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The Journal of Immunology, 2007, 179, 698 -707
Copyright © 2007 by The American Association of Immunologists, Inc.
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Intercellular Adhesion Molecule-1 Deficiency Attenuates the Development of Skin Fibrosis in Tight-Skin Mice1

Yukiyo Matsushita*, Minoru Hasegawa2,*, Takashi Matsushita*, Manabu Fujimoto*, Mayuka Horikawa*, Tomoyuki Fujita*, Ayako Kawasuji*, Fumihide Ogawa{dagger}, Douglas A. Steeber{ddagger}, Thomas F. Tedder§, Kazuhiko Takehara* and Shinichi Sato{dagger}

* Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; {dagger} Department of Dermatology, Nagasaki University Graduate School of Biomedical Science, Nagasaki, Japan; {ddagger} Department of Biological Sciences, University of Wisconsin, Milwaukee, WI 53201; and § Department of Immunology, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The tight-skin (TSK/+) mouse, a genetic model for systemic sclerosis, develops cutaneous fibrosis. Although a fibrillin 1 gene mutation and immunological abnormalities have been demonstrated, the roles of adhesion molecules have not been investigated. To directly assess roles of adhesion molecules in skin fibrosis, TSK/+ mice lacking L-selectin and/or ICAM-1 were generated. The deficiency of ICAM-1, but not L-selectin, significantly suppressed (~48%) the development of skin sclerosis in TSK/+ mice. Similarly, ICAM-1 antisense oligonucleotides inhibited skin fibrosis in TSK/+ mice. Although T cell infiltration was modest into the skin of TSK/+ mice, ICAM-1 deficiency down-regulated this migration, which is consistent with the established roles of endothelial ICAM-1 in leukocyte infiltration. In addition, altered phenotype or function of skin fibroblasts was remarkable and dependent on ICAM-1 expression in TSK/+ mice. ICAM-1 expression was augmented on TSK/+ dermal fibroblasts stimulated with IL-4. Although growth or collagen synthesis of TSK/+ fibroblasts cultured with IL-4 was up-regulated, it was suppressed by the loss or blocking of ICAM-1. Collagen expression was dependent on the strain of fibroblasts, but not on the strain of cocultured T cells. Thus, our findings indicate that ICAM-1 expression contributes to the development of skin fibrosis in TSK/+ mice, especially via ICAM-1 expressed on skin fibroblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic sclerosis (SSc)3 is an autoimmune disease characterized by excessive extracellular matrix (ECM) deposition in the skin and other visceral organs. Although the molecular basis for SSc is unknown, there are a number of studies concerning the pathogenetic mechanisms of immune activation and tissue fibrosis in SSc (1, 2, 3). The maintenance of ECM structure and function occurs via a remodeling process, involving regulated phases of degradation and synthesis of ECM components, such as collagen and fibronectin, by fibroblasts. Thereby, overdeposition of ECM results in tissue fibrosis in SSc. Most of the infiltrating cells in the skin of SSc patients are activated CD4+ T cells (4). Skin fibroblasts from SSc patients are activated and produce excessive ECM by TGF-beta and other cytokines in the context of Th2 cytokine-predominant immune response (5).

The tight-skin (TSK) mouse, a genetic model for human SSc, was originally identified as a spontaneous mutation that results in increased synthesis and accumulation of collagen and other ECM proteins in the skin (6). Although homozygous mice die in utero, heterozygous (TSK/+) mice survive, but develop cutaneous fibrosis. TSK/+ mice produce autoantibodies against SSc-specific target autoantigens, including topoisomerase I, fibrillin 1, and RNA polymerase I (7, 8, 9). Although a tandem duplication within the fibrillin 1 gene and the production of a large fibrillin 1 protein are found in TSK/+ mice (10, 11, 12), the role of the fibrillin 1 gene in the genesis of tissue hyperplasia and autoimmunity remains unsolved. Nonetheless, it is likely that CD4+ T cells are involved in the skin fibrosis of TSK/+ mice, because CD4-deficient TSK/+ mice demonstrate a marked reduction of skin fibrosis (13). Although various cytokines produced by immune-activated cells modulate the ECM synthesis by fibroblasts, TGF-beta and Th2-type cytokines, such as IL-4 and IL-6, have been considered as key molecules for tissue fibrosis in TSK/+ mice (14, 15, 16, 17, 18).

In general, leukocyte recruitment into inflammatory sites is achieved using constitutive or inducible expression of multiple adhesion molecules (19, 20, 21). L-selectin (CD62L), which primarily mediates leukocyte capture and rolling on the endothelium, is constitutively expressed by most leukocytes (22). ICAM-1 (CD54) is a member of the Ig superfamily that is constitutively expressed not only on endothelial cells, but also on a subset of leukocytes, fibroblasts, and epithelial cells (23). It can be up-regulated transcriptionally by several proinflammatory cytokines, such as TNF-{alpha}, IFN-{gamma}, and IL-1 (23). ICAM-1 is an inducible transmembrane receptor, which forms the counterreceptor for the leukocyte beta2 integrins. The ICAM-1/beta2 integrin interactions predominantly mediate firm adhesion and transmigration of leukocytes at sites of inflammation (19, 24). Elevated serum L-selectin levels (25) or circulating ICAM-1 levels (26, 27) have been reported in patients with SSc. Furthermore, augmented ICAM-1 expression could be detected on endothelial cells or fibroblasts in the skin from early SSc patients (28, 29). Nevertheless, the role of adhesion molecules, including L-selectin or ICAM-1, has not been investigated in TSK/+ mice.

To directly assess roles of ICAM-1 and L-selectin in TSK/+ mice, TSK/+ mice lacking L-selectin, ICAM-1, or both adhesion molecules were generated. The results of this study suggest that ICAM-1, but not L-selectin, significantly contributes to the development of skin fibrosis, especially via ICAM-1 expression on skin fibroblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

ICAM-1–/– (30) mice and TSK/+ (6) mice were purchased from The Jackson Laboratory. L-selectin–/– (31) mice and mice lacking both ICAM-1 and L-selectin (24) were generated, as described previously. All mice were backcrossed five to seven generations onto C57BL/6 genetic background. ICAM-1–/–, TSK/+, ICAM-1–/– TSK/+, and wild-type mice were generated by crossing ICAM-1+/– TSK/+ parents. L-selectin–/– TSK/+ and L-selectin–/– ICAM-1–/– TSK/+ mice were generated by crossing L-selectin–/– ICAM-1+/– TSK/+ parents. To verify the TSK/+ genotype, PCR amplification of a partially duplicated fibrillin 1 gene was conducted using genomic DNA from each mouse, as described (32). Similarly, lack of ICAM-1 expression was verified by PCR amplification using genomic DNA (24). L-selectin deficiency was confirmed by flow cytometric analysis of blood leukocytes stained with PE-conjugated anti-L-selectin mAb (MEL14; Beckman Coulter). All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. Twelve-week-old female mice were used, unless otherwise indicated in these experiments. The Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science approved all studies and procedures.

Histopathological assessment of skin fibrosis

Morphologic characteristics of skin sections from mutant mice were compared with wild-type mice under a light microscope. All skin sections were taken from the para-midline, lower back region (the same anatomic site to minimize regional variations in thickness) as full thickness sections extending down to the body wall musculature. Tissues were fixed in 10% formaldehyde solution for 24 h and embedded in paraffin. Sections were stained with H&E. Hypodermal thickness, which was defined as the thickness of a s.c. loose connective tissue layer (i.e., the hypodermis or superficial fascia) beneath the panniculus carnosus, was measured for multiple transverse perpendicular sections using an ocular micrometer. Dermal thickness defined as the thickness of skin from the top of the granular layer to the junction between the dermis and s.c. fat was also examined. Ten random measurements were taken per section. Two investigators in a blinded fashion examined all of the sections independently.

Determination of hydroxyproline content in the skin tissue

Hydroxyproline is a modified amino acid uniquely found as a high percentage of collagen. Punch biopsies (6 mm) from shaved dorsal skin samples were treated with chloroform/methanol (2:1 v/v) to remove the fat, and were dried by centrifugation under vacuum. Dried samples were weighed, acid hydrolyzed for 24 h at 110°C, dried, redissolved in 200 µl of water, and filtered through millipore filters. Sample aliquots of 20 µl were diluted 10-fold and used for amino acid composition analysis in an Amino Acid Analyzer (Hewlett-Packard).

