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*
Department of Immunology,
Rappaport Family Institute for Research in the Medical Sciences, and
Department of Morphological Sciences, Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel; and
§
Department of Hematology Rambam Medical Center, Haifa, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1),
monocyte-chemotactic protein-1 (MCP-1), MIP-1ß, and RANTES, were
performed on brain samples from EAE rats to evaluate mRNA transcription
at different stages of disease. Each PCR product was then used as a
construct for naked DNA vaccination. The subsequent in vivo immune
response to MIP-1 or MCP-1 DNA vaccines prevented EAE, even if
disease was induced 2 mo after administration of naked DNA vaccines. In
contrast, administration of the MIP-1ß naked DNA significantly
aggravated the disease. Generation of in vivo immune response to RANTES
naked DNA had no notable effect on EAE. MIP-1, MCP-1, and MIP-1ß
mRNA transcription in EAE brains peaked at the onset of disease and
declined during its remission, whereas RANTES transcription increased
in EAE brains only following recovery. Immunization of CFA without the
encephalitogenic epitope did not elicit the anti-C-C chemokine
regulatory response in DNA-vaccinated rats. Thus, modulation of EAE
with C-C chemokine DNA vaccines is dependent on targeting chemokines
that are highly transcribed at the site of inflammation at the onset of
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines are chemoattractants that mediate leukocyte attraction and
recruitment at the site of inflammation. As so, they are likely to be
key mediators in the recruitment of the secondary influx of leukocytes
at an inflamed target organ. This has motivated us to use the novel
technology of naked DNA vaccination (8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and explore the therapeutic
potential of anti-chemokine immunotherapy in EAE. Based on the
positions of the first two cysteines, the chemokines can be divided
into four highly conserved but distinct supergene families C-C, C-X-C,
C, and the newly discovered C-X3-C (18, 19). The C-C family is involved
primarily in the activation of endothelium and in the chemoattraction
of T cells and monocytes to the site of inflammation (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). The
protective competence of anti-C-C chemokine-based immunotherapy has
been demonstrated by Karpus et al., who blocked EAE in mice by
immunizing them with rabbit anti-mouse polyclonal Abs against
macrophage-inflammatory protein-1 (MIP-1) (33), and very recently
by Gong et al., who used an antagonist of monocyte-chemoattractant
protein-1 (MCP-1) to inhibit arthritis in the MRL-lpr mouse
model (34). In another study, Berman et al. used in situ
hybridization to demonstrate the dominant expression of MCP-1 in rat
EAE brain (35). In the current study, we have cloned each of the major
C-C chemokines, MCP-1, MIP-1, MIP-1ß, and RANTES, from EAE brains
into an eukaryotic expression vector and determined their capacity to
block EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female Lewis rats, approximately 6 wk old, were purchased from Harlan (Jerusalem, Israel) and maintained under specific pathogen-free conditions in our animal facility.
Peptide Ags
Myelin basic protein (MBP) p68–86, Y G S L P Q K S Q R S Q D E N P V, was synthesized on a MilliGen 9050 peptide synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC. Structure was confirmed by amino acid analysis and mass spectroscopy. Only peptides that were >95% pure were used in our study.
Immunizations and active disease induction
Rats were immunized s.c. in the hind footpads with 0.1 ml of MBP epitope 68–86 (p68–86) dissolved in PBS (1.5 mg/ml) and emulsified with an equal volume of CFA (in CFA supplemented with 4 mg/ml heat-killed Mycobacterium tuberculosis H37Ra in oil) (Difco, Detroit, MI). Rats were then monitored daily for clinical signs by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb paralysis; and 3, front and hind limb paralysis.
