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-Inducible Protein 10 Suppresses Ongoing Adjuvant Arthritis1
Departments of
* Immunology and
Morphological Sciences, and
Rappaport Family Institute for Research in the Medical Sciences, Bruce Rappaport Faculty of Medicine, Haifa, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein 10 (IP-10) is a CXC chemokine that is
thought to manifest a proinflammatory role because it stimulates the
directional migration of activated T cells, particularly Th1 cells. It
is an open question whether this chemokine is also directly involved in
T cell polarization. We show here that during the course of
adjuvant-induced arthritis the immune system mounts a notable Ab titer
against self-IP-10. Upon the administration of naked DNA encoding
IP-10, this titer rapidly accelerates to provide protective immunity.
Self-specific Ab to IP-10 developed in protected animals, as well as
neutralizing Ab to IP-10 that we have generated in rabbits, could
inhibit leukocyte migration, alter the in vivo and in vitro Th1/Th2
balance toward low IFN-, low TNF-
, high IL-4-producing T cells,
and adoptively transfer disease suppression. This not only demonstrates
the pivotal role of this chemokine in T cell polarization during
experimentally induced arthritis but also suggests a practical way to
interfere in the regulation of disease to provide protective immunity.
From the basic science perspective, this study challenges the paradigm
of in vivo redundancy. After all, we did not neutralize the activity of
other chemokines that bind CXCR3 (i.e., macrophage-induced gene
and IFN-inducible T cell chemoattractant) and yet significantly
blocked not only adjuvant-induced arthritis but also the in vivo
competence to mount delayed-type
hypersensitivity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1). Thereafter, Gong et
al. (12) used an antagonist of monocyte chemoattractant
protein 1 (MCP-1) to inhibit arthritis in the MRL/lpr mouse
model. Later, Barnes et al. (13) used antihuman RANTES to
ameliorate AA in the Lewis rat. Last, in a very recent study Fife et
al. (14) used anti-IFN--inducible protein 10
(IP-10) Ab to inhibit experimental autoimmune encephalomyelitis. We have used an alternative approach to generate antichemokine protective immunity. The basic idea was to clone various chemokine/cytokine, encoding genes into plasmid vectors with a strong CMV promoter and a repeated immunostimulatory sequence (i.e., CpG motif) that serves as a DNA adjuvant (15, 16, 17, 18) and then to use them as vaccines against the product of each inserted gene (19, 20, 21, 22, 23, 24). In these studies, we also demonstrated that this response includes T-dependent Ab production and generation of immunological memory that is turned on during an autoimmune condition to provide protective immunity (19, 20, 21, 22, 23, 24). This can potentially provide a patient with an ongoing autoimmune condition a powerful tool with which the immune system would restrain its own harmful activities (19, 20, 21, 22, 23, 24).
We have found much interest in exploring the role of the CXC chemokine IP-10 in the regulation of T cell-mediated autoimmunity. Our interest in this particular chemokine flows from recent studies demonstrating its ability to stimulates the directional migration of activated T cells, particularly Th1 cells (25, 26, 27), including those of human T cells in SCID mice (28), and from other studies suggesting a possible role for IP-10 in T cell-mediated autoimmunity (14, 29, 30, 31, 32). The current study explores IP-10-encoding DNA vaccination as a potential way to interfere in T cell polarization and provide long-lasting protective immunity against experimentally induced arthritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female Lewis rats, 
6 wk old, were purchased from Harlan
(Jerusalem, Israel) and maintained under clean conditions in our animal
facility.
