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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 to HSP60 and,
concomitantly, to down-regulation of IFN- secretion to the P180-188
epitope of HSP65. There were also variable changes in the profiles of
IL-10 secretion to different Ags. However, vaccination with pHSP60 or
pHSP65 enhanced the production of TGF
1 to both HSP60 and HSP65
epitopes. Our results support a regulatory role for HSP60
autoreactivity in AA and demonstrate that this control mechanism can be
activated by DNA vaccination with both HSP60 or
HSP65. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this work we studied the effect on AA of DNA vaccination using vaccines encoding for human HSP60 (pHSP60) or mycobacterial HSP65 (pHSP65). Human HSP60 is 97% identical to rat HSP60 at the amino acid level; thus, pHSP60 is rich in self-epitopes. We found that both DNA vaccines inhibited AA. We then studied both T cell proliferation and cytokine production to several Ags known to be associated with the disease. Our strategy was to document the immune effects of DNA vaccination on the immune reactions to the Ags that develop at the peak of AA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female Lewis rats were raised 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 rats were 1 mo old at the start of the experiments.
Ags, peptides, and adjuvants
Mt strain H37Ra was obtained from Difco (Detroit, MI). Purified recombinant HSP65 of Mt was kindly provided by Dr. R. van der Zee (Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht, The Netherlands). Recombinant HSP60 was prepared as described (12). The HSP65-derived peptide P176-190 used in this work was EESNTFGLQLELTEG (13); this peptide includes the P180-188 epitope. Peptides were synthesized by a standard F-moc procedure. The peptides were purified by reversed-phase HPLC and their compositions were confirmed by amino acid analysis. Con A and OVA were purchased from Sigma (Rehovot, Israel). IFA was purchased from Difco. The purified protein derivative (PPD) of Mt was provided by the Statens Seruminstitut (Copenhagen, Denmark).
DNA plasmids
The vector containing the human HSP60 gene (pHSP60) has been described (14). A construct coding for the 14.5-kDa isoform of murine myelin basic protein (pMBP) was kindly provided by Dr. L. Steinman (Department of Neurological Sciences, Stanford University, Stanford, CA). The construct encoding Mycobacterium leprae HSP65 (pHSP65) was kindly provided by Dr. D. Lowrie (Medical Research Council, London, U.K.) and has been shown to be effective in controlling AA (7). The empty vector pcDNA3 (14) was used as a DNA vaccination control.
Plasmid DNA was prepared in large scale using the alkaline lysis method
of Qiagen Plasmid Mega Prep (Qiagen, Santa Clarina, CA). Plasmid
DNA was ethanol precipitated and resuspended in sterile PBS.
Spectrophotometric analysis revealed 260/280 nm ratios 
1.80. Purity
of DNA preparations was confirmed on a 1% agarose gel. Endotoxin
levels were checked by Limulus amebocyte lysate and were
found always to be under acceptable levels for in vivo use (<0.02
EU/µg DNA).
Groups of rats were injected i.m. with 200 µl of 10 mM cardiotoxin (Sigma) into the quadriceps using a sterile 27-gauge syringe, witted with a plastic collar to limit needle penetration to 2 mm. The test DNA vaccine (150 µl of 1 mg/ml) was administered i.m. three times: 5, 19, and 33 days after the cardiotoxin. AA was induced 12 days after the last injection of DNA.
Detection of HSP60
Blood samples were collected at day 12 after the last injection of DNA, and serum was prepared as previously described (15) and kept at -20°C until used. HSP60 was quantified by a capture ELISA as previously reported by Rea et al. (16). ELISA plates (Maxisorp; Nunc, Roskilde, Denmark) were coated overnight at 4°C with the murine mAb LK1, specific for human HSP60 (17). Nonspecific binding was blocked by incubation with 1% BSA for 3 h at room temperature (RT), and serum samples or recombinant HSP60 were added and incubated overnight at 4°C. After washing the plates, bound HSP60 was detected using purified anti-human HSP60 rabbit polyclonal Abs (provided by Dr. G. Nussbaum, Department of Immunology, Weizmann Institute of Science, Rehovot, Israel) followed by an anti-rabbit murine mAb conjugated to alkaline phosphatase (Sigma). The plates were washed, alkaline phosphatase substrate (Sigma) was added, and samples were read at 405 nm. HSP60 levels in serum are expressed as micrograms per milliliter based on a calibration curve constructed using recombinant human HSP60 as a standard; the lower limit of detection was 20 ng/ml.