T cell lines
At 9 days after induction of active EAE, draining lymph node cells were cultured (12 x 106/ml) for 3 days in stimulation medium that includes DMEM (Life Technologies, Gaithersburg, MD) supplemented with 2-ME (5 x 10-5 M), L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 µ/ml), streptomycin (100 µg/ml), 1% syngeneic serum, and 20 to 30 µg/ml of the immunizing epitope; washed; and resuspended in resting medium, which was identical to the stimulation medium without syngeneic serum and with the addition of 10% FCS (Life Technologies) and 12.5% supernatant of Con A-stimulated splenocytes as a source of T cell growth factors. Con A supernatant was prepared as described elsewhere (36). After 5 to 7 days in resting medium, the cells (5 x 105/ml) were activated for 3 days in the presence of irradiated (2500 rad) syngeneic thymocytes (12 x 106/ml) and 10 to 20 µg/ml of p68–86. The activated T cells were then either used for induction of transferred EAE or resuspended in resting medium for additional growing cycles.
Induction of transferred EAE
Transferred EAE was induced by immunizing Lewis rats (i.p.) with 107 in vitro-activated (day 3) L68–86 cells.
RT-PCR analysis
RT-PCR analysis, verified by Southern blotting, was
utilized on brain samples, according to the protocol we described
elsewhere, with some modifications (3). Rats were euthanized by
CO2 narcosis. Brain samples containing mainly the midbrain
and brain stem were obtained after perfusion of the rat with 160 to 180
ml of ice-cold PBS injected into the left ventricle following an
incision in the right atrium. Each sample was homogenized. Total RNA
was extracted using the Tri-Zol procedure (Life Technologies),
according to the manufacturer’s protocol. mRNA was then isolated using
a mRNA isolation (kit 1741985; Boheringer Mannheim, Mannheim,
Germany), and reverse transcribed into first strand cDNA exactly as we
have described in detail elsewhere (3). First strand cDNA was then
subjected to 35 cycles of PCR amplification using specific
oligonucleotide primers that we designed based on the published
sequence of each chemokine (NCBI accession numbers: rat MIP-1,
U06435; rat MIP-1ß, U06434; rat RANTES, U06436; and rat MCP-1,
M57441), as follows: rat MIP-1 sense,
5'-ATGAAGGTCTCCACCACTGCCCTTGC-3'; rat MIP-1 antisense,
5'-TCAGGCATTCAGTTCCAGCTCAGTG-3'; rat MIP-1ß sense,
5'-ATGAAGCTCTGCGTGTCTGCCTTC-3'; rat MIP-1ß antisense,
5'-TCAGTTCAACTCCAAGTCATTCAC-3'; rat RANTES sense,
5'-ATGAAGATCTCTGCAGCTGCATCC-3'; rat RANTES antisense,
5'-CTAGCTCATCTCCAAATAGTTG-3'; rat MCP-1 sense,
5'-ATGCAGGTCTCTGTCACGCTTCTGGGC-3'; and rat MCP-1 antisense,
5'-CTAGTTCTCTGTCATACTGGTCAC.
All RNA samples were calibrated to ß-actin: rat ß-actin sense, 5'-CATCGTGGGCCGCTCTAGGCA-3'; rat ß-actin antisense, 5'-CCGGCCAGCCAAGTCCAGACG-3'.
The cycle profile was: denaturation at 95°C for 60 s, annealing
at 55°C for 60 s, and elongation at 72°C for 60 s.
Amplified products were subjected to electrophoresis, transferred to
nylon membranes (MagnaGraph nylon transfer membrane, msi; Westborough,
MA), fixed with UV light (120 mjoules), and hybridized with
106 cpm/ml of -32P-labeled DNA fragments
encoding the full-length PCR product of each C-C chemokine and of
ß-actin (random priming; Amersham, Arlington Heights, IL). PCR
products were used as probes only after each PCR product was cloned and
its sequence was verified as described below.
Cloning and sequencing of PCR products
Each of the amplified PCR products described above was cloned into a pUC57/T vector (T-cloning kit K1212; MBI Fermentas, Vilnius, Lithuania) and transformed to Escherichia coli, according to the manufacturer’s protocol. Each clone was then sequenced (Sequenase version 2; United States Biochemical, Cleveland, OH) according to the manufacturer’s protocol. PCR products were selected to be used as constructs for naked DNA vaccination only after cloning and sequence verification.
