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Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1,
and IL-6) (4, 5), and T cells (mainly IFN-
)
(6) which contribute to generate the joint damage. In this
sense, although the role of T cells in the pathogenesis of arthritis is
not clear, it has been discovered that there is a polarization of their
response to a Th1 one, suggesting that an unbalance of the T response
toward a self-Ag could be also important in the development of
arthritis (6, 7, 8, 9). As a result of all these facts, the
synovium, which in normal conditions is a fragile bilayer membrane
covering the cartilage and bone in the joint, is transformed into a
thick invasive one that destroys its whole structure, creating an
instability and deformation of the tissue that explains the stiffness
and ankylosis experienced by the patient in the last steps of the
disease (10, 11).
Currently, the main treatment used in RA is drug therapy
(12). First-line agents are primarily used to relieve the
symptoms. Second-line agents (also called disease-modifying agents)
appear to have an effect on the underlying process. Because many
patients do not respond to the traditional antirheumatic drugs, and
some of these treatments show harmful side effects, considerable effort
is being focused on developing new drugs. In this sense, the blockade
of TNF- (13) and IL-6 (14) with mAbs seems
to be efficient to ameliorate the synovial inflammation in RA, although
a long term treatment with these Abs may result in an increased
incidence of other autoimmune diseases. Because a specific causative
agent or Ag has not been identified yet, bypassing the potential Ag and
targeting the cytokine unbalance could represent a solid way to
control RA.
Pituitary adenylate cyclase-activating polypeptide (PACAP), a 38-aa
neuropeptide belonging to the secretin-glucagon-vasoactive intestinal
peptide (VIP) family, exerts important functions in the nervous and
immune systems (15). One of the most interesting functions
of PACAP in the immune system is the antiinflammatory function, which
is exerted by its capacity to decrease proinflammatory cytokines like
TNF- and IL-6 and by the increase of antiinflammatory cytokines like
IL-10 (16, 17, 18, 19, 20, 21, 22, 23, 24). Thus, by modifying the levels of these
cytokines, it has been proved that PACAP prevents death from lethal
endotoxemia in mice (25). Also, PACAP can redirect a Th1
cell response to a Th2 response (26). In view of these
findings, PACAP appears to be a potential therapeutic compound for RA,
regulating the two main causes of the disease, the inflammatory and the
autoimmune responses. We have used the collagen II-induced arthritis
(CIA) model in DBA/1 mice (27, 28), which resembles many
of the clinical, histological, and immunological features of human RA,
to study the effect of an exogenous injection of PACAP, expecting a
remission of the symptoms of the disorder, by modulating the levels of
macrophage and T cell-secreted cytokines. Our results show that PACAP
administration is beneficial at both the pathological and the clinical
level and is a promising candidate for the treatment of RA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male DBA/1J mice 6–10 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME).
Materials
Native bovine type II collagen (CII), DNase type IV, polymyxin B
sulfate, and avidin peroxidase were purchased from Sigma (St. Louis,
MO). CFA was obtained from Difco (Detroit, MI). Calipers were obtained
from Kroeplin (Schluchtern, Germany). PACAP was purchased from
Calbiochem (La Jolla, CA). Ab pairs (capture/biotinylated) for IL-4 and
IFN- ELISAs (BVD4-1D11/BVD6-24G2, and R4-6A2/XMG1.2 respectively),
as well as biotin-conjugated goat anti-mouse IgG, IgG1, or IgG2a,
ELISPOT kits, and the Riboquant MultiProbe RNase Protection Assay
System were obtained from BD PharMingen (San Diego, CA). Matrix
metalloproteinase 2 (MMP-2) standards were obtained from Chemicon
International (Temecula, CA). Collagenase A was purchased from
Boehringer Mannheim (Mannheim, Germany). RPMI 1640 and FCS for tissue
cultures were purchased from BioWhittaker (Verviers, Belgium). The
tissue tearer was obtained from Biospec Products (Bartlesville,
OK). [-32P]dATP (3000 Ci/mmol) was purchased
from Amersham (Arlington, IL). X-ray films were obtained from Kodak
(Rochester, NY), and PhosphorImager SI was from Molecular Dynamics
(Sunnyvale, CA).
Induction of CIA
To study the effect of PACAP on arthritis, we used the experimental model of CIA. Briefly, CII, dissolved in 0.05 M acetic acid at 4°C overnight, was emulsified with an equal volume of CFA. Mice were injected intradermally at the base of the tail with 0.15 ml of the emulsion containing 200 µg CII and then boosted i.p. with 200 µg CII in PBS 21 days after the primary immunization.