AA induction and assessment
Heat-killed Mt strain H37Ra (Difco) was finely ground using a pestle and mortar, and was suspended to a final concentration of 10 mg/ml in IFA. Test rats were injected at the base of the tail with a total of 100 µl of the Mt suspension. The day of AA induction was designated as day 0. Disease severity was assessed by direct observation of all four limbs in each animal. A relative score between 0 and 4 was assigned to each limb based on the degree of joint inflammation, redness, and deformity; thus, the maximum possible score for an individual animal was 16. Arthritis was also quantified by measuring hind limb diameter with a caliper. Measurements were taken on the day of the induction of AA and 26 days later, at the peak of AA; the results are presented as the mean ± SE of the difference between the values for hind limb diameter taken on days 0 and 26.
Joint histology
The rats were killed at day 26, the peak of AA, and their hind paws were removed and fixed in formol saline for 48 h, decalcified in 5% formic acid with formol saline, and stained with H&E (7). Sixteen individual joints were analyzed in each group. The stained sections were scored using a semiquantitative scale (18, 19) that separately measures synovial inflammation (0–4) and bone and cartilage erosions (0–4). The results are shown as the mean score ± SE each for synovial inflammation and for bone and cartilage erosion.
T cell proliferation
T cell proliferation was measured at day 26, the peak of AA. Draining lymph node (DLN) cells (prepared from inguinal and popliteal lymph nodes) were cultured in quadruplicate in round-bottom microtiter wells (Costar, Cambridge, MA) in 200 µl of stimulation medium at 2 x 105 cells per well with or without Ag. Stimulation medium was made up of DMEM (Life Technologies, Paisley, U.K.) supplemented with 5 10-5 M 2-ME (Sigma), 2 mM L-glutamine (Biological Industries, Kibbutz Beit Haemek, Israel), 1 mM sodium pyruvate (Sigma), 100 U/ml penicillin (Biological Industries), 100 µg/ml streptomycin (Biological Industries), 1% v/v nonessential amino acids (Bio Lab, Jerusalem, Israel), and 1% v/v autologous serum. The T cell mitogen Con A was used at a concentration of 1.25 µg/ml as a positive control for T cell proliferation. Cultures were incubated for 96 h at 37°C in a humidified atmosphere of 7.5% CO2. T cell responses were detected by the incorporation of 1 µCi/well [methyl-3H]thymidine (Amersham Pharmacia Biotech, Little Chalfont, U.K.) added to the wells for the last 18 h. 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. In the case of DLN cells taken from pHSP60- or pHSP65-treated rats, the results are also expressed as the percentage of increase in the proliferation compared with the proliferation seen in DLN cells isolated from pcDNA3-treated rats. The percentage of increase in the proliferation for each Ag was calculated as 100 x (SI in the pHSP60 or pHSP65 group - SI in the pcDNA3 group)/SI in the pcDNA3 group.
Cytokine assays
Supernatants were collected after 72 h of stimulation with
test Ags. Rat IL-10 and IFN- were quantified in the culture
supernatants by ELISA using the OPTEIA kit (BD PharMingen, San Diego,
CA) with some modifications. Briefly, ELISA plates (Maxisorp; Nunc)
were coated overnight at 4°C with anti-rat cytokine monoclonal
capture Abs. Nonspecific binding was blocked by incubation with 1% BSA
for 1 h at RT, and culture supernatants or recombinant cytokines
were added for 2 h at RT. After washing the plates, biotinylated
detection Abs were added for 1 h at RT, then extensively washed
and incubated with streptavidin conjugated to alkaline phosphatase
(Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at RT.