Immunohistochemistry

For immunohistochemistry, paraffin-embedded tissue sections of dorsal skin were acetone fixed and then incubated with 10% normal rabbit serum in PBS (10 min, 37°C) to block nonspecific staining. Sections were then incubated with rat mAbs specific for mouse CD3 (Serotec). Rat IgG (Southern Biotechnology Associates) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 min, 37°C) with a biotinylated rabbit anti-rat IgG and then HRP-conjugated avidin-biotin complexes (Vectastain ABC kit; Vector Laboratories). Sections were developed with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide and then counterstained with methyl green.

Fibroblast culture

Skin samples of 1 cm3 were taken from para-midline, lower back region. To obtain fibroblasts, the tissue was cut into 1-mm3 pieces, placed in sterile plastic dishes, and cultured in DMEM (Invitrogen Life Technologies) containing 10% heat-inactivated FCS, 100 U/ml penicillin (Invitrogen Life Technologies), and 100 µg/ml streptomycin (Invitrogen Life Technologies), and cultured at 37°C in a 5% CO2 humidified atmosphere. After 2–3 wk of incubation, the outgrowth of fibroblasts was detached by brief trypsin treatment and recultured in the medium. Confluent cultures of fibroblasts were serum starved for 12 h and then cultured with or without 10 ng/ml murine rIL-4 (R&D Systems) for 24 h. The monolayers were washed and the cells were used immediately in experiments, as indicated. All experiments used fibroblasts between passages 2 and 5, depending on the number of cells obtained initially from the tissue samples. Cultured fibroblasts were adherent to the dish and maintained the typical spindle-shaped aspect. The purity of fibroblasts confirmed with flow cytometry was >99% with no leukocytes found in the harvested cells (data not shown). In each experiment, all the cell lines were examined at the same time and under the same conditions of culture (e.g., cell density, passage, days after plating).

RNA isolation and real-time RT-PCR

Total RNA was isolated from deep-frozen full-thickness dorsal skin sections or cultured dermal fibroblasts using Qiagen RNeasy spin columns (Qiagen) and digested with DNaseI (Qiagen) to remove chromosomal DNA in accordance with manufacturer’s protocols. RNA was reverse transcribed into cDNA using the Reverse Transcription System (Promega). Transcript levels were quantified using a real-time PCR method, according to the manufacturer’s instructions (Applied Biosystems). Sequence-specific primers and probes were designed by Pre-Developed TaqMan Assay Reagents or TaqMan Gene Expression Assays (Applied Biosystems). Real-time PCR (1 cycle of 50°C for 2 min, 95°C for 10 min; 40 cycles of 92°C for 15 s, 60°C for 60 s) was performed on an ABI Prism 7000 Sequence Detector (Applied Biosystems). GAPDH transcript levels were used as controls to normalize mRNA levels. The relative expression of target transcript PCR products was determined using the {Delta}{Delta}Ct method (33). Fold induction = 2–[{Delta}{Delta}Ct], where Ct = the threshold cycle, i.e., the cycle number at which the sample’s relative fluorescence rises above the background fluorescence and {Delta}{Delta}Ct = (Ct gene of interest (unknown sample) – Ct GAPDH (unknown sample)) – (Ct gene of interest (calibrator sample) – Ct GAPDH (calibrator sample)). Each sample was run in triplicate, with the mean Ct used in the equation.

Flow cytometry

Abs used in this study included FITC-conjugated anti-ICAM-1 mAb (BD Biosciences), FITC-conjugated anti-CD4 mAb (BD Biosciences), PE-conjugated anti-CD44 mAb (BD Biosciences), PE-conjugated anti-CD69 mAb (BD Biosciences), PE-conjugated anti-L-selectin mAb (Beckman Coulter), and PE-conjugated anti-p150,95 mAb (eBioscience). Single-cell suspensions of cultured skin fibroblasts or CD4+ T cells were incubated with the Abs for 30 min at 4°C. The cells were washed and fixed with 1% paraformaldehyde in PBS. Fibroblasts were gated on the basis of size and granularity.

For intracellular IL-4 and IL-6 staining of CD4+ T cells, splenic CD4+ T cells were purified (>99% CD4+) by positive selection with anti-CD4 Ab-coated magnetic beads (Miltenyi Biotec). Purified splenic CD4+ T cells were incubated for 72 h and then stimulated with 25 ng/ml PMA and 1 µg/ml ionomycin for 4 h. Brefeldin A (10 µg/ml) was also added when the stimulation of T cells was initiated. The cells were stained with anti-CD4 mAb and then washed and treated with FACS-permeabilizing solution for 10 min at room temperature. These cells were incubated for 30 min in the dark with anti-IL-4 mAb (BD Biosciences) and anti-IL-6 mAb (BD Biosciences).

The levels of fluorescence were measured using a FACScan flow cytometer (BD Biosciences), analyzing data from 105 cells. Positive and negative populations of cells were determined using unreactive isotype-matched mAbs (BD Biosciences) as controls for background staining.

Proliferation assay of skin fibroblasts

Cultured skin fibroblasts (1.2 x 104/well) were seeded into a 96-well plate. Fibroblasts were serum starved for 12 h and then cultured for 24 h with 10 ng/ml murine rIL-4 (R&D Systems) or without IL-4. For the ICAM-1-blocking study, graded concentrations of anti-ICAM-1 Ab (R&D Systems) that blocks cell adhesion were added in addition to 10 ng/ml murine rIL-4. Proliferation of cultured skin fibroblasts was quantified by a colorimetric BrdU cell proliferation ELISA kit (Roche Applied Science). Briefly, after 24-h incubation, BrdU (10 µM) was added to each well and incubated for 24 h. Proliferating cells took up BrdU and incorporated it into their DNA during S phase. The cells were fixed and denatured, and BrdU-labeled DNA was detected using a peroxidase-conjugated anti-BrdU Ab. After addition of tetramethylbenzidine substrate, the degree of proliferation was quantified by measuring absorbance at 450 nm with the reference wavelength at 690 nm. Simultaneously, mRNA expression of collagen and cytokines was analyzed by real-time RT-PCR assay. Total RNA was isolated from fibroblasts shortly after 24 h of incubation with IL-4 and anti-ICAM-1 Ab.

Lymphocyte-fibroblast cocultures

Purified splenic CD4+ T cells (1 x 106/well) from wild-type, TSK/+, or ICAM-1–/– TSK/+ mice were seeded on top of cultured skin fibroblasts (0.5 x 106/well) from wild-type, TSK/+, or ICAM-1–/– TSK/+ mice in 6-well tissue culture plates in a final volume of 2 ml of supplemented RPMI 1640 and incubated for 72 h. After incubation, cocultured CD4+ T cells or fibroblasts were harvested and analyzed by real-time RT-PCR or flow cytometry.

ICAM-1 antisense study

All oligonucleotides used in this study were phosphorothioate oligodeoxynucleotides. Antisense oligodeoxynucleotides were manufactured by Biognostik, as previously described (34). ICAM-1 antisense oligodeoxynucleotides were made against the 3'-untranslated region of the murine ICAM-1 gene (35). A scrambled oligonucleotide was used as a sequence control, because the A + T/C + G ratio was similar to ICAM-1 antisense oligodeoxynucleotides. The sequence for the ICAM-1 antisense oligodeoxynucleotide was 5'-TGCATCCCCCAGGCCACCAT-3'. The sequences for the scrambled oligodeoxynucleotides were 5'-CAGCCATGGTTCCCCCCAAC-3' and 5'-TCGCATCGACCCGCCCACTA-3'. Four-week-old female TSK/+ and wild-type mice were used in this study. TSK/+ and wild-type mice received an i.p. injection of either ICAM-1 antisense or scrambled oligodeoxynucleotides at a dose of 1 µg/g body weight everyday for 14 days. At the end of the treatment period, the animals were sacrificed and skin was removed for analysis.

Antinuclear Ab (ANA) analysis and ELISA for autoantibodies

ANAs were assessed by indirect immunofluorescence staining using sera diluted 1/50 and HEp-2 substrate cells (Medical & Biological Laboratories), as described (36). ANAs were detected using FITC-conjugated goat F(ab')2 Ab specific for mouse IgG + IgM + IgA (Southern Biotechnology Associates). Anti-topoisomerase I Ab levels were quantified using ELISA kits according to the manufacturer’s protocol (Medical & Biological Laboratories).