Assessment of CIA
For the analysis of mice, conducted every other day, signs of
arthritis onset were monitored using as representative parameters paw
swelling and clinical score. The study was conducted in a blinded
manner by two independent examiners who determined the level of paw
swelling by measuring the thickness of the affected hind paws with 0-
to 10-mm calipers. Arthritis symptoms were assessed by using a scoring
system (grade 0, no swelling; grade 1, slight swelling and erythema;
grade 2, pronounced edema; grade 3, joint rigidity and ankylosis). Each
limb was observed and graded with a maximum possible score of 12 per
animal. Group comparisons were performed using the
2 test for disease incidence and an unpaired,
two-tailed Student t test for arthritis scores.
Treatment protocols
Treatment with PACAP began when the secondary immunization was performed, and it was administered i.p. at the specified doses either daily or every other day until day 35 after primary immunization. In some cases, PACAP was added at different times in pulses after the onset of the disease. A control consisting in a group of mice injected with PBS alone, was used in each experiment.
Histopathology
Forty-five days after the first immunization, mice were sacrificed by cervical dislocation, and two independent experimenters randomly collected the hind paws from five to nine animals. Paws were fixed with 10% paraformaldehyde, decalcified in 5% formic acid, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin-eosin-safranin O. Histopathological changes were scored in a blinded manner, using the following parameters. Infiltration of cells was scored on a scale of 0–3, depending on the amount of inflammatory cells in the synovial cavity (exudate) and synovial tissue (infiltrate). Cartilage destruction was graded on a scale from 0 to 3, from the appearance of dead chondrocytes (empty lacunae) to the complete loss of the joint cartilage. Bone erosion was graded on a scale of 0–3, from a normal appearance to completely eroded cortical bone structure.
mRNA analysis
A tissue tearer was used to homogenize mice joints, and total
RNA was isolated with the Ultraspec (Biotecx Laboratories, Houston, TX)
RNA reagent as recommended by the manufacturer. The Riboquant
MultiProbe RNase Protection Assay System was used on 2.5–5 µg RNA
following the manufacturer’s instructions for RNase protection assays.
A set of cytokine-chemokine templates and a template for the
housekeeping gene GAPDH were supplied by each commercial
kit. In vitro transcription from these cDNA templates was necessary to
synthesize [-32P]UTP-labeled antisense RNA
probes, being purified by phenol-chloroform extraction and ethanol
precipitation and hybridizing with the RNA samples at 56°C overnight
(unhybridized ssRNA was digested by RNase treatment). dsRNA
purified by phenol-chloroform extraction and ethanol precipitation was
electrophoresed on a 5% denaturing polyacrylamide gel, which was then
dried and exposed to x-ray films. The signal was quantitated in a
PhosphorImager SI.
The levels of TNF-, inducible NO synthase (iNOS), and MMP-2 mRNA
were determined by Northern blot analysis according to standard
methods. The probes for murine TNF-, iNOS, and GAPDH were generated
by RT-PCR as described previously (20, 21, 29), using
oligonucleotides that were end-labeled by a T4 polynucleotide kinase.
The membranes were exposed to x-ray films, and the signal was
quantitated in a PhosphorImager SI.
Zymography
Synovial culture supernatants were resolved as zymograph samples in 0.05 M Tris-HCl (pH 7.4), 5 mM CaCl2, 1% SDS, 5% glycerol and subjected to electrophoresis in 10% SDS-polyacrylamide gels into which gelatin (1 mg/ml) had been cross-linked (29, 30). After electrophoresis, the gels were washed with 2.5% Triton X-100 for 1 h. The gelatinolytic reaction was induced by incubating the gels in the reaction buffer (50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 5 mM CaCl2, 0.02% Na3N, 1 mM ZnCl2) at 37°C for an appropriate time (usually 24 h). Gels were stained and destained with 0.125% Coomassie brilliant blue and 10% acetic acid, 10% methanol, respectively. The stained gels were digitized, and the zones of proteolysis, corresponding to the presence of proteases in the gel, were quantitated using NIH image (National Institutes of Health, Bethesda, MD). Identification of the 72- and 62-kDa forms of MMP-2 was determined by comparison of the migratory position of the band with known MMP-2 standards.
