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Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion,
compatible with a shift from a Th1-like toward a Th2-like autoimmune
response. Our results extend the role of specific hsp60
immunomodulation in the control of 
cell autoimmunity and
demonstrate that immunoregulatory networks activated by specific phsp60
vaccination can spread to other Ags targeted during the progression of
diabetes, like insulin and GAD. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells of the pancreas (1). Nonobese
diabetic (NOD) mice can develop two types of autoimmune diabetes,
spontaneous diabetes and cyclophosphamide-accelerated diabetes
(CAD). Spontaneous autoimmune diabetes is usually detectable as overt hyperglycemia in female NOD mice, first appearing after the third month of life (2, 3), and is characterized by increased Th1 cell responses to several autoantigens, including the 60-kDa heat shock protein (hsp60) (4), glutamic acid decarboxylase (GAD) (5, 6), and insulin (7). Ag-specific therapies can halt the progression of spontaneous NOD diabetes (8); among them are treatment with the mycobacterial 65-kDa heat shock protein (hsp65) (9) or the mammalian hsp60 (10, 11). Spontaneous NOD diabetes is also inhibited by viral (12, 13), parasitic (14), or bacterial (15) infection or by the administration of bacterial molecules known to signal, directly or indirectly, through Toll-like receptors (TLRs) (16), such as LPS (17, 18) or immunostimulatory CpG DNA motifs (19).
A single injection of cyclophosphamide can synchronize and accelerate IDDM in NOD mice through a mechanism that involves the death of various cell populations (20), including regulatory cells (21). When compared with spontaneous NOD diabetes, CAD stands as a stronger variant of autoimmune diabetes; from the large list of agents that modulate the spontaneous disease (22), only a few of them can affect CAD (23, 24, 25, 26, 27).
In this work, we studied the effect of DNA vaccination (28) on CAD with constructs encoding the antigens hsp60 (phsp60) or hsp65 (phsp65). Animals treated with phsp60 were protected from CAD; treatment with phsp65, an empty vector, or CpG-positive oligonucleotides did not protect. The phsp60-treated mice showed a modulation of the T cell responses to hsp60, GAD, and insulin, suggesting that phsp60 vaccination activates regulatory networks that control the autoimmune response against other Ags associated with diabetes, besides hsp60 itself.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female mice of the NOD/LtJ strain were raised from breeders kindly supplied by Dr. E. Leiter of The Jackson Laboratory, and maintained under pathogen-free conditions in the Animal Breeding Center of this institute. Experiments were conducted under the supervision and guidelines of the Animal Welfare Committee. The mice were 1 mo old at the start of the experiments.
DNA plasmids and CpG
The pcDNA3 (Invitrogen, Leek, The Netherlands) vectors encoding
human hsp60 (phsp60) (19) or Mycobacterium
leprae hsp65 (phsp65) (29) have been previously
described and shown to be immunogenic in mice. Dr. D. Lowrie (Medical
Research Council, London, U.K.) kindly provided the phsp65 construct.
Plasmid DNA was prepared in large scale using the alkaline lysis method
of Qiagen Plasmid Mega Prep (Qiagen, Santa Clara, CA). Plasmid DNA was
precipitated with ethanol and resuspended in sterile PBS.
Spectrophotometric analysis revealed 260:280 nm ratios 
1.80. The
purity of DNA preparations was confirmed on a 1% agarose gel.
Endotoxin levels were checked by Limulus amebocyte lysate
and were always under acceptable levels for in vivo use (<0.02
endotoxin U/µg DNA).
The phosphorothioate-stabilized oligonucleotides used in these studies were synthesized in the Oligonucleotide Synthesis Unit of this institute as previously described (19). Oligonucleotide CpG, 5'-TCCATAACGTTGCA-AACGTTCTG-3'; oligonucleotide GpC, 5'-TCCATAAGCTTGCAAAGCTTCTG-3'.
NOD females were injected with 100 µl of 10 mM cardiotoxin (Sigma-Aldrich, Rehovot, Israel) into the tibialis anterior muscle. After 5 and 12 days, the mice were injected with 100 µl (1 µg/µl) of the desired DNA vaccine, with 100 µl (1 µg/µl) of the oligonucleotides bearing CpG or GpC motifs, or with 100 µl of PBS as controls. Diabetes was accelerated by a single injection of 200 mg/kg cyclophosphamide (Sigma-Aldrich) given 12 days after the last injection of DNA, at the age of 8 wk.