Immunizations and active disease induction
Rats were immunized s.c. in the base tail with 0.1 ml of CFA (supplemented with 10 mg/ml heat-killed Mycobacterium tuberculosis H37Ra in oil; Difco, Detroit, MI). Rats were then monitored for clinical signs daily by an observer blind to the treatment protocol. Severity of the disease was quantified subjectively by scoring each limb on a scale of 0–4 to indicate the severity of peripheral joint swelling and erythema: 0, no signs of disease; 1, disease evident in a small number of distal joints of the limb; 2, disease evident in all of distal joints of the limb; 3, disease evident in all of the limb; and 4, severe disease evident in all of the limb. The arthritic clinical score was determined as the sum of the scores of all four limbs from each animal (0–16 limbs). In addition to clinical evaluation in all experiments, severity of disease was also assessed histologically as described below.
Cloning of rat IP-10
IP-10-specific oligonucleotide primers were designed based on its published sequence (National Center for Biotechnology Information accession number U22520) as follows: rat IP-10 sense, 5'-CATGAACCCAAGTGCTGCTGTCGT-3'; and rat IP-10 antisense, 5'-TTACGGAGCTCTTTTAGACCTTCT-3'. RT-PCR was then applied on mRNA form the inflamed AA joint. PCR products were 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; Upstate Biotechnology, 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 as we previously described (22). Sequenced PCR products of rat IP-10 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-Aldrich, St. Louis, MO) was repeatedly injected into the tibia’s anterior muscle of 4- to 6-wk-old female Lewis rats (10 µM/leg). One week following injection, rats were injected with 100 µg DNA in PBS. Four to 5 days after the first immunization, one rat from the group previously subjected to IP-10 DNA vaccination was sacrificed and transcription of IP-10 was verified using RT-PCR on tibia anterior muscle samples. For treatment of ongoing disease, AA rats were subjected to the administration of 500 µg DNA in PBS into the tibia’s anterior muscle (no cardiotoxin was used).
Production and purification of rIP-10
PCR product was recloned into a pQE expression vector, expressed in E. coli (Qiagen) and then purified by an NI-NTA-supper flow affinity purification of 6xHis proteins (Qiagen). After purification, the purity of rIP-10 was verified by gel electrophoresis followed by sequencing (N terminus) by our sequencing services unit.
Western blot analysis for IP-10-specific Ab in DNA-vaccinated rats
Our recombinant rat IP-10, produced as described above, and commercially available recombinant mouse IP-10 (PeproTech, Rocky Hill, NJ) were each subjected to Western blot analysis according to the protocol described in details elsewhere (33), with the minor modification of using a 12% (rather than 8%) running gel. IgG from IP-10 DNA-vaccinated rats or IgG from normal rat serum (final concentration of 1/500 each) was used as primary Ab. Goat anti-rat biotin-conjugated Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a second step, followed by streptavidin HRP (Jackson ImmunoResearch Laboratories), Western blotting Luminol Reagent kit (Santa Cruz Biotechnology, Santa Cruz, CA) was then used as a substrate.
Evaluation of anti-IP-10 Ab titer in sera of DNA-vaccinated rats
A direct ELISA has been used to determine the anti-IP-10 Ab titer in DNA-vaccinated rats. The rIP-10, which we have produced, was 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. Goat anti-rat IgG alkaline phosphatase-conjugated Ab (Sigma-Aldrich) was used as a labeled Ab. p-Nitrophenyl phosphate (Sigma-Aldrich) was used as a soluble alkaline phosphatase substrate. Results are shown as log 2 - Ab titer ± SE.
Cyanogen bromide (CNBr) purification of IP-10-specific Ab
Recombinant rat IP-10 (5 mg) was bound to a CNBr-activated Sepharose column according to the manufacturer’s instructions (catalog no. 17-0820-01; Pharmacia Biotech, Uppsala, Sweden). IP-10-specific Ab from sera (IgG fraction) of DNA-vaccinated rats were loaded on the column and then eluted by an acidic elution buffer (glycine, pH 2.5). Isotype determination of the purified Ab (ELISA) revealed that purified Ab is mostly of the IgG2a isotype (data not shown).