In vitro study of T cell function
Mice were sacrificed at day 28 after primary immunization.
Single spleen cell suspensions were prepared and cultured in 96-well
flat-bottom microtiter plates at a density of 1 x
106 cells/ml (200 µl/well) in complete medium
in the presence or absence of different concentrations of
heat-inactivated CII. For proliferation assays, cells were
cultured at 37°C in 5% CO2 for 4 days, and 1
µCi/well [3H]TdR was added in culture for the
last 18 h. Cells were harvested, and
[3H]TdR uptake was measured using a 
scintillation counter. For IFN- and IL-4 production, cells were
cultured for 72 h, and supernatants were harvested and analyzed
for IFN- and IL-4 by sandwich ELISA using Ab pairs as previously
described (26). ELISPOT was used after 24 h culture
according to the supplier’s protocol to determine the frequency of
CII-specific T cells producing IFN- or IL-4 as previously described
(25). Purified protein derivative (PPD, 30 µg) was
injected intradermally in the CII-CFA emulsion as a recall Ag control,
and in vitro T cell function after culture stimulation with 10 µg/ml
PPD was assayed as described above.
Culture of synoviocytes
Mice were sacrificed at day 30 after primary immunization, the
rear limbs were removed, and the synovial membrane of the knee joints
was carefully separated from the bone and cartilage by microscopic
dissection. Digestion of the synovial tissue in the presence of 33
µg/ml polymyxin B sulfate was done with collagenase A (1 mg/ml) and
DNase type IV (150 µg/ml) at 37°C for 20 min, as previously
described for human synovial tissue (31). To prepare a
single-cell suspension, the digested tissue was passed through a nylon
mesh. After being washed extensively, and cells were cultured in
24-well plates at a density of 2 x 106
cells/ml (1 ml/well) in complete medium (RPMI 1640, 2 mM glutamine, 100
U/ml penicillin, 100 µg/ml streptomycin, 40 µM
2-mercaptoethanol, and 10% heat-inactivated
FCS) with or without PACAP at different concentrations. Supernatants
were collected after 24 h for chemokines, IL-1, and TNF-
analysis or 72 h for IL-10 and IL-12 analysis and stored at
-20°C until cytokine and chemokine determination. Sandwich ELISA was
performed to measure the levels of immunoreactive IL-12p40, IL-1,
IL-10, and chemokines as previously described (20, 21, 22).
The WEHI 164 cell line assay (32) was used to determine
the levels of bioactive TNF-.
Arthritis by adoptive transfer
DBA/1J mice were immunized with CII and CFA in the presence or absence of PACAP. After 14 days from the immunization, T cells were isolated from the mice spleens as previously described (26). Whole spleen cells (5 x 107 cells) or purified T cells (3 x 107 cells) were resuspended in complete medium and injected i.v. into naive DBA/1 mice. Arthritis development was observed in recipient mice as described above.
Measurement of serum anti-CII Ab levels
Serum samples were collected for the detection of anti-CII IgG, IgG1, and IgG2a Ab levels by ELISA 25, 35, and 45 days after primary immunization, as previously described (33). Briefly, ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 10 µg/ml native bovine CII in PBS and washed with PBS-0.05% Tween 20, and nonspecific protein-binding sites were blocked with 3% BSA-PBS for 2 h at room temperature. After a washing, samples in serial dilutions from 1/100 to 1/105 were added and incubated for 2 h at room temperature. After three washes, biotin-conjugated goat anti-mouse IgG, IgG1, or IgG2a was added and incubated at room temperature for 1 h, followed after extensive washing by avidin-peroxidase, and the plates were developed using ABTS as substrate. The OD was measured with a microplate reader. A standard serum, i.e., mixture of sera from arthritic mice, was added to each plate in serial dilutions, and a standard curve was generated to design arbitrary units of total IgG, IgG1, and IgG2a anti-CII Abs.
Flow cytometric analysis
Flow cytometry was performed for the analysis of intracellular
cytokines (IL-4 and IFN-) and surface Ags (CD3, CD4, and CD8) in
synovial cells as previously described by Kusaba et al.
(34).
Statistical analysis
To compare nonparametric data for statistical significance, we
applied the Mann-Whitney U test on all clinical results and
cell culture experiments. The 2 test was used
to analyze histological data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the effect of PACAP on arthritis, we used the CIA experimental model. DBA/1 mice were immunized with bovine CII in complete adjuvant, boosted with CII, and monitored for the occurrence of clinical signs of arthritis. Several doses of PACAP were given i.p. in two pulses, daily, or on alternate days for 2 wk after the onset of the disease.
