells from IL-12-deficient compared with control
mice bound P-selectin/Ig fusion protein (Fig. 6). The increase in P-selectin
ligand-positive CD4+ and
CD8+ cells was 2- and 3-fold, respectively.
These results suggest a compensatory mechanism for type 1 T cell
recruitment in the pancreas of IL-12-deficient NOD mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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80% in lymph nodes of IL-12-deficient NOD mice,
confirming results obtained in other IL-12-deficient mouse strains
(5). In addition, Th1 responses to the self-Ag IA-2 are
undetectable in IL-12-deficient NOD in contrast to control mice.
However, rather unexpectedly, IDDM develops equally well in
IL-12-deficient and wild-type NOD mice, and in both it is associated
with pancreas-infiltrating type 1 T cells only. Thus, Th1 development
is impaired in peripheral lymphoid organs of IL-12-deficient NOD mice,
but IL-12 appears dispensable for pancreatic infiltration of
IFN--producing cells and IDDM development. In contrast,
pancreas-infiltrating CD4+ cells from IL-12
antagonist-treated NOD mice are skewed to a Th2 phenotype, which was
found to be associated with protection from IDDM (21).
These findings suggest that the development of pathogenic Th1 cells can
be inhibited when a Th2-type regulation is induced and that the
incapacity to generate this regulatory pathway may contribute to IDDM
development in IL-12-deficient NOD mice. The impaired Th1 development in IL-12-deficient mice is usually associated with a propensity to develop Th2 responses (5). For example, wild-type 129/Sv/Ev mice develop a Th1-dominated immune response and are resistant to L. major, whereas mice lacking IL-12 mount a polarized Th2 response and become susceptible to the infection (43). However, IL-12 deficiency is not always paralleled by an expansion of Th2 cells (8). Inhibition of Th1 favors the establishment of Th2-promoting conditions (44), but if these conditions are intrinsically defective, development of Th2 cells will be precluded. Th2-promoting factors include the predisposition to produce IL-4. Since NOD mice have a defect in IL-4 production (45), this could explain why IL-12-deficient NOD mice fail to demonstrate an appreciable induction of Th2 responses.
The contrasting results obtained in IL-12(p40)2-treated and IL-12-deficient NOD mice suggest that IL-12 or (p40)2 may be involved in Th2 cell generation. IL-12p75 has been shown to contribute to Th2 cell development (46). In addition, a direct or indirect role of (p40)2 itself could also explain the enhancement of Th2 responses observed after (p40)2 administration in NOD mice. IL-12 is composed of two covalently associated chains, p40 and p35. Upon activation, cells secreting the biologically active IL-12p75 heterodimer also secrete a large excess of monomeric p40 as well as (p40)2 (47, 48). IL-12(p40)2 strongly inhibits IL-12 activities, and thus represents a natural antagonist (49), although an agonist role has also been hypothesized (50). Analysis of IDDM development in IL-12p40- and in IL-12p35-deficient NOD mice could have been useful to distinguish between the role of endogenous IL-12p75 and monomeric or homodimeric p40. Unfortunately, the backcross of the IL-12p35-deficient mice to the NOD background is uninformative due to the close linkage of several Idd loci on chromosome 3, where the IL-12p35 gene is located (51).
Spontaneous IDDM in NOD mice is unique among the autoimmune disease
models so far examined because of its capacity to develop as
efficiently in IL-12-deficient and in control mice. IL-12 deficiency
consistently leads to decreased autoantigen-specific Th1 responses in
induced autoimmune diseases, such as CIA, EAMG, EAU, and EAE. However,
the concomitant induction of Th2-type responses or other
immunoregulatory pathways is variable (14, 15, 16, 17).