In vitro chemotaxis assay
The in vitro Boyden chamber chemotaxis assay was conducted as we have previously described (22). A total of 1.2x106 purified protein derivative (PPD)-specific T cells, selected as described elsewhere (24), was added to the upper well. Commercially available (Chemicon International, Temecula, CA) IP-10 (200 ng/ml) was used as a chemoattractant. fMLF (10-7 M; Sigma-Aldrich) was used as a positive control for chemoattraction. Purified Abs (IgG purification) were added at a concentration of 10 µg/ml. Result are shown as mean of triplicates ± SE.
Delayed-type hypersensitivity (DTH)
Rats were immunized intradermally into the dorsal surface of the
ear with 10 µg PPD (Pasteur Merieux Connaught, Toronto, Ontario,
Canada) in 25 µl of PBS (or with PBS alone). Ear thickness was
measured using a caliper (Lange Skinfold Caliper; Cambridge Scientific
Industries, Cambridge, MA). The ear thickness at time 0 was subtracted
from the 24-h measurement to give the amount of 
ear swelling in
response to PPD (34).
T cell separation and activation with anti-CD3+ Ab
T cells were positively selected using a MACS magnetic cell sorting kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. FITC-anti-CD4 (OX-35, catalog no. 22024D; BD PharMingen, San Diego, CA) was used as a first Ab and anti-FITC magnetic beads (catalog no.130-048-701; Miltenyi Biotec) were used as a second step. Purity of separation was confirmed by FACS analysis using an Ab specific for CD4+ T cells (W3/25; Serotec, Oxford, U.K.). Purified anti-rat CD3 (catalog no. 220100; BD PharMingen) at the concentration of 10 µg/ml was coated to 96-well plates for 1.5 h and washed twice. A total of 2 x 105 CD4+ T cells was added to each well and incubated for 72 h in full medium enriched with Con A supernatant as a source of IL-2 (35).
Cytokine determination in cultured primary spleen cells
The protein level of various cytokines was determined using
semi-ELISA kits: 1) IFN-, rabbit anti-rat IFN- polyclonal Ab
(CY-048; Innogenetics, Gent, Belgium) as a capture Ab,
biotinylated mouse anti-rat mAb (CY-106 clone BD-1; Innogenetics)
as a detection Ab, and alkaline phosphatase-streptavidin (catalog no.
43-4322; Zymed Laboratories, San Francisco, CA) with rat rIFN- as a
standard (catalog no. 3281SA; Life Technologies, Grand Island, NY); 2)
TNF-, commercial semi-ELISA kit for the detection of rat TNF-
(catalog no. 80-3807-00; Genzyme, Cambridge, MA); 3) IL-4, mouse
anti-rat IL-4 mAb (24050D OX-81; BD PharMingen) as a capture Ab and
rabbit anti-rat IL-4 biotin-conjugated polyclonal Ab (2411-2D; BD
PharMingen) as second Ab. Recombinant rat IL-4, purchased from R&D
Systems (504-RL; R&D Systems, Minneapolis, MN), was used as a standard;
and 4) IL-10, commercial semi-ELISA kits for the detection of rat IL-10
(BD PharMingen).
FACS analysis
FACS analysis was conducted according to the basic protocol we
used before (20). Before being subjected to intracellular
staining, cells were suspended with PMA (50 ng/ml; Sigma-Aldrich),
ionomycin (0.2 mM; Sigma-Aldrich), and monensin (0.2 mM; Sigma-Aldrich)
for 5 h. For intracellular staining, PE-labeled mouse anti-rat
IFN- mAb (BioSource International, Nivelles, Belgium), PE-labeled
mouse anti-rat TNF- mAb (BioSource International), and
FITC-labeled mouse anti-rat IL-4 mAb (BioSource International) were
used. Cells were analyzed using a FACSCalibur (BD Biosciences, Mountain
View, CA). Data were collected for 10,000 events and analyzed using a
CellQuest program (BD Biosciences).