The i.p. administration of PACAP resulted in a dose-dependent
improvement of the clinical symptoms and paw swelling (Fig. 1
), with a direct decreasing effect on
the incidence and the clinical score of the disease. Daily or every
other day administration of PACAP offered the best protection against
disease, although a single administration at the onset of disease was
enough to significantly ameliorate the pathologic signs of arthritis.
No remission in the therapeutic effect was observed after 2 wk of
cessation of PACAP administration on day 35 (Fig. 1, A and
C), suggesting that after a short period of PACAP treatment
no additional neuropeptide is necessary to maintain the protection from
the disease.
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Because few differences were observed between the 5- and 10-nmol doses and between PACAP administration daily and every other day, all additional experiments were conducted at the 5-nmol dose on alternate days.
Histopathological analysis of joints shows that CIA-characterized
chronic inflammation of synovial tissue (synoviocyte proliferation and
leukocyte infiltration), pannus formation, cartilage destruction, and
bone erosion were completely abrogated by treatment with PACAP (Fig. 2).
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). Therefore, PACAP
prevents adoptive transfer of arthritis.
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The mechanisms responsible for the observed decrease in incidence
and severity of CIA following PACAP treatment were next investigated.
One of the causes of the joint damage in arthritis is an excessive
inflammatory response (1). Because PACAP has been reported
to be a potent anti-inflammatory molecule (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), we
tested whether PACAP was able to affect the expression and production
of these proinflammatory agents in arthritic joints. Analysis of paws
by RNase protection assay and Northern blot shows that PACAP inhibited
CIA-induced mRNA expression of the proinflammatory factors TNF-,
IL-6, IL-12, iNOS, IL-18, IL-1, and IL-1, as well as of several
chemokines, such as RANTES, monocyte chemoattractant protein (MCP)-1,
macrophage-inhibitory protein (MIP)-1, MIP-1, and MIP-2 (Fig. 5A). However, mRNA levels of
the anti-inflammatory cytokines IL-10 and IL-1R antagonist (IL-1Ra)
were significantly increased on treatment of arthritic mice with PACAP
(Fig. 5A). Similar results were obtained with primary
synovial membrane cultures, derived from the knees of arthritic mice.
In vitro treatment of synovial cells with PACAP inhibited, in a
dose-dependent manner, spontaneous release of IL-1, TNF-, IL-12,
RANTES, MIP-1, MCP-1, and MIP-2 and significantly increased
production of IL-10 (Fig. 5B). Inhibition in chemokine
production is correlated by the fact that PACAP inhibits chemotactic
activity of both synovial T cells and macrophages from CIA mice (not
shown). Together, these results indicate that treatment with PACAP
reduces the inflammatory response characteristic of arthritis by
down-regulating and up-regulating the expression of proinflammatory and
anti-inflammatory agents, respectively, in inflamed joints.
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MMPs have been assigned pivotal roles in the depletion of
proteoglycan and collagen observed in the joints, which leads to the
cartilage and bone erosion in patients with RA. We further tested
whether PACAP regulates MMP production by arthritic synovial cells.
PACAP treatment significantly inhibited mRNA expression of the
gelatinase MMP-2 in paws of arthritic mice (Fig. 6A). This effect was
correlated with a PACAP-induced decrease on MMP-2 gelatinase activity
on synovial membrane cultures from arthritic mice (Fig. 6B).
In contrast, MMP-9 and MMP-13 production was not affected by the
treatment with PACAP (not shown). The inhibitory effect of PACAP on
MMP-2 could be directly related, at least partially, to the
PACAP-mediated inhibition of cartilage destruction and bone
erosion.
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Because one of the main characteristics of CIA is
hypercellularity, and PACAP has several immunomodulatory effects,
including inhibition of T cell proliferation and regulation of Th1-Th2
balance (26, 36), we investigated whether impaired T cell
functions in PACAP-treated mice lead to CIA inhibition. We first tested
the effect of PACAP treatment in CII-specific proliferative responses
of spleen cells from CIA mice. Whereas spleen cells from control mice
proliferated in response to CII, T cells from mice receiving PACAP
responded slightly to CII (Fig. 7A). Thus, T cell clonal
expansion toward CII is inhibited, at least partially, by PACAP, this
being one of the mechanisms that this neuropeptide could be using to
abrogate the disease.