Interestingly, IL-12-deficient mice are only partially protected from
CIA and EAMG, whereas they appear to be completely protected from EAE
and EAU. In these cases, complete protection from autoimmunity seems
associated with an immunoregulatory circuit involving IL-10 (14, 15). Thus, an impaired development of Th1 cells may not be
sufficient for complete inhibition of an autoimmune disease, and the
induction of an immunoregulatory pathway could be necessary. This
regulation could depend more on IL-10 than IL-4, as indicated by the
observation that IL-4 transgenic mice do develop EAE, but IL-10
transgenic are completely protected (52), and by the
capacity of IL-10-producing Tr1 cells to inhibit autoimmune colitis
(53). IL-12-deficient NOD mice show a major reduction of
Ag-specific IFN-, but little enhancement of IL-4 and IL-10
secretion. Likewise, very few pancreas-infiltrating T cells produce
IL-4 or IL-10. A defective IL-4 production by NOD
CD4+ cells has been implicated in IDDM
development (45), possibly through impairment of
NK1.1+CD4+ cells that could
be involved in early IL-4 production (54), and it is
possible that immunoregulatory pathways involving IL-10 are impaired as
well. Consistent with this assumption, administration of a noncytolytic
IL-10-fusion protein completely protects NOD mice from IDDM
(55). In addition, IL-10-transduced islet-specific Th1
cells prevent IDDM transfer in NOD mice (56).
The absence of Th2-type regulation could, in part, explain the
accumulation of residual Th1 cells into the pancreas of IL-12-deficient
NOD mice. Could other cytokines, such as IFN- or IL-18, compensate
for the lack of IL-12 and induce diabetogenic Th1 cells? In the mouse,
IFN- synergizes with IL-12 for Th1 development (57).
Similar to the situation observed with IL-12, inhibition of endogenous
IFN- protects from diabetes (58, 59), but IDDM develops
in IFN--deficient NOD mice (60). In contrast to the
latter result, insulitis does not develop in IFN-R
-deficient NOD
mice (61). While there is no clear explanation at present
for the discrepancy between IFN-- and IFN-R-deficient mice,
this has been observed also in other models (62).
Recently, IFN-Rß-deficient mice were found to differ from
IFN-R-deficient mice in their ability to develop Th1 responses
(63). More detailed understanding of the IFN- signaling
pathway may explain these seemingly conflicting results. In any case,
the genetic absence of IL-12 or IFN- allows the development of
compensatory mechanisms not available in unmanipulated NOD mice, in
which IDDM can be prevented by treatment with cytokine antagonists.
A cytokine potentially able to replace IL-12 could be the
IFN--inducing factor IL-18 (64). A rise in both IL-18
and IL-12p40 mRNA levels has been detected in the adherent spleen cell
population of cyclophosphamide-treated NOD mice (65). Our
results demonstrate that IL-18 synergizes with IL-12, but is not able
to restore the production of IFN- by HEL or PPD-specific T cells
from IL-12-deficient NOD mice. Thus, IL-18 only acts on IL-12-primed
Th1-developing cells, stimulating them to produce more IFN-, but in
the absence of IL-12 is inefficient in inducing the differentiation of
Th1 cells. These data are consistent with results indicating that IL-12
is sufficient for normal Th1 development in the absence of IL-18
(35), and that IL-18 by itself does not induce Th1 cell
development (66, 67). However, mice deficient in both
IL-12 and IL-18 display a more profound impairment in the bacillus
Calmette-Guerin-induced Th1 response, as compared with IL-12-deficient
mice, suggesting that IL-12-independent Th1 development could be
induced by the cooperative action of IL-18 and other factor(s), yet
unidentified (35). However, this pathway might only
account for the residual Th1 development in IL-12-deficient NOD mice.
Although IL-18 could substitute in part for the lack of IL-12,
accumulation of diabetogenic Th1 cells in the pancreas of
IL-12-deficient NOD mice is likely to depend on alternative mechanisms.
In addition to Th1-promoting cytokines, the nature of the
autoantigen(s) and the chronicity of IDDM combined with a genetic
deficiency in immunoregulation could lead, even in the absence of
IL-12, to diabetogenic Th1 cell development in the NOD mouse.
Both CD4+ and CD8+ T cells
are necessary for IDDM development in unmanipulated NOD mice. However,
under some circumstances, either CD4+ or
CD8+ T cells alone are able to induce the disease
(68, 69). Interestingly, the cytotoxic activity of
CD8+ T cells is unaffected in IL-12-deficient
mice (5), or even increased in IFN--deficient mice
(70). Thus, it is possible that IL-12-deficient NOD mice
develop IDDM via a mechanism(s) involving predominantly
CD8+ T cells, as compared with their wild-type
littermates. A similar number of pancreas infiltrating
CD4+ and CD8+ cells was
visualized in histology or counted after their purification in
IL-12-deficient and wild-type NOD mice. However, the proportion of
pancreas infiltrating cells expressing P-selectin ligand is 2-fold
higher in CD4+ and 3-fold higher in
CD8+ T cells from IL-12-deficient as compared
with wild-type NOD mice. P-selectin ligand expression has been
associated with subsets of skin or mucosa-seeking memory/effector T
cells that produce proinflammatory cytokines (41, 42).