Histopathology
Joints were removed, fixed with 10% buffered Formalin, decalcified in 5% EDTA in buffered Formalin, embedded in paraffin, and sectioned along the midline through the metatarsal region (36). Sections were stained with H&E and analyzed by a histopathologist who was a blind observer to the experimental procedure. Evaluation was made based upon inflammatory mononuclear cell infiltrate in the synovial membrane, thickness of the synovial lining, joint space narrowing, and periosteal new bone formation. The histological score was determined as follows: 0, no evidence of disease; 1, mild lymphocytic infiltrate; 2, widespread mononuclear inflammation and thickening of the synovial lining; and 3, severe bone destruction, new bone formation, and destruction of the synovial lining (36).
Statistical analysis
Significance of differences was examined using Student’s t test. A value of p < 0.05 was considered to be significant. Mann-Whitney U sum of ranks test was used to evaluate significance of differences in mean of maximal clinical score. A value of p < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under our working conditions, AA manifests a long-lasting form of
disease that includes an acute phase, peaking around day 20, early
chronic phase that persists until days 50–60, and late chronic phase
that persists >90 days after disease induction. At this time, AA rats
display low clinical score, yet their histological examination shows a
vigorous progression of disease (21, 24). To explore the
effect of IP-10-encoding DNA vaccines of disease manifestation, rats
were subjected to four weekly injections of IP-10-encoding DNA
vaccines. Following the last vaccination, the development of
self-specific Ab titer to IP-10 was carefully monitored. Within 4 wk,
this titer regressed to background levels (data not shown). At that
time, rats previously administered IP-10-encoding construct and control
rats that were repeatedly injected either with the pcDNA3 vector alone,
pcDNA3-
-actin construct, or with PBS were subjected to the induction
of active AA. Fig. 1
represents one of
three experiments with very a similar pattern of results. All control
groups (six per group) developed a severe form of disease (mean maximal
clinical score of 12.33 ± 1.2, 12.6 ± 1.1, and 13 ±
0.66, respectively, Fig. 1). In contrast, rats subjected to the
administration of the IP-10 construct developed a significantly reduced
form of disease (mean maximal score of 7.1 ± 0.6,
p < 0.001compared with each of the control groups). A
significantly reduced form of disease was also observed in these
animals during the early chronic phase of disease (day 42, 2.33 ±
0.6 vs 6.33 ± 0.54, 5.66 ± 0.23, and 6.83 ± 1,
p < 0.001). Clinical scoring has also been verified by
measuring the changes in the degree of paw swelling, once again by an
observer blind to the experimental procedure. At all times during the
acute and early chronic phases of disease, IP-10 DNA-vaccinated rats
exhibited a marked reduction in paw swelling compared with each of
the control groups (p < 0.001, data not
shown). Additionally, representative joint sections from all
experimental groups (four animals per group) were obtained on days 30
(Fig. 1B) and 90 (Fig. 1C) and were screened for
histological inflammatory mononuclear cell infiltrate in the synovial
membrane, thickness of the synovial lining, joint space narrowing, and
periosteal new bone formation. Sections obtained from IP-10
DNA-vaccinated rats displayed a marked reduction in each of the above
parameters as compared with control and pcDNA3-treated AA rats. Thus,
IP-10-encoding DNA vaccine can serve as a powerful tool to prevent not
only the inflammatory process during the acute and early chronic phases
of AA (Fig. 1, A and B), but also the
long-lasting damage to the joint during the late chronic phase of
disease (Fig. 1C).
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We determined whether the subsequent administration of IP-10 DNA
vaccine may elicit or amplify breakdown of tolerance to self-IP-10.
