Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

Velosa Ana, Teodoro, Dos, Konno Renata, Oliveira, Katayama, Parra, Capelozzi, Yoshinari

Description

The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. Results IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p < 0.001), bronchioles (0.294 ± 0.139 vs. 0.646 ± 0.172, p < 0.001) and in the septal interstitium (0.027 ± 0.014 vs. 0.067 ± 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002) and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001). Conclusions Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2010
• Nombre de lectures: 4
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Velosa et al. Respiratory Research 2010, 11:1
http://respiratory-research.com/content/11/1/1
RESEARCH Open Access
Collagen V-induced nasal tolerance
downregulates pulmonary collagen mRNA gene
and TGF-beta expression in experimental
systemic sclerosis
1 1* 1 1 1Ana Paula P Velosa , Walcy R Teodoro , Daniel M dos Anjos , Renata Konno , Cristiane C Oliveira ,
2 3 3 1Maria LH Katayama , Edwin R Parra , Vera L Capelozzi , Natalino H Yoshinari
Abstract
Background: The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-
beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal
tolerance.
Methods: Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund’s adjuvant
(IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day) (IM-
TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III
and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining
and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for
multiple comparison when appropriate and the level of significance was determined to be p < 0.05.
Results: IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels
(0.371 ± 0.118 vs. 0.874 ± 0.282, p < 0.001), bronchioles (0.294 ± 0.139 vs. 0.646 ± 0.172, p < 0.001) and in the
septal interstitium (0.027 ± 0.014 vs. 0.067 ± 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM,
showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07
vs. 1.0 ± 0.528, p = 0.002) and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009) collagen, in addition to decreased TGF-beta
expression (p < 0.0001).
Conclusions: Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary
remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased
TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.
Background of pulmonary hypertension varies from 5% to 35% [1]. A
Progressive Systemic Sclerosis (SSc) is an autoimmune diagnosis of SSc has important prognostic implications
disease of unknown pathogenesis, characterized by the owing to the clinical course marked by inexorable dete-
increased extracellular matrix (ECM) synthesis, vascular rioration. Currently, no medical therapies have proved
remodeling and autoantibody emergence, which results to prolong life expectancy. Thus, there is great interest
in scarring in multiple organs. The lung is usually in understanding lung involvement in SSc and the
involved, and is the main cause of mortality in this dis- effects of treatment to avoid irreversible scarring and
ease [1]. Interstitial lung fibrosis, of variable intensity, decreased survival. Although the exact mechanism of
affects approximately 90% of patients, and the frequency treatment effects remains unknown, the influence of
immune inflammatory cells and their mediators is
diminished in animal models [2,3], thus affecting
* Correspondence: [e-mail]
1Rheumatology Division of the School of Medicine of the University of São
Paulo (FMUSP), São Paulo, SP, Brazil
© 2010 Velosa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.Velosa et al. Respiratory Research 2010, 11:1 Page 2 of 10
http://respiratory-research.com/content/11/1/1
collagen synthesis and degradation and interfering with and 2 months of age. The complete immunization pro-
ECM remodeling. tocol includes 4 inoculations. The first is a subcutaneous
Because ECM remodeling is thought to promote pul- (sc) injection with 1 mg of Col V isolated from human
monary restoration, a group of collagens have been tar- placenta [11-15], diluted in 1 ml of 10 mM acetic acid
geted as potentially useful indicators of ECM and added to an equal amount of complete Freund’s
remodeling [4,5]. Specifically, collagen V is a promising adjuvant (Sigma Chemical Co.; St. Louis, Missouri,
indicator [6]. Collagen V is a highly conserved molecule USA). The second inoculation occurs after 30 days and
among different animal species [4,5] and is normally the animals received an identical subcutaneous injection.
found in lung ECM, composing the heterotypic fibrils Fifteen days after the second subcutaneous injection, the