DNA vaccination
DNA vaccination was performed according to Waisman et al., with
some modifications (17). Sequenced PCR products of rat MIP-1, MCP-1,
MIP-1ß, and RANTES were transferred into a pcDNA3 vector (Invitrogen,
San Diego, CA). Large scale preparation of plasmid DNA was conducted
using Mega prep (Qiagen, Chatsworth, CA). Cardiotoxin (Sigma, St.
Louis, MO) was injected into the tibialis anterior muscle of 6- to
8-wk-old female Lewis rats (10 µM per leg). At 1 wk following
injection, rats were injected with 100 µg DNA in PBS. At 4 to 5 days
after the first immunization, one rat from each group was sacrificed,
and transcription of the relevant chemokine was verified using RT-PCR
on tibialis anterior muscle samples. Thereafter, naked DNA vaccines
were given three to five times, with intervals of 6 to 7 days between
each injection.
Production and purification of recombinant proteins
PCR products were recloned into a PQE expression vector (PQE-30, PQE-31, or PQE-32, according to the correct open reading frame), expressed in E. coli (Qiagen), and then purified by an NI-NTA-superflow affinity purification of 6xHis proteins (Qiagen). Each recombinant protein sequence has been verified (N terminus) by our sequencing services unit.
Purification of Abs
Abs from rat sera were purified using a High-Trap protein G column (Pharmacia, Piscataway, NJ), according to the manufacturer’s protocol. Then Ab titer to various chemokines was determined by an ELISA assay, as described below.
In vitro chemotaxis assay
In vitro chemotaxis assay was conducted as described previously
by one of us (37), with minor modifications (according to 38 .
Peritoneal macrophages were isolated as described previously (38) and
suspended in DMEM enriched with 1% BSA. Cell migration was evaluated
in standard Boyden chambers (Neuroprobe, Cabin John, MD). Macrophages
(1.2 x 106 cells) were added to the upper well.
Chemotactic factors FMLP (Sigma; 10-7 M), rat rMIP-1
(Chemicon International, Temecula, CA; 200 ng/ml), or rat rMCP-1
(Chemicon; 100 ng/ml) were added to the lower wells with or without
preincubation with the required Abs (10 µg/well) at 37°C for 30
min. Migration was allowed to proceed for 90 min at 37°C. The
cellulose nitrate filters (5 µm pore size) were then fixed and
stained as described previously (37). A total of 5 x400 fields were
selected randomly on each filter, and the number of migrating cells was
counted.
Evaluation of anti-chemokine Ab titer in sera of DNA-vaccinated rats
A direct ELISA assay has been utilized to determine the
anti-C-C chemokine Ab titer in DNA-vaccinated rats. Each
recombinant chemokine that we produced, as well as commercial
recombinant rat RANTES, rat MIP-1, rat MCP-1, and human MIP-1ß
(Chemicon) were coated onto 96-well ELISA plates (Nunc,
Roskilde, Denmark), at concentrations of 50 ng/well. Rat antisera, in
serial dilutions from 28 to 230 were added to
ELISA plates coated with each recombinant chemokine. Goat anti-rat
IgG alkaline phosphatase-conjugated Abs (Sigma) were used as a labeled
Ab. p-Nitrophenyl phosphate (p-NPP;
Sigma) was used as a soluble alkaline phosphatase substrate. The assay
conditions and data calculation of each test were performed according
to Reference 14. Results are shown as log2 Ab titer ±
SE. Commercial mAbs to each chemokine (Chemicon) were used as a
positive control for detected sera in each experiment.