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There is evidence that CIA is an autoimmune disorder mediated by Th1 cytokines, whereas the supply of Th2 cytokines has great benefit in the development of severe arthritis (6, 7, 8, 9, 37, 38). Because PACAP has been noticed to polarize the Th response to a Th2 response, we next studied the effect of PACAP on T cytokine production in CIA mice by using CII as a specific stimulus.
As shown in Fig. 7
B, CIA resulted in the development of
CII-specific effector T cells producing high levels of IFN- and low
levels of IL-4, showing a Th1 secretion pattern, which is one of the
characteristics of the disease. Inversely, cells isolated from
PACAP-treated mice produced large amounts of IL-4 and low levels of
IFN- (Fig. 7B). Therefore, PACAP leads the T cell
response to a Th2 response in the CIA model of arthritis. No cytokine
production by T cells was detectable in the presence of an unrelated Ag
(OVA; data not shown).
To test whether this control of Th1/Th2 cytokine balance was exerted by
regulating the in vivo generation/differentiation of CII-specific Th1
and Th2 effector cells, the frequency of Ag-reactive Th1 and Th2 cells
was assessed on the basis of IFN-- and IL-4-secreting cells,
respectively, using ELISPOT assays. Mice injected with the Ag in the
absence of PACAP developed many IFN--producing Th1 cells, and very
few IL-4-producing Th2 cells (Fig. 7C). In contrast, CIA
mice injected with PACAP generated few IFN--specific spots, while
developing high numbers of IL-4-secreting Th2 cells (Fig. 7C).
PACAP regulates CII-specific IgG1 and IgG2a Abs in CIA
Production of high levels of circulating Abs against CII is a major factor in determining susceptibility to CIA. Th1 and Th2 lymphocytes differentially affect switching to IgG2a/b and IgG1, respectively. Because the development of Ag-specific Abs requires T cell help, one mechanism of CIA inhibition by PACAP could be due to a failure to produce anti-CII Abs, particularly autoreactive IgG2a Abs that have been involved in the pathogenesis of CIA. The serum levels of total IgG or isotype-specific IgG2a and IgG1 anti-CII Abs were measured at different times after onset of arthritis.
As Th1 and Th2 cytokines modulate Ig isotype switching, we measured the
serum levels of specific anti-CII IgG isotypes as another proof of
PACAP modulation of the Th1-Th2 response in CIA. This study was
performed at different times from the onset of the induced arthritis.
CIA resulted in high levels of CII-specific IgG Abs, characterized by a
high IgG2a:IgG1 ratio (Fig. 7D). In contrast, treatment of
CIA mice with PACAP significantly reduced anti-CII-specific IgG
levels, mainly during the first days after disease onset (Fig. 7D). This inhibitory effect was accompanied by a reduction
in IgG2a:IgG1 ratio, due to inhibition and stimulation of anti-CII
IgG2a and IgG1 production, respectively (Fig. 7D), another
proof of PACAP modulation of the Th1-Th2 response in CIA.