The plates were washed, alkaline phosphatase substrate (Sigma) was
added, and samples were read at 405 nm after 30 min of incubation at
RT. Rat TGF1 was quantified using the TGF1
Emax ImmunoAssay System (Promega, Madison, WI)
according to the manufacturer’s instructions. The determinations were
made at day 26, the peak of AA. 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 TGF1, IL-10, and IFN-. In the case of DLN cells taken from
pHSP60- or pHSP65-treated rats, the results are also expressed as the
percentage of change in cytokine secretion compared with the secretion
detected in DLN isolated from pcDNA3-treated rats. The percentage of
change was calculated as 100 x (pg/ml of cytokine in the
pHSP60 or pHSP65 group - pg/ml in the pcDNA3 group)/pg/ml in the
pcDNA3 group. When no cytokine secretion was detected in DLN cells from
pcDNA3-vaccinated rats, 15 pg/ml (the lower detection limit) was used
for the calculation of the change in cytokine secretion.
Statistical significance
The InStat 2.01 program was used for statistical analysis. Student’s t test and the Mann-Whitney test were conducted to assay significant differences between the different experimental groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The features of the pHSP65 construct used in these studies have
been previously described (7). To determine whether the
injection of pHSP60 could lead in vivo to the production of HSP60,
eight rats were vaccinated three times (5, 19, and 33 days after the
pretreatment with cardiotoxin) with pHSP60 or with the empty vector
pcDNA3. A group of naive rats was also included as a control. Twelve
days after the last injection, serum samples were collected and HSP60
was quantified using a specific ELISA. Fig. 1
shows that pHSP60-vaccinated rats
manifested significantly higher levels of HSP60 in serum
(p < 0.0001) when compared with
pcDNA3-vaccinated or naive rats. These results are in accordance with
previous reports that describe the detection of the Ag encoded by DNA
vaccines in the serum of injected animals (20, 21).
Furthermore, they demonstrate that vaccination with pHSP60 leads to a
significant increase in HSP60 serum levels.
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We investigated the effects on AA of vaccination with DNA encoding
human pHSP60 compared with mycobacterial HSP65. We used a construct
encoding the full-length human HSP60 (pHSP60) and a construct encoding
the full-length HSP65 of M. leprae (pHSP65), shown in the
past to modulate AA (7). Two control constructs were used:
an empty vector (pcDNA3) and pMBP. Fig. 2A shows that vaccination with
pcDNA3 or pMBP did not have any effect on AA. In contrast, rats
immunized with pHSP60 or pHSP65 manifested a significantly milder
arthritis. Inhibition of AA was also reflected as a diminished swelling
of the ankle, as shown in Fig. 2B. Histological examination
of the joints revealed that pHSP60- and pHSP65-vaccinated rats
manifested reduced bone and cartilage erosion and synovial inflammation
(Fig. 3). The degree of protection
achieved by immunization with pHSP65 reproduces that which has been
previously reported by others (7). However, it can be seen
that pHSP60 was more effective than pHSP65 in modulating the autoimmune
process. The difference between pHSP60 and pHSP65 was statistically
significant with regard to the maximal AA score (2.3 ± 0.7 vs
7.7 ± 1.8; p = 0.02), the degree of leg swelling
(10.6 ± 3.4 vs 27.5 ± 6.4; p = 0.03), and
the synovial inflammation score (2 ± 0.2 vs 1 ± 0.2;
p = 0.005).
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To provide a background for the immunological effects of DNA
vaccination, we first established the T cell response profile of AA in
rats with unmodified disease. Twenty-six days after the induction of
AA, DLN cells were prepared from untreated rats or from rats that had
been control-treated with pcDNA3. We stimulated the DLN cells in vitro
using a collective of Ags previously known to be targeted or associated
with AA: HSP60, HSP65, PPD, and peptide P176-190 (which contains the
180-188 epitope of HSP65) (4). OVA was included as a
control Ag. We found that the results were essentially the same whether
the AA was induced in untreated rats or in rats pretreated with control
injections of pcDNA3 (data not shown). Fig. 4A depicts the results
obtained with DLN cells isolated from pcDNA3-treated animals, showing
the proliferative responses at the peak of clinical AA: day 26. The
rats showed a strong proliferative response to PPD and low but
significant responses to HSP65 and P176-190; no significant
proliferation was detected in response to stimulation with HSP60.