Statistical analysis

Data are expressed as mean values ± SEM. The Mann-Whitney U test was used for determining the level of significance of differences between sample means, and Bonferroni’s test was used for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
ICAM-1 loss reduces the development of skin fibrosis in TSK/+ mice

Skin fibrosis was assessed by histopathology of full-thickness skin sections from the back in L-selectin–/– TSK/+ mice, ICAM-1–/– TSK/+ mice, and L-selectin–/– ICAM-1–/– TSK/+ mice. The dermal thickness (the thickness from the top of the granular layer to the junction between the dermis and s.c. fat) was similar among each strain (data not shown), consistent with previous reports (6, 13, 37). The hypodermal thickness in TSK/+ mice was increased by ~7.1-fold compared with wild-type mice (p < 0.0001; Fig. 1, A and B). There was no significant difference in the hypodermal thickness among wild-type, L-selectin–/–, ICAM-1–/–, and L-selectin–/– ICAM-1–/– mice (Fig. 1A). The hypodermal thickness in L-selectin–/– TSK/+ mice was similar to that in TSK/+ mice. However, ICAM-1–/– TSK/+ and L-selectin–/– ICAM-1–/– TSK/+ mice showed only a moderate thickening of hypodermal tissue that was significantly 48 and 40% thinner than that found in TSK/+ mice, respectively (p < 0.0001; Fig. 1A). There was no significant difference in the hypodermal thickness between L-selectin–/– ICAM-1–/– TSK/+ and ICAM-1–/– TSK/+ mice.


Figure 1
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  		FIGURE 1. Skin fibrosis in dorsal skin from mutant and wild-type mice. A, Skin fibrosis was assessed by quantitatively measuring hypodermal thickness. The hypodermal thickness was measured under a light microscope as the thickness of the hypodermis or superficial fascia beneath the panniculus carnosus. Horizontal bars represent mean hypodermal thickness in each group. B, Representative histological sections stained with H&E are shown (magnification x40). An asterisk indicates the hypodermal thickness. These results represent those obtained with 10 mice of each genotype. C, Skin fibrosis was also assessed by skin hydroxyproline content. The quantity of hydroxyproline is expressed as µg/10 mg skin samples. Results from each mouse are represented as single dots. Horizontal bars represent mean hydroxyproline content. D, The change in hypodermal thickness (mean ± SEM) in mutant and wild-type mice during 16 wk after birth. These results represent those obtained with at least five mice of each genotype. {dagger}, p < 0.05 vs TSK/+ mice. E, Representative immunohistochemical sections stained with anti-mouse CD3 mAb are shown. CD3+ T cell infiltration around small vessels was found in the soft fibrous tissue beneath the panniculus carnosus (indicated by asterisks in B) of TSK/+ mice (magnification x200). These results represent those obtained with five mice of each genotype. F, The number of CD3+ T cells in the soft fibrous tissue beneath the panniculus carnosus. Mean number of CD3+ T cells was determined by counting 10 areas around a small vessel.

 
Cutaneous fibrosis was also assessed by quantifying the hydroxyproline content of 10-mg skin samples from mutant and wild-type mice (Fig. 1C). Although the hydroxyproline content in TSK/+ mice was increased by 4.3-fold relative to that in wild-type mice (p < 0.0001), ICAM-1 deficiency reduced the hydroxyproline content by 48% in TSK/+ mice (p < 0.0001). Hydroxyproline content was comparable between wild-type and ICAM-1–/– mice. Changes in hypodermal thickness were evaluated until 16 wk of age in wild-type, ICAM-1–/–, TSK/+, and ICAM-1–/– TSK/+ mice (Fig. 1D). Hypodermal thickness in TSK/+ mice increased until 12 wk of age, and then was maintained. A significant decrease in hypodermal thickness was already apparent in ICAM-1–/– TSK/+ mice compared with TSK/+ mice by 6 wk of age. L-selectin and ICAM-1 deficiency did not significantly affect the onset or severity of pulmonary emphysema in TSK/+ mice (data not shown), because their alveoli remained 3–4 times larger than in wild-type mice (32). Thus, ICAM-1 deficiency, but not L-selectin loss, reduced skin fibrosis.

ICAM-1 loss inhibits the infiltration of CD3+ T cells in TSK/+ skin

Although skin-infiltrating cells are rarely found in whole skin tissues of mice, those found are mostly distributed around the microvessels of soft fibrous tissue beneath the panniculus carnosus, the anatomical location of augmented tissue fibrosis in TSK/+ mice. Therefore, CD3+ T cell numbers in the soft fibrous tissue (indicated by asterisk in Fig. 1B) were assessed by immunohistochemistry staining. Although CD3+ T cell numbers in TSK/+ mice were significantly increased compared with wild-type mice, the numbers remained modest. However, ICAM-1-deficiency in TSK/+ mice resulted in significantly reduced numbers of infiltrating T cells (Fig. 1, E and F). Significant numbers of neutrophils or eosinophils were not detected in either wild-type or mutant mice by H&E staining (data not shown). Thus, ICAM-1 loss inhibited increased CD3+ T cell infiltration in the skin of TSK/+ mice, but the T cell number was modest even in TSK/+ mice.

Intracellular cytokines in splenic T cells

Purified splenic CD4+ T cells from TSK/+ mice included ~2-fold increased intracellular cytokine-positive cells for IL-4 and IL-6 compared with those from wild-type mice (Fig. 2). However, the frequency of intracellular cytokine-positive cells was not significantly changed by the loss of ICAM-1. Thus, CD4+ T cells from TSK/+ mice had augmented potential to produce Th2 cytokines, even in the absence of ICAM-1 expression.


Figure 2
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  		FIGURE 2. For intracellular IL-4 and IL-6 staining, purified CD4+ T cells from wild-type, ICAM-1–/–, TSK/+, ICAM-1–/–, and ICAM-1–/– TSK/+ mice were cultured for 72 h, and then stimulated with PMA and ionomycin for 4 h. Percentages of IL-4+ and IL-6+ cells are shown in the left panel. Representative histograms of intracellular IL-4 and IL-6 staining are shown in the right panel. Shaded regions in the histograms represent TSK/+ CD4+ T cells, boldface lines (open regions) represent wild-type CD4+ T cells, and thin lines (open regions) represent isotype control staining. Data are the mean ± SEM. These results represent those obtained with five mice of each group.

 
ICAM-1 deficiency down-regulates augmented Th2 cytokine expression in TSK/+ mouse skin

mRNA levels of Th2 cytokines, such as IL-4 and IL-6, were 3.1-fold (p < 0.05) and 10.4-fold (p < 0.01) higher in the skin from TSK/+ mice compared with wild-type mice, respectively (Fig. 3). By contrast, mRNA levels of Th1 cytokines, such as IFN-{gamma} and IL-2, were significantly lower in TSK/+ mice than in wild-type mice (75% decrease, p < 0.01 and 60% decrease, p < 0.05, respectively). Interestingly, IL-4 mRNA expression was hardly detected in ICAM-1–/– mice. Furthermore, augmented IL-4 expression in TSK/+ mice was remarkably down-regulated by ICAM-1 deficiency (93% decrease; p < 0.05). IL-6 mRNA levels were not significantly affected by ICAM-1 loss in wild-type mice. However, ICAM-1 loss reduced augmented IL-6 mRNA levels in TSK/+ mice (88% decrease; p < 0.01) to a similar level of wild-type or ICAM-1–/– mice. IFN-{gamma} and IL-2 mRNA levels were significantly lower in ICAM-1–/– mice than in wild-type mice (p < 0.01 and p < 0.05, respectively). However, ICAM-1 loss did not significantly affect the expression levels of IFN-{gamma} and IL-2 in TSK/+ mice. Thus, the skin from TSK/+ mice exhibited augmented IL-4 and IL-6 expression that was down-regulated by ICAM-1 deficiency, whereas it showed reduced IFN-{gamma} and IL-2 expression.


Figure 3
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  		FIGURE 3. mRNA expression of cytokines in dorsal skin from mutant and wild-type mice. RNA was isolated from dorsal skin of wild-type, ICAM-1–/–, TSK/+, ICAM-1–/–, and ICAM-1–/– TSK/+ mice. The mRNA levels of IL-4, IL-6, IFN-{gamma}, and IL-2 were analyzed by real-time RT-PCR, and normalized with the internal control GAPDH. Each sample was done in triplicate. Data indicate the mean ± SEM. These results represent those obtained with at least 10 mice of each genotype.