Therefore, T cells and, in particular, CD8+ cells
from IL-12-deficient NOD mice may have a higher diabetogenic
potential.
In conclusion, spontaneous IDDM in NOD mice is unique among autoimmune disease models for its capacity to develop in IL-12-deficient mice. Nevertheless, administration of the IL-12 antagonist (p40)2 does prevent IDDM in NOD mice (21). The following scenario could be envisioned to explain why IL-12 is dispensable for IDDM development. Th1 cell development in NOD mice, as in other mouse strains, is impaired, although not completely prevented, in the absence of IL-12. The residual Th1 development may be due in part to the cooperative action of IL-18 and other factor(s) (35). Th1 impairment is not sufficient for IDDM prevention, and the induction of a regulatory pathway is necessary for protection against Th1-mediated autoimmunity. Neutralization of endogenous IL-12 in normal NOD mice, when started at 3 wk of age, results in high numbers of pancreas-infiltrating IL-4-producing CD4+ cells and in increased IL-10-producing CD4+ cells, associated with protection from IDDM (21). Intriguingly, these Th2-type cells are not present in IL-12-deficient NOD mice, suggesting that IL-12 or (p40)2 may be involved in their generation. The lack of immune deviation to the Th2 pathway is the most obvious difference between IL-12-deficient NOD mice and wild-type NOD mice in which endogenous IL-12 has been targeted by IL-12 antagonist administration. Thus, IL-12-deficient NOD mice appear to have a genetic deficiency in developing a regulatory pathway able to counteract diabetogenic Th1 cells. In the absence of a regulatory pathway controlling Th1 responses, the residual Th1 cells accumulate in the pancreas of IL-12-deficient NOD mice to levels quantitatively and qualitatively similar to controls. T cell infiltration and accumulation may also be favored in IL-12-deficient NOD mice by increased P-selectin ligand expression on CD4+ and CD8+ cells, and this could further contribute to compensate for the defective Th1 cell pool recruitable from peripheral lymphoid organs.
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2 Current address: Cadus Pharmaceutical, Terrytown, NY 10591.
3 Address correspondence and reprint requests to Dr. Luciano Adorini, Roche Milano Ricerche, Via Olgettina 58, I-20132 Milano, Italy. E-mail address:
4 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; CIA, collagen-induced arthritis; EAU, experimental autoimmune uveitis; EAMG, experimental autoimmune myasthenia gravis; HEL, hen egg-white lysozyme; IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; PPD, protein purified derivative.
Received for publication January 11, 1999. Accepted for publication June 14, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and type 1 cytokine responses. Immunity 4:471.[Medline]
interferon. Proc. Natl. Acad. Sci. USA 81:4515. deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response. J. Immunol. 158:5997.[Abstract]
. J. Immunol. 154:3999.[Abstract]
-producing cells. J. Immunol. 158:643.[Abstract]
14-J281 transgenic nonobese diabetic mice against diabetes. J. Exp. Med. 188:1. and IFN- in IL-12-induced T helper cell-1 development. J. Immunol. 156:1442.[Abstract]
and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice. J. Clin. Invest. 87:739.
