Inhibition of the stem cell factor 248 isoform attenuates the development of pulmonary remodeling disease

Abstract

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.

Keywords: cytokines; fibrosis; mast cells.

Fig. 1.

SCF is highly expressed in IPF lungs and blood. A: ingenuity IPA was utilized to generate KIT-KITLG interaction and transcriptional activation network. The resulting network was then overlaid with gene expression data sets from IPF lung biopsies relative to normal lung explants (GSE24206). KIT-activated kinases and transcription factors are shown in large font and direct downstream targets for the activated transcription factors are shown in small fonts. Solid arrowheads indicated activation (A) or expression (E). Significantly upregulated (≥ 1.5-fold change and P ≤ 0.05) and downregulated (≥ −1.5-fold change and P ≤ 0.05) are depicted in red and green color, respectively. B: serum from normal (n = 9) or patients newly diagnosed with IPF by high-resolution computed tomography assessment (n = 41) were measured for SCF levels using a specific ELISA (R&D Systems, Rochester, MN). Levels of SCF were measured in serum collected after diagnosis. IPA, Integrated Pathway Analysis; IPF, idiopathic pulmonary fibrosis; SCF, stem cell factor.

Fig. 2.

SCF248 is highly expressed by IPF lung fibroblasts and in fibrotic mouse lungs. A: RNA was extracted from normal and IPF lung fibroblasts and subjected to quantitative PCR analyses using SCF220- and SCF248-specific primer sets. Data are expressed as fold change of SCF248 over SCF220 to compare relative levels between the isoforms in patient-derived fibroblast cell lines. Shown is the mean ± SE of three cell lines. B: full-length SCF, SCF220, and SCF248 transcript expression levels in lungs of 16-day bleomycin-treated B6 mice expressed as fold increase over control untreated mice. Data represents mean ± SE from five mice/group. IPF, idiopathic pulmonary fibrosis; SCF, stem cell factor.

Fig. 3.

Characterization of anti-SCF248 mAb. A: using ATCC cell lines CRL-2452, CRL-2453, and CRL-2454 that express no SCF, SCF220, or SCF248, respectively, the monoclonal antibody was used for flow cytometry binding assays to demonstrate specificity. B: monoclonal antibody generated to a peptide from exon 6 of SCF248 was subjected to Biacore surface plasmon resonance (SPR) analysis to the specific peptide. The data generated dose response curves that were assessed to have a fast on rate and a slow off rate with a 4.5 nM K_D binding. C: image flow cytometry photos of ATCC SCF248-expressing cells incubated for 5 or 60 min with anti-SCF248 or control IgG1 mAb coupled with Phrodo-red pH-sensitive fluorescent dye. D: mean fluorescent intensity (MFI) of ATCC SCF248-expressing cells incubated with control or anti-SCF248 mAb coupled with phrodo-red dye over time to demonstrate internalization. E: IPF patient lung fibroblast cultures incubated for 15 min with control or Anti-SCF248 mAb coupled with phrodo-red dye showing internalization only in the anti-SCF48 mAb-incubated cells. F: naïve Balb/c/J mice were injected intravenously with either control IgG (250 mg/kg), polyclonal anti-SCF (250 mg/kg), or anti-SCF248 monoclonal ab (100 mg/kg) on day 0, 2, 4, and 6. The peripheral blood was assessed for reticulocyte numbers on day 8 as an indication of reduced erythropoiesis as a % of total cells. Data represent the mean ± SE from eight mice/group. ATCC, American Type Culture Collection; IPF, idiopathic pulmonary fibrosis; SCF, stem cell factor.

Fig. 4.

Monoclonal Ab to SCF248 blocks LAD2 mast cell-induced myofibroblast activation. A: fibroblast cell lines from either nonfibrotic (“normal”) or IPF fibrotic lung biopsies were plated in 48-well plates to confluent monolayers. Control or anti-SCF248 monoclonal antibodies (10 ug/mL) were added 30 min before layering of 2 × 10⁵ LAD2 mast cells onto the monolayers for 24 h and assessed for increased matrix gene expression compared with control fibroblasts with no mast cells added to the culture. Data represent mean ± SE *P < 0.05 compared with the control ab-treated cells. B: immunohistochemistry using anti-SCF248 monoclonal antibody on tissue biopsy sections from two patients with IPF or RBILD. IgG control antibodies were used in IPF patient sections and did not show any nonspecific staining. A secondary alkaline phosphatase antibody was used to visualize the staining. IPF, idiopathic pulmonary fibrosis; RBILD, respiratory bronchiolitis-associated interstitial lung disease.

Fig. 5.

Therapeutically targeting of SCF248 using an isoform-specific mAb significantly ameliorated bleomycin-induced lung fibrosis. A: representative histology from 10-wk-old B6 mice 17 days after exposure to intratracheal bleomycin and treated with control or anti-SCF248 monoclonal antibody (20 mg/kg) on day 8 and 12 after the bleomycin challenge. B: the single left lobe was harvested from the normal and bleomycin-treated animals and assessed for hydroxyproline. C: the upper right lobe of the lung was used for isolation of mRNA and assessed for expression of the indicated genes by RT-PCR. D: mice were examined 17 days post-bleomycin treatment for pulmonary lung function using anesthetized and ventilated animals with bleomycin-treated animals given control or anti-SCF248 mAb on days 8 and 12 post-bleomycin instillation. E: mice were treated with IgG control or anti-SCF248 mAb on days 8 and 12 post-bleomycin instillation and examined for c-kit+ cell infiltration by flow cytometry on day 17. Data represent the mean ± SE from 6 to 8 mice/group. SCF, stem cell factor.

Fig. 6.

SCF248 deletion in collagen 1-expressing cells significantly reduced bleomycin-induced lung fibrosis. A: representative histology from lung bleomycin-treated 10–12-wk-old B6 SCFfl/fl Col1a-Cre-ERT mice exposed to tamoxifen (days 6–12 after bleomycin) to delete SCF expression in mesenchymal cells (myofibroblasts). Animals were harvested on day 17 after bleomycin instillation. B: the upper right lobe of the lungs from day 17 bleomycin-treated mice was processed for mRNA analysis by RT-PCR and genes expressed as fold increased over control non-bleomycin-exposed mice. C: hydroxyproline analysis was performed on the single left lobe of the animals and expressed as total hydroxyproline in the left lobe. Data represent mean ± SE from 6 to 7 mice/group. SCF, stem cell factor.