 
ICAM-1 expression is augmented on TSK/+ fibroblasts

As described above, skin-infiltrating T cells were modest and the frequency of IL-4- or IL-6-producing T cells was not changed by ICAM-1 deficiency in TSK/+ mice. Therefore, T cell infiltration into the skin via ICAM-1 on endothelial cells may not be critical in the development of skin sclerosis in TSK/+ mice. Because ICAM-1 is also expressed on fibroblasts, the phenotype of TSK/+ fibroblasts was examined. ICAM-1 expression levels on skin fibroblasts were examined in TSK/+ mice by flow cytometry analysis. Expression of CD44, a type I transmembrane protein that functions as the major cellular adhesion molecule for hyaluronic acid, was also assessed as a control for fibroblast staining. Expression levels of ICAM-1 and CD44 on TSK/+ fibroblasts were modest and similar to that of wild-type fibroblasts when skin fibroblasts were cultured without stimulation (Fig. 4, A and B). However, ICAM-1 expression levels on TSK/+ fibroblasts were significantly 97% higher than those found in wild-type fibroblasts when incubated with IL-4 (Fig. 4, A and B; p < 0.001). By contrast, the surface expression levels of CD44 were not significantly different between TSK/+ and wild-type fibroblasts following IL-4 stimulation. Thus, ICAM-1 expression levels on skin fibroblasts were remarkably augmented in the presence of IL-4 in TSK/+ mice.


Figure 4
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  		FIGURE 4. A, Surface ICAM-1 expression on fibroblasts cultured with IL-4 in TSK/+ mice. Dermal fibroblasts were extracted from dorsal skin of wild-type, ICAM-1–/–, TSK/+, ICAM-1–/–, and ICAM-1–/– TSK/+ mice. Fibroblasts from dorsal skin were cultured, and confluent fibroblasts were stimulated with 10 ng/ml murine rIL-4 for 24 h. Relative cell surface ICAM-1 and CD44 densities on cultured skin fibroblasts from TSK/+ and wild-type mice were determined by flow cytometry, comparing mean linear fluorescence intensity channel numbers. Values represent the mean (±SEM) percentage of wild-type expression levels. {square}, Indicate ICAM-1 and CD44 expression on fibroblasts without stimulation; {blacksquare}, indicate ICAM-1 and CD44 expression with IL-4 stimulation. These results represent those obtained with five mice of each genotype. B, Representative histograms of ICAM-1 and CD44 expression on fibroblasts from TSK/+ and wild-type mice. Thin lines (open regions) in the histograms represent isotype control staining of fibroblasts, boldface lines (open regions) represent unstimulated fibroblasts, and shaded regions represent IL-4-stimulated fibroblasts. C, mRNA expression of COL1A2 and TGF-beta in dermal fibroblasts from mutant and wild-type mice. Dermal fibroblasts were extracted from dorsal skin of 3-wk-old wild-type, ICAM-1–/–, TSK/+, or ICAM-1–/– TSK/+ mice. Dermal fibroblasts were cultured, and confluent fibroblasts were stimulated with 10 ng/ml murine rIL-4 for 24 h. Total RNA from fibroblasts was extracted and reverse transcribed to cDNA, and mRNA expression of COL1A2 and TGF-beta was analyzed by real-time RT-PCR and normalized with the internal control GAPDH. {square}, Indicate relative mRNA expression on fibroblasts without stimulation; {blacksquare}, indicate mRNA expression with IL-4 stimulation. D, Proliferation of dermal fibroblasts in wild-type, ICAM-1–/–, TSK/+, or ICAM-1–/– TSK/+ mice. Cultured fibroblasts were serum starved for 12 h and then cultured for 24 h with murine rIL-4 (10 ng/ml, {blacksquare}) or without IL-4 ({square}). After 24-h incubation, BrdU (10 µM) was added to each well and incubated for 24 h. BrdU incorporation in proliferating cells was quantified by ELISA. Each sample was done in triplicate. Data are the mean ± SEM. These results represent those obtained with at least five mice of each genotype.

 
ICAM-1 deficiency reduces collagen and TGF-beta mRNA expression in TSK/+ fibroblasts

We assessed how ICAM-1 loss affected collagen synthesis by fibroblasts in TSK/+ mice. Type I collagen, the major fiber-forming collagen of the skin, is the product of the pro{alpha}1 (I) collagen and pro{alpha}2 (I) collagen (COL1A2) genes. COL1A2 and TGF-beta mRNA expression was quantified by real-time RT-PCR in cultured skin fibroblasts (Fig. 4C). COL1A2 and TGF-beta mRNA levels in TSK/+ fibroblasts were not significantly different from wild-type fibroblasts when cultured without stimulation. In addition, these mRNA expression levels were not influenced by ICAM-1 deficiency. By contrast, when fibroblasts were incubated with IL-4, mRNA levels of COL1A2 and TGF-beta were 3.3-fold (p < 0.005) and 3.4-fold (p < 0.0001) higher in TSK/+ mice than in wild type, respectively. COL1A2 and TGF-beta mRNA levels in ICAM-1–/– fibroblasts remained similar to those found in wild-type mice. However, ICAM-1 deficiency in TSK/+ fibroblasts significantly decreased COL1A2 and TGF-beta mRNA expression (68% decrease, p < 0.005 and 52% decrease, p < 0.01, respectively) to a similar level of wild-type fibroblasts. Thus, increased mRNA levels of collagen protein and TGF-beta in TSK/+ fibroblasts following IL-4 stimulation were eliminated by ICAM-1 deficiency.

Loss or blockade of ICAM-1 reduces fibroblast proliferation

Spontaneous proliferation of skin fibroblasts was significantly increased in TSK/+ mice compared with wild-type mice (Fig. 4D). However, ICAM-1 loss significantly decreased the proliferation in wild-type and TSK/+ mice. Fibroblasts from each strain cultured with IL-4 were induced to proliferate (Fig. 4D). However, skin fibroblasts from TSK/+ mice cultured with IL-4 proliferated twice as strongly as those from wild-type mice (Fig. 4D). Furthermore, fibroblasts from ICAM-1–/– TSK/+ mice cultured with IL-4 showed comparable proliferation with fibroblasts from wild-type mice.

To confirm the possibility that ICAM-1 expression directly enhanced fibroblast proliferation, we examined whether proliferation of skin fibroblasts from TSK/+ mice was affected by the addition of anti-ICAM-1-blocking Abs in the medium, including IL-4. Anti-ICAM-1-blocking Abs significantly suppressed fibroblast proliferation in a dose-dependent manner in TSK/+ and wild-type mice. The addition of blocking Ab did not affect the proliferation in ICAM-1–/– or ICAM-1–/– TSK/+ mice. Similarly, mRNA expression of COL1A2, TGF-beta, and IL-6 was augmented in skin fibroblasts from TSK/+ mice relative to those from wild-type mice (Fig. 5B). Treatment with anti-ICAM-1-blocking Ab significantly suppressed COL1A2, TGF-beta, and IL-6 mRNA expression in fibroblasts from both TSK/+ and wild-type mice. These findings indicate that ICAM-1 expression on fibroblasts directly enhances fibroblast proliferation and ability to produce collagen or fibrogenic cytokines.


Figure 5
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  		FIGURE 5. The effect of ICAM-1 blocking on cultured skin fibroblasts. Cultured fibroblasts were serum starved for 12 h and then cultured for 24 h with murine rIL-4 (10 ng/ml) and anti-ICAM-1 Ab (0, 0.1, 1, or 10 µg/ml). A, After 24-h incubation, BrdU (10 µM) was added to each well and incubated for 24 h. BrdU incorporation in proliferating cells was quantified by ELISA. B, Total RNA was isolated from fibroblasts shortly after 24-h incubation with IL-4 and anti-ICAM-1 Ab. The mRNA levels of COL1A2, TGF-beta, and IL-6 were analyzed by real-time RT-PCR, and normalized with the internal control GAPDH. Each sample was done in triplicate. Data are the mean ± SEM. These results represent those obtained with at least five mice of each genotype. {dagger}, p < 0.01 vs TSK/+ fibroblasts without anti-ICAM-1 Ab. *, p < 0.01 vs wild-type fibroblasts without anti-ICAM-1 Ab.

 
Coculture of T cells and fibroblasts

To assess the interaction between skin fibroblasts and lymphocytes via ICAM-1 expression on fibroblasts, we cocultured splenic CD4+ T cells and dermal fibroblasts. mRNA expression levels of COL1A2 were not significantly affected by the strain of cocultured CD4+ T cells (Fig. 6A). That is, COL1A2 expression in wild-type fibroblasts cocultured with TSK/+ T cells was comparable with that of wild-type fibroblasts cocultured with wild-type T cells. However, TSK/+ fibroblasts cocultured with CD4+ T cells from wild-type, TSK/+, or TSK/+ ICAM-1–/– mice showed significantly higher COL1A2 expression. Expression levels of CD44 and ICAM-1 on wild-type fibroblasts were not significantly changed by incubating with CD4+ T cells from TSK/+ mice (data not shown). Thus, augmented mRNA expression of collagen in TSK/+ mice was dependent on TSK/+ fibroblasts, but not significantly affected by cocultured CD4+ T cells from other strains.