PACAP decreases Th-suppressor ratio in the synovial tissue
Arthritic joints present an elevated Th-suppressor ratio in
comparison with the blood and spleen T cell ratios. Therefore, we next
investigated whether this ratio in synovial cells can be affected by
PACAP treatment of ongoing CIA. As Fig. 8A shows, PACAP-treated mice
showed a significantly lower CII-specific proliferative response of
synoviocytes than that in control arthritic mice. PACAP treatment of
CIA mice reduced the Th (CD4)-T suppressor (CD8) ratio from 11
(controls) to 
4.4 (Fig. 8B, top). Because T suppressor
numbers were not significantly affected, although Th numbers are lower
in PACAP-treated mice than in controls (Fig. 8B, bottom),
the CD4:CD8 ratio decrease seems to be due to impaired development of
Th cells, rather than to an increase in the number of T suppressor
cells. This relative decrease of synovial Th cells mediated by PACAP
was due to a preferential diminution of IFN-- vs IL-4-producing CD4
cells (Fig. 8B), probably by inhibiting development of
CII-specific Th1 and/or their entry into joints.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main action of PACAP in relation to this chronic disease is its
capacity to modulate the levels of several soluble factors including
proinflammatory, anti-inflammatory cytokines, and chemokines, which
deregulation triggers an excessive immune response leading to the
damage of the joint (15, 16, 17, 18, 19, 20, 21, 22, 23). On the one hand,
PACAP function is mediated by its effect on the inflammatory component
of the disease by down-modulating the cytokines, chemokines, and
destructive enzymes that are massively released by the activated
fibroblasts and macrophages in the altered synovial tissue
(1, 2, 3, 4, 5). Thus, this neuropeptide not only acts directly,
reducing the levels of proinflammatory mediators like TNF-, IL-6,
IL-1, iNOS, and IL18, but also increases the amounts of
antiinflammatory cytokines like IL-10 and IL-1Ra. In this sense, the
down-regulation of TNF- is crucial to explain the healing of the
affected mice because this cytokine is considered to have a critical
role in the development of inflammatory diseases like RA or Crohn’s
disease in that it promotes the increase of the production of most
other proinflammatory mediators (1, 2, 3, 4, 5). PACAP also has
antirheumatic effects because it reduces the levels of chemokines like
RANTES, MIP-1, MIP-1, MIP-2, and MCP which are responsible for
the hypercellularity of the synovial membrane of CIA mice. Other
soluble mediators like MMPs (MMP-2), which contribute to destruction of
the joint structure by attacking the conjunctive support
(10), are also down-regulated by PACAP. The main action of
PACAP as an anti-inflammatory factor on all these mediators is
carried on macrophages (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 39). Thus, this cellular
type might be the main target of the antiinflammatory action of this
neuropeptide in the synovial tissue. As a result of the reduction of
the levels of all of these harmful soluble factors in RA, there is a
clear remission of the chronic inflammation of the joint of the
affected mice.
The effect of PACAP is not restricted to the mediators produced by
synoviocytes; it also affects the cytokines released by the infiltrated
T cells. There is strong evidence that the majority of the T cells in
the inflamed tissues in RA as in many other autoimmune diseases show a
Th1 cytokine pattern (6, 7, 8). In fact, certain factors that
promote a Th2 response instead of a Th1 response are beneficial for
patients with these disorders. Moreover, the Ig isotype switching that
is directed by Th1 or Th2 cytokines in a different way (i.e., IFN-
and IL-4 induce IgG2a and IgG1 synthesis, respectively) is another
marker of this disease that shows high anti-CII IgG2a circulating
levels (40). PACAP has been previously shown to induce a
Th2 response by stimulating the release of Th2 cytokines and inhibiting
the production of Th1 cytokines (22). Moreover, PACAP
preferentially induces the expression of costimulatory molecules
related to Th2 differentiation (i.e., B7.2) in APC like macrophages
after Ag stimulation (26). An additional mechanism could
be the preferential PACAP-induced prevention of clonal deletion of Th2
against Th1 cells after antigenic stimulation, resulting in the
generation of Th2 effector and memory
cells.4 The data of
the present report regarding these cytokines (reduction of IFN-
levels and increase of IL-4 in treated mice) and the levels of the
different Ig isotypes (reduction of IgG2a but increase of IgG1 in
PACAP-treated mice) confirm the Th2-inducing response of PACAP,
possibly being one of the factors that contribute to the remission of
the disease, blocking the autoimmune component of this disease.
All in all, the effects of PACAP in the CIA model suggest it as a therapeutic agent that could be possibly used in the treatment of the human RA. It has been reported that endogenous neuropeptides and hormones like calcitonin-gene related peptide and melanocyte-stimulating hormone, which are released in the immune microenvironment, have beneficial effects on different arthritic disorders (30, 41). Recently, VIP, a neuropeptide that is structurally related to PACAP and shares its receptors and many of the functions that PACAP exerts in the immune system, has been demonstrated to have an important down-regulative action on arthritis (42). Therefore, as in other inflammatory diseases, all these endogenously produced peptides, probably including PACAP, are increased in the arthritic mice, acting as a natural antiarthritic attempt to reduce the excessive response of the immune system.
The fact that PACAP exerts most of its actions in immune system by increasing intracellular cAMP levels (18, 19, 20, 21, 22, 23, 24, 36) suggests that the activation of this pathway by PACAP is the mechanism of action mainly involved in its antiarthritic effect. In this sense, other cAMP-inducing agents, such as rolipram and other type IV phosphodiesterase inhibitors, have been shown to prevent CIA and are currently searched as alternative therapies for arthritis (43, 44, 45).
