MIP-4 is Induced by Bleomycin and Stimulates Cell Migration Partially via Nir-1 Receptor

Abstract

Background: CC-chemokine ligand 18 also known as MIP-4 is a chemokine with roles in inflammation and immune responses. It has been shown that MIP-4 is involved in the development of several diseases including lung fibrosis and cancer. How exactly MIP-4 is regulated and exerts its role in lung fibrosis remains unclear. Therefore, in the present study, we examined how MIP-4 is regulated and whether it acts via its potential receptor Nir-1.

Materials and methods: A549 cells were grown and maintained in DMEM : F12 (1 : 1) and supplemented with 10% FBS and 1000 U of penicillin/streptomycin and maintained as recommended by the manufacturer (ATCC). Cell migration and invasion, immunohistochemistry (IHC), Western blot, qPCR, and siRNA Nir-1 were used to determine MIP-4 regulation and its role in cell migration.

Results: Cell migration was increased following stimulation of cells with recombinant (r) MIP-4 and bleomycin (BLM), whereas quenching rMIP-4 with its antibody (Ab) or addition of the Ab to BLM or H₂O₂ diminished rMIP-4-induced cell migration. Along with cell migration, rMIP-4, BLM, and H₂O₂ induced the formation of actin filaments dynamic structures whereas costimulation with MIP-4 Ab limited BLM- and H₂O₂-induced effects. MIP-4 mRNA and protein were increased by BLM and H₂O₂, and the addition of its Ab significantly reduced treatments effect. Experiments with siRNA investigating whether Nir-1 is a potential MIR-4 receptor indicated that the inhibition of Nir-1 decreased cell migration/invasion but did not totally inhibit rMIP-4-induced cell migration.

Conclusion: Therefore, our data indicate that MIP-4 is regulated by BLM and H₂O₂ and costimulation with its Ab limits the effects on MIP-4 and that the Nir-1 receptor partially mediates MIP-4's effects on increased cell migration. These data also evidenced that MIP-4 is regulated by fibrotic and oxidative stimuli and that quenching MIP-4 with its Ab or therapeutically targeting the Nir-1 receptor may partially limit MIP-4 effects under fibrotic or oxidative stimulation.

Figure 1

Cell migration in wound-healing assay. (a) The cell monolayer was wounded using scratch wound assay and exposed to (A) MIP-4 antibody (Ab), (B) rMIP-4, (C) BLM (30 µg/ml), (D) BLM + Ab, (E) H₂O₂ (100 µM), and (F) H₂O₂ + Ab. Cell migration towards wound healing was observed after 24, 48, or 72 h as described in materials and methods. One representative micrograph of scratch assay is shown. (b) Scratch assay quantification showing counted cells migrating towards wound healing. Data are presented as the mean ± SEM, n = 3. Statistically significant, ^∗Ab (control) vs. treatment (trt) at respective time point, ^#trt vs. trt + Ab, t-test, ANOVA, p ≤ 0.05.

Figure 2

Actin filaments remodeling in A549 cells exposed to (a) MIP-4 Ab, (b) rMIP-4, (c) BLM (40 µg/ml), (d) BLM + Ab, (e) H₂O₂ (100 µM), and (f) H₂O₂ + Ab after 72 h as described in materials and methods. One representative micrograph is shown. Green = FITC-phalloidin staining of actin filaments; blue = DAPI staining of nuclei. Fluorescent microscopy was used to analyze the integrity of actin filament. Arrow = stress fiber (SF); broken arrow = focal adhesion (FA); arrow head = filopodium.

Figure 3

Mip-4 mRNA levels in the cells treated with: PBS or control (C), MIP-4 Ab (5 µg/ml), rMIP-4 (1 µg/mL), rMIP-4 + Ab (5 µg/ml), BLM (40 µg/ml), BLM + Ab, H₂O₂ (100 µM), H₂O₂ + Ab for 24 h as described in Materials and Methods. The data are presented as mRNA fold change normalized to 18S relative to control sample. The data represent the mean ± SEM, n = 3. Statistically significant at p < 0.05, ANOVA, t-test, n = 3, ^∗vs. control, ^#vs. BLM.

Figure 4

MIP-4 immunohistochemistry detection using MIP-4 immunofluorescence (green) and DAPI (nucleus). The cells were treated with (a) control, (b) MIP-4 Ab, (c) rMIP-4, (d) rMIP + Ab, (e) BLM, (f) BLM + Ab, (g) H₂O₂, and (h) H₂O₂ + Ab. The arrow points to immunofluorescence of MIP-4.

Figure 5

MIP-4 expression analysis. (a) MIP-4 protein level was determined by Western blot analysis. The protein amounts were quantified and normalized to β-actin in A549 cells treated as indicated in the figure. One representative blot is shown. (b) MIP-4 intensity analysis normalized to β-actin and relative to control untreated samples. The data represent the mean ± SEM, n = 3. Statistically significant: ^∗vs. control, ^#vs. treatment with Ab, ANOVA, t-test, p ≤ 0.05.

Figure 6

Analysis of cell invasion. (a) The cells were cultured in Boyden chamber inserts and treated as follows: (A) control, (B) siRNA Nir-1, (C) siRNA Nir-1 + rMIP-4, (D) rMIP-4, (E) H₂O₂ (100 µM), and (F) siRNA Nir-  + H₂O₂ for 24 h and analyzed as described in materials and methods. Photomicrographs of invasive cells. (b) The cells were stained and quantified at OD560 nm after stained solution extraction according to the protocol. Data are OD560 nm relative to control cells. Statistically significant at p < 0.05, ANOVA, t-test, n = 3, ^∗vs. control, ^#vs. control and all treatments.