Figure 6
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  		FIGURE 6. Coculture of lymphocytes and skin fibroblasts. A, Purified splenic CD4+ T cells from wild-type, TSK/+, or ICAM-1–/– TSK/+ mice were cocultured with fibroblasts from wild-type or TSK/+ mice. The mRNA levels of COL1A2 were analyzed by real-time RT-PCR and normalized with the internal control GAPDH. Each sample was done in triplicate. B, Purified splenic CD4+ T cells from wild-type and TSK/+ mice were cultured without fibroblasts. In addition, CD4+ T cells from wild-type mice were cocultured with fibroblasts from wild-type, TSK/+, or ICAM-1–/– TSK/+ mice. Surface expression of CD44 on CD4+ T cells was determined by flow cytometry, comparing mean fluorescence intensity (MFI). Data are the mean ± SEM. Representative histograms of CD44 expression on wild-type CD4+ T cells cocultured with wild-type, TSK/+, or ICAM-1–/– TSK/+ fibroblasts were also shown. Shaded regions in the histograms represent wild-type CD4+ T cells that were cocultured with TSK/+ fibroblasts, boldface lines (open regions) represent wild-type CD4+ T cells cultured with wild-type fibroblasts, and thin lines (open regions) represent wild-type CD4+ T cells cultured with ICAM-1–/– TSK/+ fibroblasts. These results represent those obtained with at least five sets of each combination.

 
Activated and memory/effector T cells demonstrate increased surface expression of CD44 (38). Cultured splenic CD4+ T cells from TSK/+ mice showed ~2-fold increased expression of CD44, compared with wild-type mice (Fig. 6B). Interestingly, CD4+ T cells from wild-type mice showed increased CD44 expression that was comparable to that of TSK/+ mice after 72 h of coculture with TSK/+ fibroblasts. CD44 expression on wild-type CD4+ T cells was not changed after coculture with fibroblasts from ICAM-1–/– TSK/+ mice. However, expression level of another activation marker, CD69, on T cells was comparable between wild-type and TSK/+ mice and was not significantly changed by coculture with fibroblasts from other strains (data not shown). Additionally, ICAM-1 and L-selectin on cultured CD4+ T cells were comparable between wild-type and TSK/+ mice, and were not significantly affected by the addition of TSK/+ or ICAM-1–/– TSK/+ fibroblasts (data not shown). Thus, CD4+ T cells from TSK/+ mice had a specific T cell phenotype represented by increased CD44 expression. Furthermore, our data suggest that dermal fibroblasts from TSK/+ mice can influence CD4+ T cell phenotype via ICAM-1 expression.

ICAM-1 loss does not affect autoantibody production in TSK/+ mice

Antinuclear Abs (ANAs) in mutant and wild-type mice were determined by indirect immunofluorescence staining using HEp-2 cells as the substrate. ANAs with a homogenous chromosomal staining pattern were detected in 36% (11 of 30) of TSK/+ mice, which was similar to that in ICAM-1–/– TSK/+ mice (33%, 5 of 15). By contrast, ANAs were rarely detectable in ICAM-1–/– (7%, 1 of 15) and wild-type (7%, 2 of 30) mice. Because a homogenous staining pattern generally results from a variety of autoantibodies, including Abs against topoisomerase I, autoantibody specificities were assessed by ELISA (Fig. 7). TSK/+ mice had significantly elevated IgG or IgM Abs reactive with topoisomerase I relative to wild-type mice (p < 0.05 and p < 0.001, respectively). Furthermore, ICAM-1–/– TSK/+ mice had significantly higher IgG topoisomerase I Ab levels than ICAM-1–/– mice (p < 0.005). There was no significant difference between TSK/+ and ICAM-1–/– TSK/+ mice. Thus, ICAM-1 loss had no direct effect on autoantibody production in TSK/+ mice.


Figure 7
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  		FIGURE 7. Anti-topoisomerase I Ab levels in sera from mutant and wild-type mice. Sera were collected from wild-type, ICAM-1–/–, TSK/+, ICAM-1–/–, and ICAM-1–/– TSK/+ mice. Relative IgG and IgM anti-topoisomerase I Ab levels were determined by ELISA. Horizontal bars represent mean ODs.

 
ICAM-1 antisense oligonucleotides prevent the development of skin fibrosis in TSK/+ mice

To further clarify the role of ICAM-1 in the development of skin fibrosis in TSK/+ mice, a blocking study using ICAM-1 antisense oligonucleotides was performed. Phosphorothioate oligodeoxynucleotides that hybridize against the 3'-untranslated region of the murine ICAM-1 gene were used (35). Before the blocking study, we confirmed that ICAM-1 mRNA expression was significantly decreased (~78%) by treatment with ICAM-1 antisense oligonucleotides, but not with scrambled control oligonucleotides in the skin from TSK/+ mice (Fig. 8A). Before treatment, ICAM-1 mRNA levels in the skin from TSK/+ mice were similar to those in the skin from wild-type mice. Four-week-old female TSK/+ and wild-type mice received either ICAM-1 antisense or scramble oligodeoxynucleotides every day for 14 days. During this period, the hypodermal thickness increases by ~2 times in TSK/+ mice, as demonstrated in Fig. 1D. However, injection of ICAM-1 antisense oligonucleotides significantly suppressed the development of skin fibrosis relative to TSK/+ mice treated with scrambled control oligonucleotides (p < 0.001; Fig. 8, B and C). Treatment with scrambled control oligonucleotides did not affect skin fibrosis in TSK/+ mice (Fig. 8B). Therefore, the effect of ICAM-1 antisense oligonucleotides was sequence specific and was consistent with an antisense mechanism. This treatment did not significantly affect autoantibody production or the onset or severity of pulmonary emphysema in TSK/+ mice (data not shown). Thus, the development of skin fibrosis in TSK/+ mice was attenuated by treatment with ICAM-1 antisense oligonucleotides.


Figure 8
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  		FIGURE 8. ICAM-1 antisense oligodeoxynucleotide treatment in TSK/+ mice. Fibrosis of dorsal skin from 6-wk-old TSK/+ and wild-type mice treated with ICAM-1 antisense oligodeoxynucleotides (antisense oligo) or control oligodeoxynucleotides (CTL 1 and 2). A, RNA was isolated from dorsal skin of 6-wk-old TSK/+ and wild-type mice treated with ICAM-1 antisense or control oligodeoxynucleotides. The mRNA levels of ICAM-1 were analyzed by real-time RT-PCR and normalized with the internal control GAPDH. Each sample was done in triplicate. Data are the mean ± SEM. B, Four-week-old female TSK/+ and wild-type mice received an i.p. injection of oligodeoxynucleotides everyday for 14 days. Skin fibrosis was assessed by quantitatively measuring hypodermal thickness. The hypodermal thickness was measured under a light microscope as the thickness of the hypodermis or superficial fascia beneath the panniculus carnosus. Horizontal bars represent mean hypodermal thickness. C, Representative H&E-stained histological sections are shown (x40). An asterisk indicates the hypodermal thickness. These results represent those obtained with four mice of each genotype.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This is the first study to reveal roles of adhesion molecules in the development of skin sclerosis in TSK/+ mice. Specifically, ICAM-1, but not L-selectin, was critical for the development of skin fibrosis in TSK/+ mice (Fig. 1, A–D). The development of skin fibrosis in TSK/+ mice was also prevented by treatment with antisense oligonucleotides that block selectively ICAM-1 expression (Fig. 8). Thus, our findings indicate that ICAM-1 has critical roles in the development of skin fibrosis in TSK/+ mice.