Lewis rats were subjected to four weekly injections of the IP-10 DNA
construct as described in Fig. 1
. One month after the last
immunization, when IP-10-specific Ab titer maintained a baseline level,
the rats were injected with CFA to induce active AA. At different times
serum from representative rats were analyzed for the presence of Ab to
IP-10. At first, SDS-PAGE (under reducing conditions) followed by
Western blot analysis confirmed that the Ab produced in IP-10-encoding
DNA-vaccinated rats binds not only our recombinant rat IP-10, but also
the commercially available mouse IP-10 fragment (Fig. 2A). These Abs did not bind
C-C chemokines including MCP-1, MIP-1, or RANTES (data not shown).
Then the kinetics of Ab production along the course of disease was
measured (Fig. 2B). Rats with developing AA displayed a
significant Ab titer to IP-10 (at the onset of disease
log2 Ab titer of 9 ± 0.4 in AA rats vs
6 ± 0 in control rats, p < 0.01) even without
DNA vaccination. This titer was dramatically accelerated just before
the onset of disease in DNA-vaccinated rats (Fig. 2B,
log2 Ab titer of 16.5 ± 0.66 in
DNA-vaccinated rats vs 9 ± 0.4 in control AA rats,
p < 0.001), possibly, to provide protective immunity.
To further characterize the function of these Abs, we have tested their
ability to affect the migration properties of spleen T cells from AA
rats (Table I), on the ability of IP-10
to induce T cell polarization (Table II),
and, most importantly, their competence to adoptively transfer to AA
resistance (Fig. 3).
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After CNBr-IP-10 purification, anti-IP-10 Abs from
DNA-vaccinated AA rats were determined for their specificity (Fig. 2C) and for their competence to inhibit the migratory
properties of activated T cells (Table I). From various proinflammatory
mediators (MIP-1, MCP-1, RANTES, TNF-, macrophage-induced gene
(MIG), IFN-inducible T cell chemoattractant (ITAC)), these purified
Abs specifically bound IP-10 (Fig. 2C,
log2 Ab titer of 22 ± 0.9 vs 6 ± 0.4,
6 ± 0.4, 5 ± 0.4, 6 ± 0.4, 5 ± 0.4, and 6
± 0.4, p < 0.001). To evaluate their competence to
inhibit the migratory properties of activated T cells, spleen T cells
from AA rats were activated (in vitro) with PPD, and subjected to an in
vitro migration assay in a Boyden chamber. Recombinant mouse IP-10
(PeproTech) significantly enhanced the migratory properties of
these cells (Table I, 141 ± 20 vs 76 ± 2, p
< 0.001), so did fMLF (Table I, p < 0.001).
Anti-IP-10 Ab could entirely reverse the chemoattraction properties of
IP-10 (80.1 ± 5 in the presence of anti-IP-10 Ab and 76
± 2 in their absence), but had no effect on fMLF-induced attraction
(Table I). Thus, the inhibitory effect of the Ab is chemokine
specific.
To determine whether IP-10 directs T cell polarization,
CD4+ T cells were purified from naive Lewis rats
and then subjected to anti-CD3-induced activation in IL-2-enriched
medium. Under these conditions, naive T cells undergoing activation
produced a Th2 cytokine profile with high levels of IL-4 (Table II,
112 ± 11 pg/ml) and low levels of IFN- (1.2 ± 0.3
ng/ml). The addition of IP-10 redirected T cell polarization into Th1
(Table II, IFN-, 2.8 ± 0.4 ng/ml; IL-4 15 ± 2 pg/ml,
p < 0.001 for each compression). Addition of
anti-IP-10 Ab from DNA-vaccinated AA rats reversed this effect
(Table II). Thus, IP-10 polarizes T cells into Th1. A function that can
be effectively reversed by neutralizing Ab produced in DNA-vaccinated
protected animals.
IP-10-specific Ab produced in DNA-vaccinated rats transfer protective immunity and alter the in vivo polarization of T cells
The in vitro characteristics of anti-IP-10 Ab purified from
protected rats (Tables I and II) suggests that these Abs may
participate in the regulation of disease in DNA-vaccinated rats. To
determine this possibility, we have detected their ability to
adoptively transfer AA resistance (Fig. 3A). Beginning 2
days after the onset of disease, AA rats were administered, every other
day, either purified anti-IP-10 Ab, IgG from naive rats, or PBS.