Hyperglycemia
Blood glucose was measured weekly. A mouse was considered diabetic when its blood glucose level was >13 mM on two consecutive examinations, tested using a Beckman Glucose Analyzer II (Beckman Instruments, Brea, CA).
Pancreas histology
Mice from each treatment group were killed 1 mo after the injection of cyclophosphamide, at the age of 12 wk. The pancreata were fixed in 10% buffered formalin, cut, and stained by standard H&E; the average degree of insulitis was assessed over 20 islets scored per pancreas. Each islet was classified as: clear, if no infiltrate was detected; mildly infiltrated, if peri-insulitis or an intra-islet infiltrate occupied <25% of the islet; or infiltrated or heavily infiltrated, if 25–50%, or >50% of the islet was occupied by inflammatory cells.
Peptides and Ags
The peptides used in this study are listed in Table I
. The peptides were synthesized by a
standard F-moc procedure and purified by reverse phase HPLC, and their
compositions were confirmed by amino acid analysis. Peptide p277 was
stabilized by substituting two cysteines at positions 442 and 447 for
valines. These substitutions do not affect the immunological properties
of p277 (11). Insulin, GAD, OVA, and Con A were purchased
from Sigma-Aldrich. Recombinant hsp60 was prepared as described
(19).
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Groups of 3–4 female NOD mice were sacrificed 4 wk after the acceleration of diabetes with cyclophosphamide, their spleens were removed, and the T cell-proliferative responses to Con A or test Ags were studied. Cultures were incubated for 72 h at 37°C in a humidified atmosphere with 7.5% CO2. T cell responses were detected by the incorporation of [methyl-3H]thymidine (Amersham Biosciences, Little Chalfont, U.K.; 1 µCi/well) added to the wells for the last 18 h of incubation. The stimulation index (SI) was computed as the ratio of the mean cpm of Ag- or mitogen-containing wells to control wells cultured with medium alone.
Cytokine assays
Supernatants were collected after 72 h of stimulation with
test Ags, Con A, or medium alone. Murine IL-4, IL-5, IL-10, and IFN-
were quantitated in the culture supernatants with ELISA, using
appropriate paired Abs from BD PharMingen (San Diego, CA) (IL-4, IL-10,
and IFN-) or Endogen (Woburn, MA) (IL-5) with some modification.
Briefly, ELISA plates (Maxisorp; Nunc, Roskilde, Denmark) were coated
overnight at 4°C with anti-mouse cytokine monoclonal capture Abs.
Nonspecific binding was blocked by incubation with 1% BSA for 1 h
at room temperature, and culture supernatants or recombinant cytokines
were incubated overnight at 4°C. After the plates were washed,
biotinylated detection Abs were added for 1 h at room temperature,
then extensively washed, and incubated with streptavidin conjugated to
alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove,
PA) for 30 min at room temperature. The plates were washed,
Sigma-Aldrich alkaline phosphatase substrate was added, and samples
were read at 405 nm after 30 min of incubation at room temperature.
Cytokine levels in supernatants are expressed as picograms per
milliliter based on calibration curves constructed using recombinant
cytokines as standards. The lower limits of detection for the
experiments described in this paper were 15 pg/ml for IL-5, IL-10, and
IFN-.
Statistical significance
The InStat 2.01 program was used for statistical analysis.
Student’s t test and the 
2 test were
conducted to assay significant differences between experimental and
control groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We treated 4-wk-old female NOD mice with phsp60, with phsp65, with the empty vector containing CpG motifs (pcDNA3), or with CpG or GpC oligonucleotides. A control group was treated with PBS. Each group consisted of 15 mice. Cyclophosphamide was injected 12 days after the last injection of DNA, and glucose levels were measured at weekly intervals.
Vaccination with phsp60 led to significant protection from CAD. In
contrast, the progression of diabetes after administration of
cyclophosphamide was the same in the mice treated with PBS, pcDNA3,
phsp65, or CpG- or GpC-bearing oligonucleotides (Fig. 1A). Fig. 1B
depicts the results obtained on histological examination of the
pancreas: phsp60 vaccination led to a significant increase in the
number of islets free of insulitis 30 days after the injection of
cyclophosphamide, together with a significant decrease in the numbers
of infiltrated and heavily infiltrated islets.