There are several possible mechanisms by which ICAM-1 could be mediating skin sclerosis in TSK/+ mice. First, ICAM-1 expressed on endothelial cells may be important for leukocyte infiltration and subsequent cytokine production. T cells and their cytokine production have been suggested to contribute to the development of skin fibrosis in TSK/+ mice. TSK/+ mice deficient in CD4+ T cells show a marked reduction in skin fibrosis (13). Furthermore, Th2 cytokines, but not Th1 cytokines, are critical for the development of cutaneous fibrosis in TSK/+ mice (17, 18, 32, 39, 40). Consistent with these previous findings, cytokine balance is skewed to Th2 cytokines such as IL-4 and IL-6 rather than Th1 in the skin of TSK/+ mice (Fig. 3). The number of CD3+ T cells was increased in the fibrotic skin region of TSK/+ mice compared with wild-type mice (Fig. 1, E and F). ICAM-1 deficiency decreased T cell infiltration in the skin of TSK/+ mice to a similar level of wild-type mice (Fig. 1, E and F), indicating that ICAM-1 plays a central role in T cell infiltration into the skin of TSK/+ mice. However, it remains possible that other adhesion molecules, including selectins or VCAM-1, may contribute to this process in cooperation with ICAM-1. These infiltrating CD3+ T cells are most likely the main source of IL-4, because IL-4 is produced by T cells or NKT cells. However, IL-6 may be produced by a more diverse array of cells, including macrophages and fibroblasts, in addition to T cells. CD4+ T cells from TSK/+ mice had potential to produce more Th2 cytokines such as IL-4 and IL-6 than those from wild-type mice (Fig. 2). These findings may explain why ICAM-1 deficiency reduced Th2 cytokines rather than Th1 cytokines in TSK/+ mice (Fig. 3). Thereby, endothelial ICAM-1 may be crucial for the Th2-dominant cytokine production in the TSK/+ skin by contributing to leukocyte recruitment into the skin. In addition, deficiency of adhesion molecules influences not only leukocyte trafficking, but also survival and the state of activation through their role in outside-in signaling (41). Therefore, it cannot be ruled out that down-regulated IL-4 and IL-6 expression in ICAM-1–/– TSK/+ mice may be due to a decrease in the functional state of infiltrating Th2 cells in the skin. Nonetheless, the number of infiltrating T cells was modest even in the skin of TSK/+ mice (Fig. 1, E and F). Therefore, ICAM-1 expressed on endothelial cells may not be a primary factor for the development of skin sclerosis, although IL-4 produced by infiltrating cells may promote skin sclerosis.

Second, ICAM-1 expressed on fibroblasts may be critical for skin sclerosis. Surface ICAM-1 expression on skin fibroblasts cultured with IL-4 was ~2-fold higher in TSK/+ mice than in wild-type mice (Fig. 4, A and B). Although it is not clear why ICAM-1 expression is higher on TSK/+ fibroblasts stimulated with IL-4, specific fibroblasts may be selectively expanded in TSK/+ skin. Thereby, ICAM-1 expression on fibroblasts may affect skin fibrosis by regulating the interaction with other fibroblasts or infiltrating cells in TSK/+ mice. Because L-selectin is not expressed on fibroblasts, this may be one of the possible explanations why L-selectin deficiency did not affect skin fibrosis in TSK/+ mice. Fibroblast proliferation and transcription levels of collagen and fibrogenic cytokines, including TGF-beta and IL-6, were remarkably increased in TSK/+ fibroblasts cultured with IL-4 (Figs. 4C and 5). A similar tendency in proliferation was also found in fibroblasts cultured without IL-4, although the proliferation was modest compared with those cultured with IL-4 (Fig. 4D). However, the loss or blockade of ICAM-1 significantly suppressed the proliferation and synthesis of collagen and fibrogenic cytokines in TSK/+ or wild-type fibroblasts. A possible explanation for these findings is that ICAM-1 expression affects the proliferation or collagen synthesis via modulating the effect of IL-4. Another possibility is that the interaction between fibroblasts themselves via ICAM-1 expression may be critical for fibroblast function, especially in the environment with IL-4. However, possible ligands for ICAM-1 expressed on skin fibroblasts remain unclear. Although p150,95 is a candidate, we could not detect any expression by flow cytometric analysis (data not shown). Future studies will be needed to clarify whether there are unknown ligands for ICAM-1 or a relationship between IL-4 and ICAM-1 signaling in dermal fibroblasts.

Another possibility is that interaction between fibroblasts and T cells via ICAM-1 expressed on fibroblasts may induce skin sclerosis. Several previous studies have shown that like TSK/+ mice, SSc fibroblasts exhibit increased surface ICAM-1 expression and augmented potential to bind with T cells (42, 43, 44). The augmented collagen mRNA expression is found on fibroblasts that are localized adjacent to dermal blood vessels within the lesional skin of SSc patients, suggesting the direct cellular interaction between fibroblasts and T cells (45, 46). In human dermal fibroblasts, increased ICAM-1 expression induced by IL-4 parallels the increase in ICAM-1-dependent T cell adhesion (47). Despite these findings in humans, to our knowledge, this is the first report that assessed ICAM-1 expression in TSK/+ mice. Although deficiency of ICAM-1 did not affect intracellular IL-4 and IL-6 levels in splenic CD4+ T cells (Fig. 2), we cannot exclude the possibility that ICAM-1 expressed on fibroblasts has some direct role for cytokine production in infiltrated T cells in the lesional skin. It has been demonstrated that ICAM-1 can provide T cell costimulatory signals that are independent of CD86/CD28 pathway (48). Although CD4+ T cells from TSK/+ mice showed >2-fold increase in surface expression of CD44, an activation marker of T cells (38), CD44 expression on CD4+ T cells from wild-type mice increased to a similar level by coculturing with TSK/+ fibroblasts, but not with ICAM-1–/– TSK/+ fibroblasts (Fig. 6B). This finding suggests that enhanced ICAM-1 expression on fibroblasts affects T cell activation via ICAM-1/beta2 integrin interaction. By contrast, strains of CD4+ T cells did not significantly affect collagen expression on wild-type or TSK/+ fibroblasts (Fig. 6A). That is, TSK/+ fibroblasts showed augmented collagen expression independent of the strain of cocultured CD4+ T cells. Thus, altered function of skin fibroblasts rather than infiltrating T cells is most likely the primary factor for the development of skin sclerosis in TSK/+ mice.

In the present study, despite down-regulated skin fibrosis (Figs. 1 and 8), the development of lung emphysema was not affected by the loss or blocking of ICAM-1 in TSK/+ mice (data not shown). Similar dissociation between cutaneous hyperplasia and lung emphysema has been reported in previous studies (13, 16, 17, 18, 32, 49, 50). Early studies have suggested that fibrillin 1 mutation induces skin fibrosis by altering the binding between fibrillin 1 and growth factors, such as TGF-beta or latent TGF-beta-binding protein in TSK/+ mice (51, 52). Nonetheless, the association between fibrillin 1 mutation and autoimmunity still remains unknown. The loss of ICAM-1 expression in TSK/+ mice reduced skin sclerosis independent of anti-topoisomerase I Ab production (Figs. 1, A–D, and 7). Likewise, CD4 deficiency in TSK/+ mice results in decreased cutaneous fibrosis, but does not affect anti-topoisomerase I Ab levels (13). B cell-deficient JHD–/– TSK/+ mice exhibited cutaneous hyperplasia that is indistinguishable from that of TSK/+ mice despite diminished anti-topoisomerase I Ab (11). However, other previous reports have shown an association between skin sclerosis and autoantibody production (17, 40, 50, 53). Although reasons for these discrepancies are unclear, these findings suggest that the TSK phenotype is multifactorial rather than due to a single mutation in the fibrillin 1 gene. ICAM-1 expression most likely regulates skin sclerosis independent of autoantibody production and lung emphysema in TSK/+ mice. Further studies will be needed to clarify the relationship between fibrillin 1 mutation and ICAM-1 expression.

The current study showed that the development of skin fibrosis in TSK/+ mice was significantly attenuated by treatment with antisense oligonucleotides (Fig. 8). Antisense oligonucleotides that hybridize to a specific mRNA or pre-mRNA have the potential to inhibit targeted molecule expression in vivo (54, 55). Recently, antisense oligonucleotides that inhibit ICAM-1 expression on either human (34, 56) or murine (35) cells have been generated. The murine-specific antisense oligonucleotide, ISIS 3082, was shown to inhibit selectively ICAM-1 expression in a sequence-specific manner (35). Furthermore, ISIS 3082 significantly extends the survival of heterotypic cardiac allografts and diminishes dextran sulfate sodium-induced colitis (35, 57). Therefore, antisense oligonucleotides represent a strategy for modulating cell adhesion by inhibiting the expression of adhesion molecules. Clinical trials of ICAM-1 antisense oligonucleotides have already been started in human inflammatory bowel diseases (58, 59) and rheumatoid arthritis (60). Until now, few therapies have proved to be effective for SSc in control studies. Although results from TSK/+ mice cannot be simply translated into human therapies, our findings suggest that ICAM-1 antisense oligonucleotides are potential therapeutic tools in human SSc.

