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. 2010 Jun;120(6):2049-57.
doi: 10.1172/JCI38644. Epub 2010 May 17.

Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4

Dianhua Jiang  1 , Jiurong Liang, Gabriele S Campanella, Rishu Guo, Shuang Yu, Ting Xie, Ningshan Liu, Yoosun Jung, Robert Homer, Eric B Meltzer, Yuejuan Li, Andrew M Tager, Paul F Goetinck, Andrew D Luster, Paul W Noble
Affiliations

Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4

Dianhua Jiang et al. J Clin Invest. 2010 Jun.

Abstract

Pulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4-null (Sdc4-/-) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4-/- mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis.

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Figures

Figure 1
Figure 1. Noninfectious lung injury induced syndecan-4 expression.
(A) Sdc4 mRNA was upregulated by bleomycin-induced lung injury. Bleomycin or saline was given to C57BL/6 mice intratracheally, and lungs were harvested 1, 3, and 7 days after injury. The mRNA levels in lung tissues were assessed by RT-PCR (n = 3–4). (B) Syndecan-4 cell surface expression was upregulated in lung tissues 7 days after bleomycin-induced injury. Bleomycin was given to C57BL/6 mice intratracheally, lungs were harvested 7 days after injury, and single cells were isolated by Dispase II and DNase I digestions. Cell surface expression of syndecan-4 was determined by staining cells with specific antibody to syndecan-4 (n = 6 [untreated]; 9 [bleomycin]). Flow plots showed syndecan-4 expression gated on total live cells and on leukocytes (CD45+). Fibroblasts isolated from lung tissue of mice untreated (Ctl) or treated with bleomycin (Bleo) for 7 days were also stained for syndecan-4. Negative staining with control IgG is shown in green.
Figure 2
Figure 2. Increased pulmonary fibrosis in Sdc4–/– mice.
(A) Sdc4–/– and littermate WT controls were subject to bleomycin-induced lung injury. Hydroxyproline contents in the lung were measured 14 and 21 days after injury (n = 5–10). Data are representative of 3 similar experiments. (B) Lung sections of Sdc4–/– and WT mice 21 days after injury were stained with trichrome and anti–α-SMA, showing increased collagen staining and α-SMA in Sdc4–/– mice (n = 7). Original magnification, ×100. (C) Lung sections of Sdc4–/– and WT mice before and 14 and 21 days after injury were stained for collagen I, showing increased collagen I staining in lung sections of Sdc4–/– mice (n = 7). Original magnification, ×100.
Figure 3
Figure 3. Dysregulated inflammation in Sdc4–/– mice after bleomycin lung injury.
(A) Sdc4–/– mice showed increased total inflammatory cells in BAL during a 21-day period after bleomycin-induced lung injury. (B–E) The total cell increase was a result of an increase in macrophages (B) and lymphocytes (E). (C and D) There was also a decrease in neutrophils. (A–E) n = 5–7. *P < 0.05; **P < 0.01. Similar results were obtained in 3 separate experiments.
Figure 4
Figure 4. Exogenous CXCL10 did not inhibit fibrosis in Sdc4–/– mice.
Sdc4–/– and littermate WT controls were subject to bleomycin-induced lung injury. Recombinant mouse CXCL10 or BSA buffer was given intramuscularly daily from the day of injury. Hydroxyproline contents in the lung were measured 13 days after injury (n = 8–9).
Figure 5
Figure 5. More fibroblast staining in Sdc4–/– versus WT mouse lungs.
Lung sections of Sdc4–/– and littermate WT mice before and 14 and 21 days after bleomycin injury were stained with fibroblast marker ER-TR7, showing increased fibroblast staining in lung sections of Sdc4–/– mice (n = 7). Original magnification, ×100.
Figure 6
Figure 6. CXCL10 binds to syndecan-4.