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Increasing spontaneous T cell reactivity to hsp60 has been
previously related to the progression of diabetes (4, 10),
and modulation of the hsp60-specific immune response was associated
with the control of the diabetogenic process (11). We
therefore studied the proliferative T cell response to hsp60 and to two
of its peptides, p277 and p12, by splenocytes isolated from
DNA-vaccinated mice 30 days after the administration of
cyclophosphamide. When compared with phsp65- or pcDNA3-vaccinated mice,
phsp60-treated animals showed significantly reduced T
cell-proliferative responses to hsp60 and p277 (Fig. 2). The responses to p12 were too low to
be considered significant (SI < 2). Nevertheless, the responses
to Con A were of the same magnitude in all the groups (pcDNA3, 6.8
± 1.2; phsp65, 6.6 ± 1.9; phsp60, 5.5 ± 0.7), suggesting
that the inhibition of T cell responses to hsp60 and to p277 were Ag
specific and not due to a general down-regulation of the immune
response.
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(20); hence we followed the
secretion of IFN-, IL-4, IL-5, and IL-10 as indicators of the
Th1/Th2 phenotype. The different experimental groups did not differ in
their responses to Con A, and no stimulation of cytokine secretion was
detected on stimulation with the control Ag OVA. Nevertheless, IFN-
secretion on stimulation with hsp60 was diminished in the
phsp60-treated animals (Fig. 3A). This down-regulation of
IFN- secretion was associated with an increase in IL-10 and IL-5
secretion in response to stimulation with hsp60, and also with the
hsp60-derived peptides p12 and p277 (Fig. 3, B and
C). No IL-4 was detected on Ag stimulation; however, high
levels of IL-4 were found in the supernatants of Con A-stimulated
cultures: pcDNA3, 11.9 ± 0.6 ng/ml; phsp65, 11 ± 0.9 ng/ml;
and phsp65, 11.8 ± 0.5 ng/ml. A decrease in IFN- together with
an increase in IL-10 and IL-5 in response to hsp60 stimulation is
compatible with a shift of the anti-hsp60 T cell response from what
has been termed a pathogenic Th1 to a regulatory Th2 phenotype, in an
Ag-specific manner.
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The autoimmune process leading to overt diabetes targets other Ags
besides hsp60, such as GAD (5, 6) and insulin
(7). We therefore studied T cell reactivity to insulin, to
GAD, and to two GAD-derived peptides, p34 and p35 (6), by
splenocytes isolated from DNA-vaccinated mice 30 days after the
administration of cyclophosphamide. The phsp60-treated animals showed a
significant decrease in their spontaneous proliferative response to
GAD. The proliferative responses to GAD-derived peptides p34 and p35,
or to insulin were too low to be evaluated (Fig. 4). However, splenocytes taken from
phsp60-treated mice secreted significantly lower amounts of IFN- and
higher amounts of IL-10 and IL-5 on stimulation with insulin, GAD, or
p35 (Fig. 5). Again, IL-4 was not
detected after Ag-specific stimulation. We did not detect any cytokine
secretion on in vitro stimulation with p34 or with the control Ag OVA.
Thus phsp60 vaccination induces a shift toward Th2 in the
hsp60-specific T cell response and in the T cell responses to insulin
and GAD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, we studied the susceptibility of CAD to modulation by
DNA vaccination. DNA vaccination with a construct encoding human hsp60
(phsp60), but not with a construct encoding mycobacterial hsp65
(phsp65), controlled CAD (Fig. 1
). The effective phsp60 vaccine
contained two kinds of signals: a specific Ag associated with diabetes
autoimmunity, hsp60 (2), and ligands for receptors that
stimulate the innate immune system; hsp60 through TLR-2 and TLR-4
(31); and CpG motifs through TLR-9 (32).
However, CAD, in contrast to spontaneous NOD diabetes, did not respond
to treatment with pcDNA3 (empty vector) or a CpG-containing
oligonucleotide (Fig. 1). In addition, although hsp65 too has been
shown to stimulate the innate immune response via TLR-4 and TLR-2
(31), the plasmid encoding mycobacterial hsp65 (phsp65)
did not have any significant effect on CAD progression (Fig. 1). Thus,
it is unlikely that the efficacy of the phsp60 vaccine could be based
solely on its activity on innate receptors; it is more likely that
regulatory epitopes present in the hsp60 molecule are also needed.
Indeed, the failure of phsp65 vaccination could be explained by the
lack in the hsp65 molecule of the two major hsp60 T cell epitopes found
to control spontaneous autoimmune diabetes when administered as
peptides (10, 30); p12 and p277 are not conserved in
mycobacterial hsp65 (Table II). This
hypothesis does not rule out the possibility that the effects of phsp60
in controlling CAD might involve signals contained within the hsp60
molecule for both the adaptive and innate arms of the immune system.