Only the subsequent administration of anti-IP-10 Ab led to a marked
reduction in disease severity (day 19: 6 rats per group, mean maximal
score ± SE of 11.33 ± 1.1 and 10.5 ± 1 in control and
IgG-treated rats, respectively, vs 2.17 ± 0.6 in
anti-IP-10-treated rats, p < 0.001 for each
comparison). At the peak of disease, the ability of these rats to mount
a DTH response to PPD was determined (Fig. 3B). It appears
that anti-IP-10 Ab could effectively (p <
0.0001) inhibit the ability of the immune system to mount a
proinflammatory DTH response. Inhibition of DTH could be associated
with altered migration of proinflammatory cells (Table I), altered
polarization of T cells (Table II), or both. To determine whether
neutralizing IP-10 affects T cell polarization in vivo, popliteal and
inguinal lymph node CD+ T cells were subjected to
intracellular staining of IL-4, IFN-, and TNF- (Fig. 4). Apparently the subsequent
administration of anti-IP-10 Ab redirected in vivo polarization of
CD4+ T cells into low IFN-, high IL-4 (Fig. 4, B vs A) low TNF- (Fig. 4, D
vs C)-producing cells. Very similar results were obtained
using rabbit anti-rat IP-10 polyclonal Ab which we have also
generated against our rIP-10. That is administration of these Abs using
the same protocol described above significantly suppressed AA and
blocked DTH (data not shown).
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From the practical perspective, it is important to evaluate the
competence of the IP-10 encoding naked DNA to interfere with the
progression of an ongoing disease. Lewis rats were immunized with CFA
to induce active AA and divided into four random groups. Two days after
the onset of disease (day 13) and on days 15 and 18, each group was
subjected to the administration of PBS, pcDNA3 alone, -actin
construct, or the IP-10-encoding construct (500 µg/rat). Although all
control and pcDNA3-treated rats continued to
develop severe AA, those exposed to the
IP-10 DNA vaccine exhibited a marked reduction of disease severity
(Fig. 5 A, day 24, mean maximal score of 11.25
± 1, 11.5 ± 0.9, and 12 ± 0.95, respectively, in rats
treated with either PBS, -actin construct, or pcDNA3 alone vs
3.8 ± 0.25, p < 0.001 for the comparison of
IP-10 DNA-vaccinated rats to each control group). The rapid production
of anti-IP-10 Ab in rats treated with IP-10-encoding DNA vaccines
(Fig. 5B) suggests an amplification of a pre-existing
response can explain, in part, the rapid beneficial effect of the
therapy. The notable reduction maximal clinical score in rats treated
with empty plasmid or -actin-encoding plasmid (Fig. 5A)
could not be attributed to elicitation of anti-IP-10 Ab titer (Fig. 5B) and is probably associated with the nature of the
plasmid itself. Clinical scores were confirmed histologically (Fig. 5C), including the chronic phase (day 60) at which the
massive cartilage loss, bone erosion, and periosteal new bone formation
characterized control and pcDNA3-treated rats were entirely absent in
joint sections of rats treated with IP-10 naked DNA
vaccine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously demonstrated that naked DNA vaccines encoding
self-proinflammatory mediators, particularly soluble cytokines and
chemokines, may be effectively used to breakdown immunological
tolerance to their gene products, resulting in the generation of
anti-self-immunity (19, 20, 21, 22, 23, 24). The current study defines
a significant anti-self-response to IP-10 in rats immunized with
CFA to induce active AA (Fig. 2B). We believe that this
response is part of a regulatory mechanism in which the immune response
mounts protective immunity against self proinflammatory mediators to
restrain other activities against self (i.e., autoimmune diseases).