In conclusion, immune cell infiltration via ICAM-1 expression on endothelial cells and subsequent production of cytokines such as IL-4 may be a trigger or stimulate fibroblast proliferation and collagen synthesis in TSK/+ mice. However, our findings more strongly indicate that altered function of TSK/+ skin fibroblasts is the primary cause of skin sclerosis, and ICAM-1 expression on fibroblasts is critical for fibroblast function.


   Acknowledgments
 
We thank M. Matsubara and Y. Yamada for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant-in-aid from the Ministry of Health and Welfare of Japan (to M.H. and S.S.), Kanazawa University (to M.H.), Nagasaki University (to S.S.), a project research for Japan Rheumatism Foundation (to S.S.), the Arthritis Foundation (to T.F.T.), and National Institutes of Health (CA105001, CA96547, and AI56363 to T.F.T.). Back

2 Address correspondence and reprint requests to Dr. Minoru Hasegawa, Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail address: minoruha{at}derma.m.kanazawa-u.ac.jp Back

3 Abbreviations used in this paper: SSc, systemic sclerosis; ANA, antinuclear Ab; Ct, threshold cycle; ECM, extracellular matrix; TSK, tight skin. Back

Received for publication June 20, 2006. Accepted for publication April 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sato, S., M. Fujimoto, M. Hasegawa, K. Takehara, T. F. Tedder. 2004. Altered B lymphocyte function induces systemic autoimmunity in systemic sclerosis. Mol. Immunol. 41: 1123-1133. [Medline]
  2. Simms, R. W., J. H. Korn. 2002. Cytokine directed therapy in scleroderma: rationale, current status, and the future. Curr. Opin. Rheumatol. 14: 717-722. [Medline]
  3. Derk, C. T., S. A. Jimenez. 2003. Systemic sclerosis: current views of its pathogenesis. Autoimmun. Rev. 2: 181-191. [Medline]
  4. Roumm, A. D., T. L. Whiteside, T. A. Medsger, Jr, G. P. Rodnan. 1984. Lymphocytes in the skin of patients with progressive systemic sclerosis: quantification, subtyping, and clinical correlations. Arthritis Rheum. 27: 645-653. [Medline]
  5. Varga, J., J. H. Korn. 2004. Pathogenesis: emphasis on human data. P. J. Clements, Jr, and D. E. Furst, Jr, eds. Systemic Sclerosis 63-98. Williams & Wilkins, Philadelphia.
  6. Green, M. C., H. O. Sweet, L. E. Bunker. 1976. Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton. Am. J. Pathol. 82: 493-512. [Abstract]
  7. Bona, C., N. Rothfield. 1994. Autoantibodies in scleroderma and tightskin mice. Curr. Opin. Immunol. 6: 931-937. [Medline]
  8. Murai, C., S. Saito, K. N. Kasturi, C. A. Bona. 1998. Spontaneous occurrence of anti-fibrillin-1 autoantibodies in tight-skin mice. Autoimmunity 28: 151-155. [Medline]
  9. Tan, F. K., D. N. Stivers, M. W. Foster, R. Chakraborty, R. F. Howard, D. M. Milewicz, F. C. Arnett. 1998. Association of microsatellite markers near the fibrillin 1 gene on human chromosome 15q with scleroderma in a Native American population. Arthritis Rheum. 41: 1729-1737. [Medline]
  10. Siracusa, L. D., R. McGrath, Q. Ma, J. J. Moskow, J. Manne, P. J. Christner, A. M. Buchberg, S. A. Jimenez. 1996. A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Res. 6: 300-131. [Abstract/Free Full Text]
  11. Kasturi, K. N., A. Hatakeyama, C. Murai, R. Gordon, R. G. Phelps, C. A. Bona. 1997. B-cell deficiency does not abrogate development of cutaneous hyperplasia in mice inheriting the defective fibrillin-1 gene. J. Autoimmun. 10: 505-517. [Medline]
  12. Lemaire, R., J. H. Korn, W. P. Schiemann, R. Lafyatis. 2004. Fibulin-2 and fibulin-5 alterations in tsk mice associated with disorganized hypodermal elastic fibers and skin tethering. J. Invest. Dermatol. 123: 1063-1069. [Medline]
  13. Wallace, V. A., S. Kondo, T. Kono, Z. Xing, E. Timms, C. Furlonger, E. Keystone, J. Gauldie, D. N. Sauder, T. W. Mak, et al 1994. A role for CD4+ T cells in the pathogenesis of skin fibrosis in tight skin mice. Eur. J. Immunol. 24: 1463-1466. [Medline]
  14. Wynn, T. A.. 2004. Fibrotic disease and the TH1/TH2 paradigm. Nat. Rev. Immunol. 4: 583-594. [Medline]
  15. Roberts, A. B., M. B. Sporn. 1993. Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). Growth Factors 8: 1-9. [Medline]
  16. Saito, E., M. Fujimoto, M. Hasegawa, K. Komura, Y. Hamaguchi, Y. Kaburagi, T. Nagaoka, K. Takehara, T. F. Tedder, S. Sato. 2002. CD19-dependent B lymphocyte signaling thresholds influence skin fibrosis and autoimmunity in the tight-skin mouse. J. Clin. Invest. 109: 1453-1462. [Medline]
  17. Ong, C., C. Wong, C. R. Roberts, H. S. Teh, F. R. Jirik. 1998. Anti-IL-4 treatment prevents dermal collagen deposition in the tight-skin mouse model of scleroderma. Eur. J. Immunol. 28: 2619-2629. [Medline]
  18. Kodera, T., T. L. McGaha, R. Phelps, W. E. Paul, C. A. Bona. 2002. Disrupting the IL-4 gene rescues mice homozygous for the tight-skin mutation from embryonic death and diminishes TGF-beta production by fibroblasts. Proc. Natl. Acad. Sci. USA 99: 3800-3805. [Abstract/Free Full Text]
  19. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301-314. [Medline]
  20. Ley, K., G. S. Kansas. 2004. Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation. Nat. Rev. Immunol. 4: 325-335. [Medline]
  21. Luster, A. D., R. Alon, U. H. von Andrian. 2005. Immune cell migration in inflammation: present and future therapeutic targets. Nat. Immunol. 6: 1182-1190. [Medline]
  22. Tedder, T. F., D. A. Steeber, P. Pizcueta. 1995. L-selectin deficient mice have impaired leukocyte recruitment into inflammatory sites. J. Exp. Med. 181: 2259-2264. [Abstract/Free Full Text]
  23. Dustin, M. L., R. Rothlein, A. K. Bhan, C. A. Dinarello, T. A. Springer. 1986. Induction by IL 1 and interferon-{gamma}: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J. Immunol. 137: 245-253. [Abstract]
  24. Steeber, D. A., M. A. Campbell, A. Basit, K. Ley, T. F. Tedder. 1998. Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression. Proc. Natl. Acad. Sci. USA 95: 7562-7567. [Abstract/Free Full Text]
  25. Shimada, Y., M. Hasegawa, K. Takehara, S. Sato. 2001. Elevated serum L-selectin levels and decreased L-selectin expression on CD8+ lymphocytes in systemic sclerosis. Clin. Exp. Immunol. 124: 474-479. [Medline]
  26. Ihn, H., S. Sato, M. Fujimoto, K. Kikuchi, T. Kadono, K. Tamaki, K. Takehara. 1997. Circulating intercellular adhesion molecule-1 in the sera of patients with systemic sclerosis: enhancement by inflammatory cytokines. Br. J. Rheumatol. 36: 1270-1275. [Abstract/Free Full Text]
  27. Sfikakis, P. P., J. Tesar, H. Baraf, R. Lipnick, G. Klipple, G. C. Tsokos. 1993. Circulating intercellular adhesion molecule-1 in patients with systemic sclerosis. Clin. Immunol. Immunopathol. 68: 88-92. [Medline]
  28. Gruschwitz, M. S., G. Vieth. 1997. Up-regulation of class II major histocompatibility complex and intercellular adhesion molecule 1 expression on scleroderma fibroblasts and endothelial cells by interferon-{gamma} and tumor necrosis factor {alpha} in the early disease stage. Arthritis Rheum. 40: 540-550. [Medline]