with types I and III collagen. Collagen V is a minor col- rabbits received one reinforcement dose of 1 mg of Col
lagen fraction not normally exposed in the tissues V plus 1 ml incomplete Freund’s adjuvant intramuscu-
[7-10], retaining the amino- and carboxy-terminals, larly (third inoculation). Finally, a second identical rein-
making it quite immunogenic. forcement (fourth inoculation) is administrated after
Previously, we discovered an experimental model of another 15 days [11-15]. The control group (N = 6) was
SScbyimmunizinghealthyNew Zealand rabbits with inoculated with Freund’s adjuvant diluted in 10 mM of
humancollagenVemulsifiedwithFreund’s adjuvant. acetic acid, following the same protocol of the immu-
This resulted in intense inflammation of the lung and nized animals.
progressive ECM remodeling of the septal and broncho- Collagen V-Induced Nasal Tolerance
vascular axis [11]. The examination of other organs Nasal tolerance was induced in a group of six collagen
usually affected in SSc, such as skin, esophagus, kidney, V-immunized animals, through the nasal administration
heart and synovial membrane, showed identical and of daily doses of 25 μg of collagen V diluted in 25 μlof
intense ECM remodeling [12-14]. In addition, several 10 mM acetic acid (IM-TOL). The nasal tolerance
immunological alterations were observed, such as the induction was initiated 150 days after the first immuni-
presence of types I, III and IV anti-collagen antibodies, zation, and conducted for 60 days. Another group of six
circulating immune complexes, and the emergence of immunized animals (IM) was not tolerated. The control
antinuclear antibodies (ANA) and anti-Scl-70 antibodies group (n = 6), inoculated with Freund’sadjuvant(CT-
[15]. Based on Sakkas’s works [16,17] suggesting that FA) was tolerated by nasal route with collagen V,
SSc pathogenesis is related to the activation of T cells initiated 150 days after immunization. All animals were
by still unidentified antigens, we postulated that collagen sacrificed at 210 days.
V usually found hidden between collagen I and III in The animal procedures were approved by the Ethics
heterotypic fibers, but exposed in our experimental Committee in Research, CAPPesq of the Clinical Board
model, could be one of the antigens responsible for trig- of the School of Medicine, University of São Paulo, as
gering the T-dependent response (Th2). The activated stated in Protocol of Research number 268/05.
Th2 cells and the IL-4 and IL-17 cytokines generated by Collagenous Fibers Histomorphometric Analysis
their activation would explain the SSc triad: increased To characterize the collagenous fibers in peribroncho-
ECM synthesis, vascular remodeling and autoantibody vascular and septal pulmonary interstitium, Masson’s
production [17]. These alterations associated with the trichrome was used to stain the collagen-containing
immunogenic role of collagen V make our experimental fibers in blue. Also, the Picrosirius staining method [19]
model important to test tolerance induction in the treat- observed under polarized light was used to intensify the
ment of SSc. Considering that we have already demon- normal birefringence of collagenous fibers and to deter-
strated the efficacy of nasal tolerance with collagen V in mine the location of collagen-containing fibers. The
skin remodeling of animals with SSc [18], in the present number of collagen fibers in lungs was determined by
study we evaluated the amount of collagen deposition, an image analysis system in an optical microscope
mRNA collagen synthesis and TGF-beta expression in equipped with a light polarizer coupled to an image ana-
pulmonary septal and bronchovascular interstitium of lyzer. The system consisted of a Q-Color 5 camera,
rabbits after collagen V-induced nasal tolerance in coupled to an Olympus microscope, from which the
experimental SSc. It was hypothesized that collagen V- images could be visualized on the monitor. The images
induced nasal tolerance decreases the density of pul- were processed through a digital system installed in a
monary perivascular and septal collagenous fibers. computer (Pentium 4, 300 Mhz) using the Image-Pro-
Plus, version 6.0 software. The enhancement of collagen
Methods birefringence promoted by the Picrosirius polarization
Collagen V Immunization method is specific for collagenous structures composed
Experimental SSc was induced in healthy New Zealand of aggregates of orientated molecules. The threshold for
female rabbits (N = 12) with a mean weight of 2.50 Kg collagenous fibers was established for each slide afterVelosa et al. Respiratory Rese