Administration of the actin-encoding construct had no significant
effect on the development of this titer (Fig. 2B) that was
not sufficient to provide protective immunity (Fig. 1). This titer
rapidly accelerated in IP-10-encoding DNA-vaccinated rats (Fig. 1),
even when vaccines were administered after the onset of disease (Fig. 5), leading to AA suppression. In a very recent study, we have
demonstrated that administration of TNF--encoding DNA vaccine at the
peak of a full-blown experimentally induced arthritis can lead to a
rapid production of neutralizing Ab to self-TNF- and thus reverse
this long-lasting disease (21). In both studies, we could
not observe development of anti-self-Ab response to regulatory gene
products such as IL-10-, IL-4-, or IL-18-binding protein (data not
shown). It is possible that TNF- and IP-10 are among few
proinflammatory mediators against which the immune system mounts a
"natural" autoimmune response as a part of its attempt to restrain
autoimmunity. Because these Abs include IgG isotypes, it likely that
the response is a T-dependent one. To further elaborate this
interesting issue, anti-IP-10 Ab generated in AA rats that were not
subjected to IP-10-encoding DNA vaccines were purified from blood sera
(day 14) and evaluated for their neutralizing properties. These Abs, at
a concentration of 10 µg/ml, could significantly inhibit T cell
migration induced by 100 ng/ml rIP-10 (155 ± 12 without addition
of Ab, 148 ± 17 in the presence of total IgG from naive rats, and
84 ± 8 in the presence of Ab from AA rats, p <
0.001). This suggests that during the course of AA the immune system
mounts an immune response against IP-10 as a part of its attempt to
restrain the autoimmune condition. This may also explain why naked DNA
vaccines encoding IP-10 are so effective. After all, it probably
augments a pre-existing response that, to begin with, is not sufficient
to entirely suppress the development of an autoimmune condition.
Interestingly, targeting the function of a single proinflammatory
mediator suppresses a disease regulated by various cytokines and
chemokines (13, 43, 44, 45, 46, 47, 48, 49, 50). These results are very surprising
since different chemokines bind and activate common chemokine
receptors. This is also true for CXC chemokines (i.e., ITAC, MIG, and
IP-10 bind the same receptor). Two possible explanations should be
considered: 1) Despite competition on common receptors, the absence of
one chemokine could be overcome by others in an in vivo system. 2) The
clinical readout of disease resides from a balance between the activity
of various proinflammatory cytokines/chemokines such as TNF-, IL-1,
RANTES, MCP-1, and regulatory cytokines like IL-10 and IL-4. Thus,
blockade of the function of one proinflammatory mediator would
significantly alter this balance. Our results showing that blockade of
IP-10 not only alters the clinical balance of disease, but also
inhibits DTH (Fig. 3B) challenges the paradigm of chemokine
redundancy.
Recent studies demonstrated the ability of IP-10 to stimulate the
directional migration of activated Th1 cells (25, 26, 27).
Neutralizing the in vivo activity of IP-10 could alter the in vivo
Th1/Th2 balance (Fig. 4) either due to an increase in selective
accumulation of effector T cells at the autoimmune site and/or
following a direct effect of IP-10 on T cell polarization. It is indeed
an open question whether IP-10 can directly drive T cell polarization
into Th1. The current study demonstrates the ability of IP-10 to
directly drive naive T cells into Th1 (Table II). It is unclear whether
alteration of the Th1/Th2 balance toward Th2 may provide protection
from AA since IL-4-producing Th2 cells are regulatory cells, or because
alteration of this balance preferentially polarizes Ag-specific T cells
that produce a very low level of TNF- in vivo (Fig. 4, D
vs C). Hence, it is clear that targeted DNA vaccines
encoding IP-10 can be used to effectively circumvent potential pro-Th1
polarization of T cells resulting from CpG-mediated activation of cells
of the innate immune system (18), probably via Toll
receptor 9 (17). This is an alternative way to the one
recently suggested by Garren et al. (51) who used
IL-4-encoding DNA vaccines to redirect T cell polarization.