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B shows the T cell proliferative responses measured 26
days after the induction of AA in DLN cells isolated from rats
vaccinated with pHSP60 or pHSP65. The results presented in Fig. 4B are the mean ± SE of three independent experiments
and are expressed as the percentage of increase in reactivity relative
to the proliferation obtained using cells from pcDNA3-treated rats. In
addition, Table I shows the SI values of
a representative experiment. The pHSP60- or pHSP65-vaccinated rats,
with reduced AA (see Fig. 2), showed markedly increased proliferative
responses to the mycobacterial Ags PPD and HSP65 and to human HSP60.
The response to P178-190 peptide was only slightly increased relative
to the other Ags. The increase in the proliferation to HSP60, HSP65,
and PPD was stronger in pHSP60-treated rats than in those treated with
pHSP65. Thus, vaccination with pHSP60, which inhibits AA, can actually
augment T cell proliferation to HSP65 as well as to HSP60 itself;
likewise, HSP65 vaccination augments T cell proliferation to
HSP60 and to itself.
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AA immune response in vaccinated rats: effects on cytokine secretion
Cytokine release upon in vitro stimulation was studied to further
characterize the T cell responses of DNA-treated rats 26 days after AA
induction. DLN cells isolated from untreated rats (data not shown) or
from rats pretreated with control injections of pcDNA3 gave essentially
the same results (shown in Table I). Note the cytokine profile in
unmodified AA: although the proliferative response to P176-190 was
quite low, this peptide induced the release of IFN- to at least the
same levels as those achieved by stimulation with PPD. IFN- was
secreted to a lower extent in response to HSP65; very low secretion was
detected upon stimulation with HSP60. IL-10 and TGF1 were detected
only upon activation with PPD. Thus, induction of AA up-regulates
IFN- secretion in response to mycobacterial Ags, but the T cells in
these animals do not appear to respond to HSP60. The question was how
the cytokine profile might be modified by effective DNA vaccination
with pHSP60 or pHSP65.
The mean values of cytokine secretion obtained in three independent
experiments done using cells isolated from rats treated with pHSP60 or
pHSP65 are presented in Fig. 5, as the
percentage of change relative to the amount of cytokines secreted from
cells taken from control-treated rats. In addition, Table I shows the
results corresponding to a representative experiment, expressed as
picograms per milliliter. The effect of DNA vaccination on IFN-
secretion depended on the stimulating Ag. DLN cells from pHSP60- and
pHSP65-vaccinated rats secreted significantly lower amounts of IFN-
upon stimulation with P176-190 than did rats with unmodified AA;
secretion was reduced by half. Remarkably, IFN- secretion was
relatively increased in response to HSP60 after vaccination with pHSP60
or pHSP65. Thus, inhibition of AA was associated with a reduction in
IFN- secretion to P176-190 and with an increase in IFN- secretion
to HSP60.
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B).
In contrast to the Ag-dependent changes in IFN- and IL-10 secretion,
markedly increased amounts of TGF1 were produced by the cells of
vaccinated rats in response to stimulation with all the Ags tested:
mycobacterial Ags HSP65, PPD, P176-190, and HSP60 (Fig. 5C).
In summary, inhibition of AA by vaccination with specific DNA vaccines
was associated with three observations: first, decreased secretion of
IFN- upon stimulation with the HSP65 peptide P178-190. Second,
increased secretion of IFN- in response to stimulation with HSP60.