In view of these findings, pharmacological studies to determine the correct dose, route of administration, and possible side effects could be of special interest because arthritis has become a frequent disease in the world population and without many expectations of cure with the current alternatives. The studies performed in mice do not show any harmful secondary effects, supporting the idea that PACAP could be investigated as a drug for the treatment of arthritis. Moreover, although a supply of PACAP in daily or a every other day doses is more successful, a single injection in the beginning or even in the later stages of the illness is efficient enough to prevent its worsening. All of these facts should be taken into account in future studies on the use of PACAP in the treatment of RA. Based on our results, PACAP is a promising candidate for the development of treatment of RA and other chronic inflammatory diseases and autoimmune disorders.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mario Delgado, Department of Cell Biology, Faculty of Biology, Complutense University, Madrid 28040, Spain. E-mail address: mariodm{at}eucmos.sim.ucm.es
3 Abbreviations used in this paper: RA, rheumatoid arthritis; PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal peptide; CIA, collagen II-induced arthritis; CII, type II collagen; MMP-2, matrix metalloproteinase 2; IL-1Ra, IL-1R antagonist; iNOS, inducible NO synthase; PPD, purified protein derivative; MCP, monocyte chemoattractant protein; MIP, macrophage-inhibitory protein.
4 M. Delgado and D. Ganea. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide enhance the in vivo generation of memory Th2 cells by inhibiting the peripheral deletion of antigen-specific effectors. Submitted for publication.
Received for publication March 12, 2001. Accepted for publication July 16, 2001.
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 R.P. GOMARIZ, Y. JUARRANZ, C. ABAD, A. ARRANZ, J. LECETA, and C. MARTINEZ VIP-PACAP System in Immunity: New Insights for Multitarget Therapy Ann. N.Y. Acad. Sci., July 1, 2006; 1070(1): 51 - 74. [Abstract] [Full Text] [PDF]
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 M. Delgado, A. Chorny, E. Gonzalez-Rey, and D. Ganea Vasoactive intestinal peptide generates CD4+CD25+ regulatory T cells in vivo J. Leukoc. Biol., December 1, 2005; 78(6): 1327 - 1338. [Abstract] [Full Text] [PDF]
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C. Martinez, Y. Juarranz, C. Abad, A. Arranz, B. G. Miguel, F. Rosignoli, J. Leceta, and R. P. Gomariz Analysis of the role of the PAC1 receptor in neutrophil recruitment, acute-phase response, and nitric oxide production in septic shock J. Leukoc. Biol., May 1, 2005; 77(5): 729 - 738. [Abstract] [Full Text] [PDF]
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 H. Kato, A. Ito, J. Kawanokuchi, S. Jin, T. Mizuno, K. Ojika, R. Ueda, and A. Suzumura Pituitary adenylate cyclase-activating polypeptide (PACAP) ameliorates experimental autoimmune encephalomyelitis by suppressing the functions of antigen presenting cells Multiple Sclerosis, December 1, 2004; 10(6): 651 - 659. [Abstract] [PDF]
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 M. Steinhoff, S. Stander, S. Seeliger, J. C. Ansel, M. Schmelz, and T. Luger Modern Aspects of Cutaneous Neurogenic Inflammation Arch Dermatol, November 1, 2003; 139(11): 1479 - 1488. [Abstract] [Full Text] [PDF]
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M. DELGADO, C. ABAD, C. MARTINEZ, M. G. JUARRANZ, J. LECETA, D. GANEA, and R. P. GOMARIZ PACAP in Immunity and Inflammation Ann. N.Y. Acad. Sci., May 1, 2003; 992(1): 141 - 157. [Abstract] [Full Text] [PDF]
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) mice was assessed. Results are expressed
as the mean ± SD from three separate experiments (eight
mice/group per experiment); p < 0.001 vs control
for both 5-nmol treatments after PACAP administration.
, control; 
, PACAP). Spleen cells from
PPD/CFA-immunized CIA mice stimulated with PPD Ag (10 µg/ml) were
used for assessed Ag specificity (
, control; 
, PACAP). A pool of
three unimmunized DBA/1 spleens were used for assessment of the basal
response. A, Proliferative response was determined after
4 days of culture by [3H]TdR incorporation and was
expressed as a stimulation index ((CII-specific proliferation -
unstimulated proliferation)/unstimulated proliferation).
B, After 72 h culture, IL-4 and IFN-