A major disadvantage in treating chronic diseases with xenogenic neutralizing Abs lies in their immunogenicity. This has motivated investigators to develop chimeric humanized Abs and mAbs engineered with human Ig H and L chain yeast artificial chromosome (52). However, following repeated immunization, these engineered Abs can trigger allotypic responses. The therapeutic strategy suggested here has an advantage over the above methods since it resulted in the generation of immunity to autologous Ags that accelerate during the course of an autoimmune condition in accordance with disease progression. Even though this makes this type of therapy a very promising means of treatment for RA and possibly other T cell-mediated autoimmune diseases (19, 22), one should be also aware of its limitations. Patients developing a chronic form of disease will have to spend the rest of their lives with Abs to their own IP-10. We therefore believe that such vaccines would be used in humans only after Ab/soluble receptor therapy has been explored.
We have recently demonstrated that RA patients, but not patients
suffering from osteoarthritis, mount a significant Ab titer against
TNF- (G. Wildbaum, A. M. Nahir, S. Youssef, Y. Geron, A.
Admon, and N. Karin, manuscript in preparation). Possible self-specific
titer to various chemokines, including IP-10, will also be determined.
Patients with RA are usually administered immunosuppressive drugs. In
light of our observations, it could be that these drugs suppress a
natural mechanism aimed to restrain the severity of disease. On the
other hand, while planning intervention in an ongoing disease by
IP-10-or TNF--encoding DNA vaccines, one should take into
consideration that such drugs might affect the efficiency of the
vaccines. Finally, the data presented in the current study, together
with our very recently published manuscript (53), may
suggest a novel role for IP-10 in the polarization of naive cells
entering an autoimmune site of inflammation (Fig. 6). This further
emphasizes the key role of this chemokine in T cell-mediated
autoimmunity.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nathan Karin, Technion-Israel Institute of Technology, Bruce Rappaport Faculty of Medicine, Rappaport Family Institute for Research in the Medical Sciences, Efron Street, P.O.B. 9697, Haifa 31096, Israel. E-mail address: nkarin{at}tx.technion.ac.il
3 Abbreviations used in this paper: RA, rheumatoid arthritis; IP-10, IFN--inducible protein 10; AA, adjuvant-induced arthritis; MIP-1, macrophage-inflammatory protein 1; MCP-1, monocyte chemoattractant protein 1; CNBr, cyanogen bromide; MIG, macrophage-induced gene; PPD, purified protein derivative; ITAC, IFN-inducible T cell chemoattractant; DTH, delayed-type hypersensitivity.
Received for publication December 19, 2001. Accepted for publication June 18, 2002.
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). Control rats that were repeatedly injected either with
the pcDNA3 vector alone (
), pcDNA3-
). One month later, all rats were subjected to the
induction of active AA. Data are from one of three independent
experiments with very a similar pattern of results. Six rats per group
were monitored for clinical manifestation of disease by observers blind
to the experimental protocol. Other rats were sacrificed at different
time points for histological evaluation. A, The mean
clinical score ± SE of six rats per group (b
< a, p < 0.001). B,
histological evaluation conducted 30 days after disease induction.
C, Histological evaluation conducted 90 days after
disease induction. At each time, joint samples from a naive rat
(a) or from two AA rats treated with PBS
(b), pcDNA3 alone (c), IP-10-encoding
construct (d), or 
),
and AA rats administrated the IP-10-encoding construct (
). Results
are given as mean Ab titer obtained from three rats per group ±
SD. C, After CNBr-IP-10 purification, our anti-IP-10
Abs were tested for specificity of binding to IP-10 compared with
other proinflammatory mediators including MIP-1
) rats and rats
treated with either empty vector (
), 