Third, augmentation of IL-10 and TGF1 secretion in response to both
mycobacterial Ags and HSP60.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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);
thus, the vaccine was functional. Although both constructs inhibited
AA, vaccination with pHSP60, which is much closer to rat self-HSP60
than is HSP65, had a significantly stronger effect (Fig. 2). Protection
from AA with both DNA constructs was associated with increased
proliferative responses to mycobacterial Ags and to HSP60 (Fig. 4B). Up-regulation of the proliferation to mycobacterial Ags
has also been found when AA was inhibited using a recombinant vaccinia
virus coding for HSP60 or HSP65, and by immunization with mycobacterial
HSP65 as a recombinant protein or a DNA vaccine (7, 13, 15, 16). Our finding of increased T cell proliferation to human
HSP60 in suppressed AA is compatible with the observation that
increased T cell proliferative reactivity to HSP65 and HSP60 is
associated with milder forms of RA and oligoarticular juvenile chronic
arthritis (10, 11).
The immune effects of DNA vaccination on AA were considerable.
Significant differences were found between control and pHSP60-treated
animals, in decreased disease both clinically (Fig. 2) and
histologically (Fig. 3). Immunologically, effective vaccination was
associated with increased T cell proliferation to specific Ags (Fig. 4B). However, how can increased T cell reactivity to
disease-associated Ags correlate with protection from AA?
Th cells have been assigned to different categories according to the
cytokines they secrete upon stimulation with their target Ags
(17). Among these categories, self-reactive Th1 cells
(defined by IFN- secretion) have been classically associated with
autoimmune pathology, while "regulatory" Th2/3 cells (secreting
IL-10 and/or TGF1) were proposed to protect against autoimmune
disease (22, 23, 24). In our experiments, protection by DNA
vaccination was associated with two different effects in the same rats:
down-regulation of IFN- secretion in response to P176-190 and
up-regulation of IFN- secretion in response to HSP60, along with
up-regulation of IL-10 and TGF1 (Table I and Fig. 5). Regarding
P176-190 the picture is clear: this peptide contains an epitope from
HSP65 that has been linked to the autoimmune process involved in AA. A
T cell clone specific to this epitope, which cross-reacts with
cartilage, could transfer arthritis to irradiated rats
(2, 3, 4). Thus, the decrease in IFN- release and increase
in IL-10 and TGF1 upon in vitro stimulation with P176-190
might reflect the effective control of pathogenic T cell clones
(3) in the DNA-vaccinated rats involving a switch from Th1
to Th2/3.
However, the induction of increased IFN- secretion in response to
"self" HSP60 does not seem to fit a simple cytokine shift
hypothesis. Indeed, other investigations suggest that IFN- secretion
might mark some regulatory T cells. A recent study by Paul et al.
(25) reported that HSP65-specific rat T cells able to
confer protection to AA cross-react with mammalian HSP60 and secrete
IL-10 and IFN-. Similar IFN--associated regulatory mechanisms
have been described to operate controlling self-reactivity after T cell
vaccination in humans (26, 27), or vaccination with
TCR-derived peptides (28) or DNA vaccines (29, 30) in experimental models of autoimmunity. In these examples,
vaccination induced a response rich in IFN- against the protective
vaccine, but this response correlated with a shift toward Th2 in the
pathogenic response to the target Ags of the pathogenic T cells.
Finally, the Tr1 regulatory cells (22, 23) first described
by Groux et al. (31) are characterized by the secretion of
IL-10, TGF1, and IFN-, but no IL-4, in response to Ag
stimulation, and have been shown to control Th1 (31) and
Th2 (32) immune responses. Hence, based on these results
we might propose that DNA vaccination with pHSP60 or pHSP65 induces a T
cell response to HSP60 that modulates the T cell response to P176-190
and other target Ags. The HSP60-specific T cells secrete both the Th1
cytokine IFN- and the Th2/3 cytokines IL-10 and TGF1. By
yet-unknown mechanisms, the regulatory HSP60 specific T cells induce a
more polarized shift to the Th2/3 cytokine profile of the autoimmune
clones responding to the P176-190 target Ag.