Histopathology
Histologic examination of hematoxylin and eosin-stained sections
of Formalin-fixed, paraffin-embedded sections of brain and the lower
thoracic and lumbar regions of the spinal cord was performed. Each
section was evaluated without knowledge of the treatment status of the
animal. The following scale was used: 0, no mononuclear cell
infiltration; 1, 1 to 5 perivascular lesions per section with minimal
parenchymal infiltration; 2, 5 to 10 perivascular lesions per section
with parenchymal infiltration; and 3, >10 perivascular lesions per
section with extensive parenchymal infiltration. The mean histologic
score ± SE was calculated for each treatment group. A
representative photomicrograph is shown in Figure 3
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Significance of differences was examined using the Student
t test. A value of p < 0.05 was considered
significant. One-way multiple range ANOVA test with significance level
of p < 0.05 was performed for multiple compression of
Ab titer to various C-C chemokines in naked DNA-vaccinated rats (Figs. 4 and 6).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rats injected with L68–86 developed transferred EAE that
persisted for 5 to 6 days (Fig. 1A). Before adoptive transfer
of disease (day 0), and at various time points, before the onset of
disease (day 3), at the day of onset (day 5), the peak (day 7),
following recovery (day 10), and 10 days after recovery (day 20),
midbrain-brain stem samples were obtained from six different rats at
each time point. From each sample, mRNA was isolated and subjected to
RT-PCR analysis using specific oligonucleotide primers that we
constructed to each chemokine. Each amplification was calibrated to
ß-actin and verified by Southern blotting analysis. This enabled
semiquantitative analysis of the dynamics of mRNA transcription of each
of the above C-C chemokines at the site of inflammation. Figure 1B shows representative results from each time point of the
experiment. An increased transcription of MIP-1, MCP-1, and MIP-1ß
mRNA in EAE brains was observed at the onset of disease (day 5). The
augmented transcription of MIP-1 and MCP-1 regressed to background
within 2 days, even though disease continued to progress to its maximal
clinical score on day 7 (Fig. 1, A and B). The
increased transcription of MIP-1ß, however, declined to its
background in correlation with recovery (Fig. 1, A and
B). Unexpectedly, RANTES transcription increased in EAE
brains only after recovery. The biologic significance of this
observation remains to be elucidated.
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MIP-1ß at the onset of disease, which declines during recovery, and
an augmented transcription of RANTES following recovery (Fig. 1
, C and D). Prevention of EAE using C-C chemokine naked DNA vaccines
Cloned PCR products of each C-C chemokine, obtained as described
above, were ligated into a pcDNA3 eukaryotic expression vector and used
as constructs for naked DNA vaccination (Fig. 2). In the first experiment (Fig. 2A), rats were subjected to three weekly injections of each
construct. Control rats were either injected with the pcDNA3 vector
alone, or with PBS. Two weeks after the last immunization, all rats
were immunized with p68–86/CFA to induce active EAE. All control
(PBS-immunized) and pcDNA3-vaccinated rats developed active disease
that persisted for 5 to 6 days (Fig. 2A, 6/6 in each group
with a maximum clinical score 2.33 ± 0.1 in control and 2 ±
0.26 in pcDNA3-immunized rats). In contrast, rats injected with either
MIP-1 or MCP-1 DNA naked DNA vaccines were resistant to EAE
(incidence of 0/6 for MIP-1 and 1/6 for MCP-1-vaccinated rats with a
maximum clinical score of 0 and 0.33 ± 0.34, respectively;
p < 0.001 for each treatment compared with either
control or pcDNA3 treatments). Thus, the subsequent in vivo immune
response to MIP-1 or MCP-1 DNA vaccines prevented EAE. In contrast,
administration of the MIP-1ß naked DNA significantly aggravated
active EAE (Fig. 2A, 6/6 in each group, maximum clinical
score 3.2 ± 0.17 compared with 2.33 ± 0.1 in control and
2 ± 0.26 in pcDNA3-immunized rats; p < 0.033 and
0.028, respectively).
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, MCP-1, and MIP-1ß mRNA transcription in EAE brains peaked
at the onset of disease and declined during its remission, whereas
RANTES transcription increased in EAE brains only following recovery
(Fig. 1). Thus, intervention in EAE development by C-C chemokine DNA
vaccines is effective provided that the related chemokine is highly
transcribed at the site of inflammation at the onset of disease.