. J. Autoimmun. 4:237.[Medline]
-interferon delays but does not prevent diabetes in NOD mice. Diabetes 45:812.[Abstract]
impacts at multiple points during the progression of autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94:13844. receptor 2 gene results in severe immune defects in mice. Proc. Natl. Acad. Sci. USA 95:8233. production by T cells. Nature 378:88.[Medline]
production and activates IRAK and NF
B. Immunity 7:571.[Medline]
genes. Science 259:1739.This article has been cited by other articles:
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 |
|
  J. H. Esensten, M. R. Lee, L. H. Glimcher, and J. A. Bluestone T-bet-Deficient NOD Mice Are Protected from Diabetes Due to Defects in Both T Cell and Innate Immune System Function J. Immunol., July 1, 2009; 183(1): 75 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Marleau, K. L. Summers, and B. Singh Differential Contributions of APC Subsets to T Cell Activation in Nonobese Diabetic Mice J. Immunol., April 15, 2008; 180(8): 5235 - 5249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Hawiger, E. Tran, W. Du, C. J. Booth, L. Wen, C. Dong, and R. A. Flavell ICOS Mediates the Development of Insulin-Dependent Diabetes Mellitus in Nonobese Diabetic Mice J. Immunol., March 1, 2008; 180(5): 3140 - 3147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Irie, B. Reck, Y. Wu, L. S. Wicker, S. Howlett, D. Rainbow, E. Feingold, and W. M. Ridgway Genome-Wide Microarray Expression Analysis of CD4+ T Cells from Nonobese Diabetic Congenic Mice Identifies Cd55 (Daf1) and Acadl as Candidate Genes for Type 1 Diabetes J. Immunol., January 15, 2008; 180(2): 1071 - 1079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Enzler, S. Gillessen, M. Dougan, J. P. Allison, D. Neuberg, D. A. Oble, M. Mihm, and G. Dranoff Functional deficiencies of granulocyte-macrophage colony stimulating factor and interleukin-3 contribute to insulitis and destruction of {beta} cells Blood, August 1, 2007; 110(3): 954 - 961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Boyton, S. Davies, C. Marden, C. Fantino, C. Reynolds, K. Portugal, H. Dewchand, and D. M. Altmann Stat4-null non-obese diabetic mice: protection from diabetes and experimental allergic encephalomyelitis, but with concomitant epitope spread Int. Immunol., September 1, 2005; 17(9): 1157 - 1165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gregori, N. Giarratana, S. Smiroldo, and L. Adorini Dynamics of Pathogenic and Suppressor T Cells in Autoimmune Diabetes Development J. Immunol., October 15, 2003; 171(8): 4040 - 4047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Trembleau, G. Penna, S. Gregori, N. Giarratana, and L. Adorini IL-12 Administration Accelerates Autoimmune Diabetes in Both Wild-Type and IFN-{gamma}-Deficient Nonobese Diabetic Mice, Revealing Pathogenic and Protective Effects of IL-12-Induced IFN-{gamma} J. Immunol., June 1, 2003; 170(11): 5491 - 5501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu and D. I. Beller Distinct Pathways for NF-{kappa}B Regulation Are Associated with Aberrant Macrophage IL-12 Production in Lupus- and Diabetes-Prone Mouse Strains J. Immunol., May 1, 2003; 170(9): 4489 - 4496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Hill, K. Van Gunst, and N. Sarvetnick Th1 and Th2 Pancreatic Inflammation Differentially Affects Homing of Islet-Reactive CD4 Cells in Nonobese Diabetic Mice J. Immunol., February 15, 2003; 170(4): 1649 - 1658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Davoodi-Semiromi, J. J. Yang, and J.-X. She IL-12p40 Is Associated With Type 1 Diabetes in Caucasian-American Families Diabetes, July 1, 2002; 51(7): 2334 - 2336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
S. Gregori, M. Casorati, S. Amuchastegui, S. Smiroldo, A. M. Davalli, and L. Adorini Regulatory T Cells Induced by 1{alpha},25-Dihydroxyvitamin D3 and Mycophenolate Mofetil Treatment Mediate Transplantation Tolerance J. Immunol., August 15, 2001; 167(4): 1945 - 1953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Induction of Experimental Autoimmune Thyroiditis in IL-12-/- Mice J. Immunol., August 1, 2001; 167(3): 1720 - 1727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F.-D. Shi, M. Flodstrom, B. Balasa, S. H. Kim, K. Van Gunst, J. L. Strominger, S. B. Wilson, and N. Sarvetnick Germ line deletion of the CD1 locus exacerbates diabetes in the NOD mouse PNAS, June 5, 2001; 98(12): 6777 - 6782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hong, E. L. Berg, and R. O. Ehrhardt Persistence of Pathogenic CD4+ Th1-Like Cells In Vivo in the Absence of IL-12 but in the Presence of Autoantigen J. Immunol., April 1, 2001; 166(7): 4765 - 4772. [Abstract] [Full Text] [PDF]
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