In our experiments, the immune response toward the P176-190 target
peptide was enriched in IL-10 and/or TGF1 and decreased in IFN-
secretors. Administration of recombinant murine IL-10 or of T cells
engineered to secrete rat IL-10 constitutively was shown to
down-regulate autoimmune arthritis (33, 34). In addition,
IL-10 can render activated T cells susceptible to the action of TGF1
(35). TGF1 is a suppressor cytokine with broad spectrum
of action (36). Although its role in the control of
experimental arthritis is just starting to be understood (37, 38), secretion of TGF1 by DLN cells taken
from pHSP60- and pHSP65-vaccinated rats might reflect the induction of
regulatory cells. Thus, the HSP60-specific regulatory T cells might
exert their effects through the secretion of IL-10 and TGF1.
But what could be the origin of the HSP60-specific regulatory T cells
in pHSP65-treated animals and how might this explain why pHSP65 is less
effective than HSP60 in vaccinating against AA? HSP65 and HSP60 are
members of a family of highly conserved proteins, and immunization with
HSP65 activates T cells cross-reactive with self-HSP60
(39). The results presented in this work indicate that
HSP60-specific T cells are indeed activated in pHSP65-vaccinated
animals upon induction of AA (Fig. 4B). Nevertheless,
vaccination with pHSP60 is apparently more effective than pHSP65 in
activating an anti-HSP60 regulatory response. Vaccination with
pHSP60 induced stronger proliferative responses to HSP60 (Fig. 4B), accompanied by the secretion of higher amounts of the
regulatory cytokines IL-10 and TGF1 upon activation with
mycobacterial Ags (Fig. 5, B and C). Thus,
although pHSP65 vaccination induced HSP60-specific T cells, the
response to HSP60 was not as strong as that induced by vaccination with
pHSP60 itself, leading to the generation of less IL-10 and/or TGF1
secretors and to a weaker control of AA by pHSP65.
In this work we demonstrate that it is feasible to control AA with a
DNA vaccine aimed to up-regulate the specific immune response directed
to self-HSP60. Our results are in agreement with the need for
controlled autoreactivity for the proper functioning of the immune
system and body homeostasis (40, 41, 42). Hence, therapies
aimed at activating built-in regulatory networks might serve as
effective tools for the management of autoimmune diseases. Indeed, Raz
et al. (43) have recently demonstrated that autoimmune
regulation can be activated in humans by vaccination with an HSP60
peptide leading to arrest of cell destruction in new-onset type 1
diabetes.
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Acknowledgments |
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Footnotes |
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2 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
3 Abbreviations used in this paper: AA, adjuvant arthritis; HSP65, 65-kDa heat shock protein; HSP60, 60-kDa heat shock protein; pHSP65, DNA vaccine encoding HSP65; pHSP60, DNA vaccine encoding HSP60; Mt, Mycobacterium tuberculosis; RA, rheumatoid arthritis; PPD, purified protein derivative; pMBP, DNA vaccine encoding murine myelin basic protein; DLN, draining lymph node; SI, stimulation index; RT, room temperature.
Received for publication April 11, 2002. Accepted for publication July 11, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and are protective in adjuvant arthritis. J. Immunol. 165:7270.8.2 DNA vaccination results in immune deviation and protection from experimental autoimmune encephalomyelitis. Int. Immunol. 13:835. response during T cell activation is modulated by IL-10. J. Immunol. 167:773.-1 (TGF-1) in autoimmune diseases. J. Autoimmun. 14:23.[Medline]
1 ameliorate disease in a collagen-induced arthritis model. Gene Ther. 4:553.[Medline]
-cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomized, double-blind, phase II trial. Lancet 358:1749.[Medline]This article has been cited by other articles:
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F. J. Quintana, P. Carmi, F. Mor, and I. R. Cohen DNA Fragments of the Human 60-kDa Heat Shock Protein (HSP60) Vaccinate Against Adjuvant Arthritis: Identification of a Regulatory HSP60 Peptide J. Immunol., October 1, 2003; 171(7): 3533 - 3541. [Abstract] [Full Text] [PDF]
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