  29. Sollberg, S., J. Peltonen, J. Uitto, S. A. Jimenez. 1992. Elevated expression of beta1 and beta2 integrins, intercellular adhesion molecule 1, and endothelial leukocyte adhesion molecule 1 in the skin of patients with systemic sclerosis of recent onset. Arthritis Rheum. 35: 290-298. [Medline]
  30. Sligh, J. E., Jr, C. M. Ballantyne, S. S. Rich, H. K. Hawkins, C. W. Smith, A. Bradley, A. L. Beaudet. 1993. Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA 90: 8529-8533. [Abstract/Free Full Text]
  31. Arbones, M. L., D. C. Ord, K. Ley, H. Radich, C. Maynard-Curry, D. J. Capon, T. F. Tedder. 1994. Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin (CD62L) deficient mice. Immunity 1: 247-260. [Medline]
  32. McGaha, T., S. Saito, R. G. Phelps, R. Gordon, N. Noben-Trauth, W. E. Paul, C. Bona. 2001. Lack of skin fibrosis in tight skin (TSK) mice with targeted mutation in the interleukin-4R{alpha} and transforming growth factor-beta genes. J. Invest. Dermatol. 116: 136-143. [Medline]
  33. Meijerink, J., C. Mandigers, L. van de Locht, E. Tonnissen, F. Goodsaid, J. Raemaekers. 2001. A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR. J. Mol. Diagn. 3: 55-61. [Abstract/Free Full Text]
  34. Chiang, M. Y., H. Chan, M. A. Zounes, S. M. Freier, W. F. Lima, C. F. Bennett. 1991. Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms. J. Biol. Chem. 266: 18162-18171. [Abstract/Free Full Text]
  35. Stepkowski, S. M., Y. Tu, T. P. Condon, C. F. Bennett. 1994. Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities. J. Immunol. 153: 5336-5346. [Abstract]
  36. Sato, S., M. Hasegawa, M. Fujimoto, T. F. Tedder, K. Takehara. 2000. Quantitative genetic variation in CD19 expression correlates with autoimmunity. J. Immunol. 165: 6635-6643. [Abstract/Free Full Text]
  37. Everett, E. T., J. L. Pablos, R. A. Harley, E. C. LeRoy, J. S. Norris. 1995. The role of mast cells in the development of skin fibrosis in tight-skin mutant mice. Comp. Biochem. Physiol. 110A: 159-165.
  38. Lesley, J., R. Hyman, P. W. Kincade. 1993. CD44 and its interaction with extracellular matrix. Adv. Immunol. 54: 271-335. [Medline]
  39. Ong, C. J., S. Ip, S. J. Teh, C. Wong, F. R. Jirik, M. J. Grusby, H. S. Teh. 1999. A role for T helper 2 cells in mediating skin fibrosis in tight-skin mice. Cell. Immunol. 196: 60-68. [Medline]
  40. Tsuji-Yamada, J., M. Nakazawa, K. Takahashi, K. Iijima, S. Hattori, K. Okuda, M. Minami, Z. Ikezawa, T. Sasaki. 2001. Effect of IL-12 encoding plasmid administration on tight-skin mouse. Biochem. Biophys. Res. Commun. 280: 707-712. [Medline]
  41. Longhurst, C. M., L. K. Jennings. 1998. Integrin-mediated signal transduction. Cell Mol. Life Sci. 54: 514-526. [Medline]
  42. Shi-wen, X., M. Panesar, R. Vancheeswaran, J. Mason, D. Haskard, C. Black, I. Olsen, D. Abraham. 1994. Expression and shedding of intercellular adhesion molecule 1 and lymphocyte function-associated antigen 3 by normal and scleroderma fibroblasts: effects of interferon-{gamma}, tumor necrosis factor {alpha}, and estrogen. Arthritis Rheum. 37: 1689-1697. [Medline]
  43. Needleman, B. W.. 1990. Increased expression of intercellular adhesion molecule 1 on the fibroblasts of scleroderma patients. Arthritis Rheum. 33: 1847-1851. [Medline]
  44. Abraham, D., S. Lupoli, A. McWhirter, C. Plater-Zyberk, T. H. Piela, J. H. Korn, I. Olsen, C. Black. 1991. Expression and function of surface antigens on scleroderma fibroblasts. Arthritis Rheum. 34: 1164-1172. [Medline]
  45. Kahari, V. M., M. Sandberg, H. Kalimo, T. Vuorio, E. Vuorio. 1988. Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization. J. Invest. Dermatol. 90: 664-670. [Medline]
  46. Scharffetter, K., B. Lankat-Buttgereit, T. Krieg. 1988. Localization of collagen mRNA in normal and scleroderma skin by in-situ hybridization. Eur. J. Clin. Invest. 18: 9-17. [Medline]
  47. Piela-Smith, T. H., G. Broketa, A. Hand, J. H. Korn. 1992. Regulation of ICAM-1 expression and function in human dermal fibroblasts by IL-4. J. Immunol. 148: 1375-1381. [Abstract]
  48. Kim, J. J., A. Tsai, L. K. Nottingham, L. Morrison, D. M. Cunning, J. Oh, D. J. Lee, K. Dang, T. Dentchev, A. A. Chalian, et al 1999. Intracellular adhesion molecule-1 modulates beta-chemokines and directly costimulates T cells in vivo. J. Clin. Invest. 103: 869-877. [Medline]
  49. Walker, M. A., R. A. Harley, F. A. DeLustro, E. C. LeRoy. 1989. Adoptive transfer of tsk skin fibrosis to +/+ recipients by tsk bone marrow and spleen cells. Proc. Soc. Exp. Biol. Med. 192: 196-200. [Medline]
  50. Hasegawa, M., Y. Hamaguchi, K. Yanaba, J. D. Bouaziz, J. Uchida, M. Fujimoto, T. Matsushita, Y. Matsushita, M. Horikawa, K. Komura, et al 2006. B-lymphocyte depletion reduces skin fibrosis and autoimmunity in the tight-skin mouse model for systemic sclerosis. Am. J. Pathol. 169: 954-966. [Abstract/Free Full Text]
  51. Saito, S., H. Nishimura, T. D. Brumeanu, S. Casares, A. C. Stan, T. Honjo, C. A. Bona. 1999. Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK) mice. Mol. Immunol. 36: 169-176. [Medline]
  52. Kielty, C. M., M. Raghunath, L. D. Siracusa, M. J. Sherratt, R. Peters, C. A. Shuttleworth, S. A. Jimenez. 1998. The tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils. J. Cell Biol. 140: 1159-1166. [Abstract/Free Full Text]
  53. Saito, S., H. Nishimura, R. G. Phelps, I. Wolf, M. Suzuki, T. Honjo, C. Bona. 2000. Induction of skin fibrosis in mice expressing a mutated fibrillin-1 gene. Mol. Med. 6: 825-836. [Medline]
  54. Crooke, S. T.. 1993. Progress toward oligonucleotide therapeutics: pharmacodynamic properties. FASEB J. 7: 533-539. [Abstract]
  55. Bennett, C. F., S. T. Crooke. 1994. Regulation of endothelial cell adhesion molecule expression with antisense oligonucleotides. Adv. Pharmacol. 28: 1-43. [Medline]
  56. Bennett, C. F., T. P. Condon, S. Grimm, H. Chan, M. Y. Chiang. 1994. Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides. J. Immunol. 152: 3530-3540. [Abstract]
  57. Bennett, C. F., D. Kornbrust, S. Henry, K. Stecker, R. Howard, S. Cooper, S. Dutson, W. Hall, H. I. Jacoby. 1997. An ICAM-1 antisense oligonucleotide prevents and reverses dextran sulfate sodium-induced colitis in mice. J. Pharmacol. Exp. Ther. 280: 988-1000. [Abstract/Free Full Text]
  58. Yacyshyn, B. R., C. Barish, J. Goff, D. Dalke, M. Gaspari, R. Yu, J. Tami, F. A. Dorr, K. L. Sewell. 2002. Dose ranging pharmacokinetic trial of high-dose alicaforsen (intercellular adhesion molecule-1 antisense oligodeoxynucleotide) (ISIS 2302) in active Crohn’s disease. Aliment Pharmacol. Ther. 16: 1761-1770. [Medline]
  59. Van Deventer, S. J., J. A. Tami, M. K. Wedel. 2004. A randomized, controlled, double blind, escalating dose study of alicaforsen enema in active ulcerative colitis. Gut 53: 1646-1651. [Abstract/Free Full Text]
  60. Maksymowych, W. P., W. D. Blackburn, Jr, J. A. Tami, W. R. Shanahan, Jr. 2002. A randomized, placebo controlled trial of an antisense oligodeoxynucleotide to intercellular adhesion molecule-1 in the treatment of severe rheumatoid arthritis. J. Rheumatol. 29: 447-453. [Abstract/Free Full Text]




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