In a subsequent experiment, each of the above constructs, as well as
pcDNA3 alone, was administered five rather than three times (Fig. 2B). As with the first experiment, MIP-1 and MCP-1 naked
DNA vaccines effectively prevented the development of active EAE
(incidence of 1/6 for each treatment with a maximum clinical score of
0.17 ± 0.17, compared with 6/6 in either control or pcDNA-treated
rats; p < 0.001 for each comparison); MIP-1ß vaccine
significantly aggravated the disease (Fig. 2B, 6/6 in each
group, maximum clinical score 3 ± 0 compared with 2 ± 0 in
control and 1.33 ± 0.21 in pcDNA3-immunized rats;
p < 0.001 for each comparison); and RANTES naked DNA
vaccination did not exhibit any notable effect on disease
manifestation. Five consecutive immunizations of pcDNA3 did, however,
notably affect disease severity (maximum score in control rats 2
± 0 compared with 1.33 ± 0.21 in pcDNA-treated rats,
p < 0.007). It is possible that numerous subsequent
immunizations of a eukaryotic vector with a viral promoter may affect
cytokine production by T cells, as has recently been suggested (39).
When active EAE attained its maximal severity (day 12, Fig. 2B), spinal cord samples of representative animals from each
group (Expt. 2) were evaluated histologically (Table I, Fig. 3).
While control EAE rats and rats previously immunized with pcDNA3 all
displayed perivascular lesions with parenchymal mononuclear cell
infiltration (Fig. 3, B and C, and Table I,
B and C; mean histologic score 2.2 ± 0.3
and 1.8 ± 0.2, respectively), rats previously immunized with
MIP-1 or MCP-1 naked DNA vaccines were either free of mononuclear
cell infiltration, or exhibited minimal parenchymal infiltration (Fig. 3, D and E, and Table I, D and
E, compared with Fig. 3, B and C, and
Table I, B and C; mean histologic score 0.2
± 0.2 and 0.4 ± 0.24 compared with 1.8 ± 0.2 and 2.2
± 0.3; p < 0.001). In contrast, rats that were
immunized with MIP-1ß naked DNA vaccines manifested an extensive
parenchymal mononuclear cell infiltration (Fig. 3F and
Table IF, mean histologic score 3 ± 0). Thus,
inhibition or exacerbation of disease by various naked DNA vaccines
could each be demonstrated histologically.
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The development of anti-self-protective immunity in
DNA-vaccinated rats was evaluated. When active EAE attained its maximal
severity (day 12; Fig. 2B), blood samples of all animals
that were sacrificed for histologic evaluation (Expt. 2, Table I, Fig. 3) were analyzed for the production of Abs against gene products of
each vaccinated DNA (Fig. 4), for the
kinetics of Ab production along the course of active disease (Fig. 5), and for the possible development of
cross-reactive immunity between various chemokines (Fig. 6).
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A, 12.25 ± 0.55 versus 7 ± 0.47; to MIP-1,
Fig. 4B, 11 ± 0.47 versus 8.25 ± 0.55; to
MIP-1ß, Fig. 4C, 11 ± 0.47 versus 7 ± 0.47; to
RANTES, Fig. 4D, 11.25 ± 0.55 versus 7 ± 0.47),
which is, however, not sufficient to prevent the development of
disease. Naked DNA vaccination, on the other hand, significantly
augmented this Ab titer (Fig. 4, A–D, 14.5 ± 0.33,
14.75 ± 0.55, 16.25 ± 0.72, and 17 ± 0.66 in rats
immunized with MCP-1, MIP-1, MIP-1ß, or RANTES constructs versus
9 ± 0.47, 8.5 ± 0.33, 8.75 ± 0.29, and 7 ± 0.47
in rats immunized with pcDNA alone, p < 0.05 for each
reciprocal comparison, and versus 7 ± 0.47, 8.25 ± 0.55,
7 ± 0.47, and 7 ± 0.47 in naive rats, p <
0.05 for each reciprocal comparison; no significant difference was
identified between rats immunized with pcDNA alone and naive controls).