Study of the functions of innate receptors in the NOD mouse is needed
to explore this issue; our results certainly do not exclude the
possibility that under different conditions TLR-mediated stimulation
might control CAD.
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) are a feature of the diabetogenic attack,
whereas regulatory Th2 responses (characterized by the secretion of
IL-4, IL-5, and IL-10) have been associated with the inhibition of
cell destruction (2). In our experiments, protection from
CAD by phsp60 vaccination was associated with a significant reduction
of T cell proliferation and of IFN- secretion by T cells responding
to hsp60 or its T cell epitope p277 (Figs. 2 and 3). Although we could
not detect Ag-specific release of IL-4, an increase in the release of
IL-5 and IL-10 was detected (Fig. 3), indicating that phsp60
vaccination shifted the diabetogenic Th1 autoimmune attack more into
the direction of a regulatory Th2 response. We (11) and
others (33, 34) have previously associated a shift in the
T cell reactivity to hsp60 with the control of the diabetogenic
response in NOD mice and in newly diagnosed human IDDM patients treated
with p277 (35). Remarkably, the immune response to
insulin, GAD, and GAD-derived peptides showed reduced proliferation and
was also shifted towards Th2 (Figs. 4 and 5), suggesting a role for
Th2-like spreading in the control of CAD by phsp60 vaccination. Tian et
al. (34) demonstrated the occurrence of Th2 spreading to
diabetes-associated Ags in NOD mice using a panel of autoantigens made
up of GAD, hsp60, and insulin. Later Tisch et al. (36)
showed that a single GAD-specific Th2 cell clone can delay the onset of
diabetes, spreading a regulatory Th2-like response to
non-cross-reactive Ags such as hsp60 or carboxypeptidase H. In our
hands, this effect is relatively cell specific, because we could
not detect the induction of Th2 immunity to the control Ag OVA (data
not shown). Also, treatment of mice (11) or humans
(35) with the hsp60 peptide p277 did not interfere with
Th1 immunity to bacterial Ags.
On the basis of these data, we might propose a three-step process by
which phsp60 triggers inhibition of CAD: (1) anti-hsp60 T cells are
shifted from Th1 to Th2 as a consequence of phsp60 vaccination, perhaps
through the activation of hsp60-specific Th2 cells from a pool of
undifferentiated T cells (33); (2) hsp60-specific
regulatory Th2 cells meet the pathogenic T cells in the islets or in
the pancreatic draining lymph nodes; and (3) the hsp60-specific
regulatory Th2 cells control the pathogenic self-reactive T cells,
either by direct T-T interactions involving the local release of
regulatory cytokines or through indirect interactions that modify local
APC function. Other explanations are also conceivable. For example, it
has been reported that the inhibition of spontaneous NOD diabetes
triggered by some immunostimulatory protocols is dependent on the
presence of IFN- (37). Furthermore, regulatory T cell
clones capable of inhibiting NOD diabetes, both spontaneous and
accelerated by cyclophosphamide, can secrete IFN- (26).
Cyclophosphamide enhances Th1 responses (20); therefore,
it is quite possible that our treatment of CAD by DNA vaccination might
also involve Th1 regulators.
The administration of the hsp60-derived peptide p277 has been recently reported to stop islet destruction in human IDDM (35). Hence, hsp60-derived DNA vaccines encoding relevant regulatory epitopes like p277 might constitute an additional method for the management of human autoimmune diabetes.
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Acknowledgments |
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Footnotes |
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2 I.R.C. is the incumbent of the Mauerberger Chair in Immunology and the Director of the Robert Koch-Minerva Center for Research in Autoimmune Diseases.
3 Address correspondence and reprint requests to Dr. Irun R. Cohen, Department of Immunology, Weizmann Institute of Science, Rehovot, 76100 Israel. E-mail address: irun.cohen{at}weizmann.ac.il
4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; hsp60, 60-kDa heat shock protein; hsp65, 65-kDa heat shock protein; GAD, glutamic acid decarboxylase; CAD, cyclophosphamide-accelerated diabetes; phsp60, DNA vaccine encoding hsp60; phsp65, DNA vaccine encoding hsp65; TLR, Toll-like receptor; SI, stimulation index.
Received for publication June 18, 2002. Accepted for publication September 6, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cell target antigen of autoimmune T cells. J. Autoimmun. 9:159.[Medline]
-cell antigens. Diabetes 46:758.[Abstract]
secretion pathway. J. Autoimmun. 13:383.[Medline]
-Cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind, Phase II trial. Lancet 358:1749.[Medline]
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