(A) Equal numbers of cells isolated from lung tissue of WT, Sdc4–/–, and Cxcr3–/– mice were incubated with 125I-labeled CXCL10. After wash, total radiolabel activity was measured. Decreased binding of 125I-labeled CXCL10 to Sdc4–/– and Cxcr3–/– cells was observed (n = 3). Similar results were obtained from 3 experiments. (B) Equal numbers of fibroblasts isolated from lung tissue of WT, Sdc4–/–, and Cxcr3–/– mice were incubated with 125I-labeled CXCL10. Decreased binding of 125I-labeled CXCL10 to Sdc4–/– fibroblasts was observed (n = 4). Similar results were obtained from 3 experiments. (C) Binding of CXCL10 to fibroblasts. Syndecan-4–bearing fibroblasts from WT lung tissue were plated to confluence in 24-well plates and incubated with 125I-labeled CXCL10 and increasing concentrations of unlabeled CXCL10 at 4°C. The amount of 125I-CXCL10 bound to cells is shown as the percentage of that in the absence of unlabeled CXLC10. (D) Crosslinking CXCL10 to syndecan-4. Biotin-labeled CXCL10 (0.5 μg) was incubated with lung tissue homogenates in the presence or absence of an excess amount of unlabeled CXCL10, crosslinked with DSS, and followed by IP with antibody specific to syndecan-4. Crosslinked syndecan-4 with biotinylated CXCL10 was visualized with streptavidin-HRP followed by enhanced chemiluminescence. Arrow denotes the CXCL10–syndecan-4 complex.
Figure 7
Figure 7. CXCL10 inhibition of BAL-induced fibroblast chemotaxis requires syndecan-4.
(A) Lung fibroblasts were isolated from Sdc4–/– and littermate control mice and plated onto fibronectin-coated Boyden chambers with a pore size of 8 μm. BAL from WT mice 5 days after bleomycin injury was added to the bottom chambers. For certain groups, recombinant CXCL10 (500 pg/ml) was also added to the top and bottom chambers (n = 12–14). Original magnification, ×100. Similar results were obtained from 5 experiments. (B) Quantitation of fibroblast migration. Fibroblasts were counted in 5 fields per slide under light microscopy with a ×40 objective lens. The total number of fibroblasts in 5 fields was expressed as a migration index (n = 3–5). (C) Lung fibroblasts were isolated from Cxcr3–/– mice and plated onto Boyden chambers. BAL from WT mice 5 days after bleomycin injury was added to the bottom chambers. For certain groups, recombinant CXCL10 was also added to the top and bottom chambers. The total number of fibroblasts in 4 fields under microscope with a ×40 objective lens was expressed as migration index (n = 4).
Figure 8
Figure 8. A critical role for the GAG-binding site of CXCL10 protein in inhibition of BAL-induced fibroblast chemotaxis.
Lung fibroblasts were isolated from WT mice (A–C) or from pulmonary fibrosis patients (D), plated onto Boyden chambers, and treated as detailed below. Fibroblast migration was quantified by microscopy with a ×40 objective lens, and fibroblasts were counted in 4 fields per slide (n = 4 per group). Data are expressed as migration index. (A and B) BAL from WT mice 5 days after bleomycin injury was added to the bottom chambers; for certain groups, recombinant native CXCL10, CXCL10 mutants R8A and CtR22A, or heparan sulfate (HS) was also added to the top and bottom chambers. (C) Some fibroblasts were treated with heparinase for 2 hours before migration assay. BAL was added to the bottom chambers, and CXCL10 was added to the top and bottom chambers. (D) Lung fibroblasts isolated from pulmonary fibrosis patients were treated with CXCL10, R8A, or CtR22A. Similar results were obtained from lung fibroblasts isolated from 3 different IPF patients.
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References

    1. Thannickal VJ, Toews GB, White ES, Lynch JP 3rd, Martinez FJ. Mechanisms of pulmonary fibrosis. Annu Rev Med. 2004;55:395–417. doi: 10.1146/annurev.med.55.091902.103810. - DOI - PubMed
    1. Noble PW, Homer RJ. Idiopathic pulmonary fibrosis: new insights into pathogenesis. Clin Chest Med. 2004;25(4):749–758, vii. doi: 10.1016/j.ccm.2004.04.003. - DOI - PubMed
    1. Kuwano K, et al. Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis. J Clin Invest. 1999;104(1):13–19. doi: 10.1172/JCI5628. - DOI - PMC - PubMed
    1. Ramos C, et al. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol. 2001;24(5):591–598. - PubMed
    1. Selman M, et al. TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment? Am J Physiol Lung Cell Mol Physiol. 2000;279(3):L562–L574. - PubMed

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