Nevertheless, the Ab titer in rats immunized with each C-C chemokine
DNA except RANTES markedly increased following induction of active EAE,
but not following an immunization with CFA without p68–86 (Fig. 4, A–D, 21.25 ± 0.98, 19.25 ± 0.73, 21.25 ±
0.99, and 20 ± 0.47 in rats immunized with MCP-1, MIP-1,
MIP-1ß, or RANTES constructs, and then with p68–86/CFA versus
14.75 ± 0.55, 13.75 ± 0.72, 15.25 ± 0.55, and
17.5 ± 1.1 in rats immunized with MCP-1, MIP-1, MIP-1ß, or
RANTES constructs, and then with CFA, p < 0.05, for
each reciprocal comparison except for the last one, 0.05 <
p < 0.1). Thus, naked DNA vaccines may serve as a
powerful technique to generate protective immunity against
autologous cytokines and provides a tool by which the immune system is
encouraged to elicit anti-self-protective immunity to restrain its
own harmful reactivity only when such a response is needed.
Sera from each of the above groups, immunized with various DNA vaccines
and then with p68–86/CFA, were analyzed for a possible development of
cross-reactive Ab titer (Fig. 6). Sera from MIP-1, MIP-1ß, and
RANTES DNA-vaccinated rats manifested a highly specific titer against
homologous Ag (p < 0.05 for the compression of
each titer to any of the other three chemokines). MCP-1-vaccinated
rats, however, exhibited a significant cross-reactive Ab titer against
MIP-1 (Fig. 6B; 21.25 ± 0.99 to self, 17 ±
0.66 to MIP-1, and 11 ± 0.47 to either MIP-1ß or RANTES;
p < 0.005 for the comparison of anti-MIP-1 with
anti-MIP-1ß or RANTES, Ab titer, and for the comparison of
anti-self with anti-MIP-1 Ab titer). Since both MCP-1 and
MIP-1 naked DNA vaccines are protective, it is possible that the
protective immunity generated by anti-MCP-1 DNA vaccination may
be mediated at least in part by reaction with MIP-1.
Since DNA vaccination elicits both cellular and humoral responses
against products of a given construct, it is difficult to know which of
these responses contributed more to the development of EAE resistance
in MCP-1 and MIP-1 DNA-vaccinated rats. To evaluate the possible
contribution of anti-self Abs to the development of EAE resistance,
12 days after active induction of EAE, when production of anti-self
Abs in naked DNA-vaccinated rats attained at its maximal titer (Fig. 5), Abs were purified (IgG fraction, protein G purification) and
evaluated for their competence to inhibit the migration of oil-induced
peritoneal macrophages in a Boyden chemotaxis chamber assay, as
previously described by one of us (37). MCP-1- and MIP-1-specific
Abs produced in MCP-1 naked DNA-vaccinated rats significantly blocked
MCP-1- and MIP-1-induced chemotaxis (Table II, 70 ± 7 and 88 ± 12 versus
185 ± 15, p < 0.001 for each comparison),
whereas MIP-1-specific Abs generated in MIP-1 naked
DNA-vaccinated rats effectively blocked MIP-1-induced chemotaxis
(63 ± 4 versus 155 ± 15, p < 0.001), and
to a much lesser extent MCP-1-induced chemotaxis (144 ± 11 versus
185 ± 15, p < 0.05, Table II). Thus, MCP-1 and
MIP-1 chemokine-specific Abs generated in naked DNA-vaccinated rats
are neutralizing Abs. These Abs were then evaluated for their
competence to provide subsequent protection from severe EAE (Fig. 7). Four days before the onset of active
EAE, rats were daily challenged (days 6–13) with 100 µg of each of
the above neutralizing Abs, or with Abs from rats that were vaccinated
with pcDNA3 alone. Repeated administration of Abs from MCP-1 and from
MIP-1 DNA-vaccinated rats provided substantial protection from
disease progression (mean maximal score of 0.66 ± 0.2 in rats
treated with purified Abs from either MCP-1 or MIP-1 DNA-vaccinated
donors versus 3.16 ± 0.2 and 3 ± 0 in rats treated with
purified Abs from PBS- or pcDNA3-treated rats, p <
0.001 for each compression). In addition, elevated levels of MCP-1- and
MIP-1-specific Abs could be observed in spinal cord fluid (SCF) of
EAE rats (day 12 of active EAE) that were previously subjected to MCP-1
or MIP-1 naked DNA vaccines (log2 Ab titer of 27 ±
3 and 18 ± 2 to MCP-1 and MIP-1 in SCF of rats administered
with MCP-1 naked DNA vaccine, and of 25 ± 3 to MIP-1 in SCF of
rats administered with MIP-1 naked DNA vaccine, compared with
12 ± 2 and 10 ± 1 in SCF of rats treated with pcDNA3 or
PBS, p < 0.01 for each comparison). Rats administered
with MIP-1 naked DNA vaccine did not generate a significant Ab titer
to MCP-1 compared with rats administered with pcDNA3 or PBS. Thus,
during the course of EAE, neutralizing Abs to MCP-1 and MIP-1
are generated in MCP-1 and MIP-1 DNA-vaccinated rats, and elevated
levels of these Abs can be identified at the site of inflammation in
the CNS, where they probably block disease progression.
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, MIP-1ß, or RANTES naked DNA
vaccines and then immunized with p68–86/CFA, as described in the
legend to Figure 2A. At different time points (0, 3, 5, 7,
10, 12, 21, 30, and 40 days after EAE induction), generation of
anti-self Ab was determined (Fig. 5, A–D). Each Ab
titer profoundly increased within 5 to 7 days of MBP p68–86/CFA
immunization (p < 0.001 compared with day 0). The increase was
simultaneous with the accelerated transcription of each chemokine mRNA
at the site of inflammation (Fig. 1), suggesting that generation of
each gene product at the site of inflammation elicits the production of
anti-self Abs. Each Ab titer peaked after the onset of disease (day
10–12) and returned to background within 40 days. Our data therefore
clearly show that transcriptional up-regulation of individual
chemokines in the CNS can provide protection from disease progression.
Finally, the competence of C-C chemokine naked DNA vaccines to render
long-lasting protective immunity against EAE was evaluated. Rats were
subjected to three weekly injections of C-C chemokine naked DNA
vaccines, as described above (Expt. 1, Fig. 2A). Two months
after last vaccine was administered, EAE was actively induced. Rats
immunized with either MIP-1 or MCP-1 DNA vaccines were highly
protected against EAE (incidence of 0/4 for each treatment, compared
with 4/4, with a maximal score of 1.25 ± 0.28 in either control
or pcDNA3-treated rats, p < 0.001 for each
comparison). MIP-1ß naked DNA vaccination, however, aggravated the
disease (incidence 4/4 with a maximal score of 2.5 ± 0.33,
p < 0.013 for each comparison). Thus, MIP-1 and
MCP-1 DNA vaccines generate long-lasting protective immunity against
autologous cytokines when such a response is needed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously have used RT-PCR verified by Southern blotting analysis
to follow the trafficking of T cells to the site of inflammation during
the course of transferred EAE and distinguished between selective and
nonselective stages in leukocyte homing to the CNS (3). Based on these
data, we have described the development of EAE as a sequential event in
which a primary influx (days 0–2) activates the blood brain barrier to
allow accumulation of a secondary influx of endogenous leukocytes and
the initiation of the disease (days 5–9) (3). Using the same
experimental system and the same strategy, we have now shown a positive
correlation in time course between the accumulation of the secondary
influx at the site of inflammation (3) and an elevated expression of
MIP-1, MCP-1, and MIP-1ß at the site of inflammation (Fig. 1A). Each of the above C-C chemokines is well known for its
competence to attract monocytes and T cells to a site of inflammation
and for its ability to elicit the expression of various adhesion
molecules that mediate the trafficking of these cells (3). Thus, the
positive correlation in time course between chemokine expression and
cell accumulation at the target organ may be explicated by the putative
biologic functions of these chemokines. Unexpectedly, RANTES
transcription augmented in EAE brains only after recovery. While
similar results were obtained previously in a murine model of disease
(40), the biologic implications of this observation are not fully
understood.
MIP-1 or MCP-1 DNA vaccines prevented EAE. MIP-1ß naked DNA
significantly aggravated the disease, and only the generation of in
vivo immune response to RANTES naked DNA had no notable effect on EAE
manifestation. Thus, intervention in EAE development by C-C chemokine
DNA vaccines was effective only for those chemokines that were highly
transcribed during the development of the inflammation. This emphasizes
the pivotal role of these chemokines in the pathogenesis of EAE. It is
possible, although still to be proved, that RANTES plays a role in the
establishment and maintenance of the resistant state following
recovery.
DNA vaccines represent a novel means of expressing Ags in vivo for the
generation of both humoral and cellular immune responses (10, 14, 41, 42, 43). This technology has proven successful in obtaining immunity
not only to foreign Ags and tumors, but also to self Ags, such as TCR V
genes (17) or autologous cytokines (42). C-C chemokines were selected
as candidates for DNA vaccination mostly because of their
well-established role in cell migration to a target organ (22, 23, 44, 45, 46, 47, 48, 49). Since DNA vaccination elicits both cellular and humoral
responses against products of a given construct (10, 14, 41, 42, 43), it is
difficult to know which of these responses contributed more to the
development of EAE resistance in MCP-1 and MIP-1 DNA-vaccinated
rats. It has, however, been shown that rabbit anti-MIP-1 Abs
were capable of blocking EAE in a murine model (33), and an antagonist
of MCP-1 markedly inhibited arthritis in the MRL-lpr mouse
(34). Under our experimental conditions, vaccination with MCP-1 DNA
elicited a significant cross-reactive immune response to MIP-1 (Fig. 6B; an Ab titer of 21.25 ± 0.99 to self, 17 ±
0.66 to MIP-1, and 11 ± 0.47 to either MIP-1ß or RANTES).
Our data clearly show that anti-chemokine Abs produced by naked DNA
vaccination are neutralizing Abs (Table II) and can provide subsequent
protection from severe EAE (Fig. 7). Thus, it is conceivable that these
Abs contribute to disease inhibition in MIP-1 and MCP-1 naked
DNA-vaccinated rats.
A major disadvantage in treating chronic diseases with xenogenic
neutralizing Abs lies in their immunogenicity. This has motivated
investigators to develop chimeric humanized Abs (reviewed in 50 ,
and mAbs engineered with human Ig heavy and light chain yeast
artificial chromosome (51). However, following repeated immunization,
these engineered Abs do trigger an apparently allotypic response. The
therapeutic strategy suggested by our study is of advantage over the
above methods since it resulted in the generation of immunity to
autologous Ag. In addition, our study reveals an unexpected, yet
extremely important, advantage in applying C-C chemokine DNA
vaccination. It appears that the immune response to each of the given
DNA constructs elicited only during the course of disease and only at
the time when the transcription of the related chemokine profoundly
elicited at the site of inflammation EAE induction (Figs. 1 and 4).
Finally, a recent study shows a coordinated chemokine up-regulation in brain and spinal cord during clinical relapse in mice with relapsing EAE (52). This emphasizes the importance of treating a disease caused by a malfunction of the immune system in distinguishing self from foreign, such as multiple sclerosis, by encouraging this system to elicit anti-self-protective immunity, and thus restrain its own harmful activity only when such a response is needed.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nathan Karin, Bruce Rappaport Faculty of Medicine, Technion, P.O.B. 9697, Haifa 31096, Israel. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; MBP, myelin basic protein; MCP, monocyte-chemotactic protein; MIP, macrophage-inflammatory protein; SCF, spinal cord fluid.
Received for publication March 9, 1998. Accepted for publication June 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4ß1 integrin. Nature 356:63.[Medline]
and TNF- production. J. Exp. Med. 180:2227.. Am. J. Pathol. 145:913.[Abstract]
(MIP-1) and MIP-1ß chemokines attract distinct populations of lymphocytes. J. Exp. Med. 177:1821. in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
, and MIP-1ß on human monocytes. Eur. J. Immunol. 25:64.[Medline]
during monocyte-endothelial cell interactions. Blood 83:1174.4ß1 and 5ß1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis. J. Cell Biol. 134:1063.- and ß-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am. J. Pathol. 150:617.[Abstract]
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