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. 2011 Apr;300(4):C896-906.
doi: 10.1152/ajpcell.00439.2010. Epub 2011 Jan 5.

Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of cytoskeletal regulators of VSMC motility

Khatuna Gabunia  1 , Surbhi Jain, Ross N England, Michael V Autieri
Affiliations

Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of cytoskeletal regulators of VSMC motility

Khatuna Gabunia et al. Am J Physiol Cell Physiol. 2011 Apr.

Abstract

Vascular smooth muscle cell (VSMC) migration is an important cellular event in multiple vascular diseases, including atherosclerosis, restenosis, and transplant vasculopathy. Little is known regarding the effects of anti-inflammatory interleukins on VSMC migration. This study tested the hypothesis that an anti-inflammatory Th2 interleukin, interleukin-19 (IL-19), could decrease VSMC motility. IL-19 significantly decreased platelet-derived growth factor (PDGF)-stimulated VSMC chemotaxis in Boyden chambers and migration in scratch wound assays. IL-19 significantly decreased VSMC spreading in response to PDGF. To determine the molecular mechanism(s) for these cellular effects, we examined the effect of IL-19 on activation of proteins that regulate VSMC cytoskeletal dynamics and locomotion. IL-19 decreased PDGF-driven activation of several cytoskeletal regulatory proteins that play an important role in smooth muscle cell motility, including heat shock protein-27 (HSP27), myosin light chain (MLC), and cofilin. IL-19 decreased PDGF activation of the Rac1 and RhoA GTPases, important integrators of migratory signals. IL-19 was unable to inhibit VSMC migration nor was able to inhibit activation of cytoskeletal regulatory proteins in VSMC transduced with a constitutively active Rac1 mutant (RacV14), suggesting that IL-19 inhibits events proximal to Rac1 activation. Together, these data are the first to indicate that IL-19 can have important inhibitory effects on VSMC motility and activation of cytoskeletal regulatory proteins. This has important implications for the use of anti-inflammatory cytokines in the treatment of vascular occlusive disease.

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Figures

Fig. 1.
Fig. 1.
Interleukin (IL)-19 reduces human vascular smooth muscle cell (hVSMC) migration. hVSMC were seeded onto the top chamber of a modified Boyden chamber with or without medium containing 0.2% BSA, with or without 100 ng/ml IL-19, or 40 ng/ml PDGF as a positive control, in the lower chamber. Some VSMC were pretreated with IL-19 4 h before trypsinization and seeding in migration chambers (4h), and in others IL-19 was added at the same time as PDGF (0). Values are means from three experiments performed in triplicate from three independent groups of VSMC. **Significant difference of 4 h pretreated from PDGF control (P < 0.01).
Fig. 2.
Fig. 2.
IL-19 reduces VSMC wound healing. A: scratch wounding in the presence or absence of 100 ng/ml IL-19, 2 μg/ml neutralizing antibody to IL-19, or antibody to IL-20 receptor. Cells were seeded in the presence of 10% FCS. After a single scratch, medium was replaced with medium plus platelet-derived growth factor (PDGF-AB) (40 ng/ml). Cells were stained with hematoxylin and are magnified ×40. This is representative of three independent experiments. B: quantitative analysis of scratch wound area from three different groups of treated VSMC. *Significant difference from 4 different slides of IL-19-treated versus untreated control (P < 0.05).
Fig. 3.
Fig. 3.
IL-19 reduces VSMC spreading. A: VSMC grown on glass coverslips were treated with IL-19 for 3 h or left untreated. Rhodamine phalloidin was added to stain filamentous actin. B: images were captured and calculated by tracing the cell and measuring the area using Image-Pro Plus software. Data shown are means ± SD of 100 cells from each population from three separate experiments. **Significant difference of IL-19 treated versus PDGF alone control (P < 0.05). Magnification ×400.
Fig. 4.
Fig. 4.
IL-19 inhibits phosphorylation of myosin light chain (MLC). A: representative immunoblot of time course of IL-19 pretreatment. To determine whether IL-19 inhibited or decreased MLC activation, VSMC were serum starved for 48 h, and after several time points of IL-19 pretreatment, VSMC were stimulated with PDGF, and lysates were examined for phosphorylation of MLC. B: inhibition was further investigated focusing on 4 h of IL-19 pretreatment and two different times of PDGF stimulation. Combined statistical analysis; values and means from four experiments using independently treated groups of VSMC (*P < 0.05 compared with non-IL-19-treated control).
Fig. 5.
Fig. 5.
IL-19 inhibits dephosphorylation of cofilin. A: representative immunoblot of time course of IL-19 pretreatment. To determine whether IL-19 inhibited or decreased MLC activation, VSMC were serum starved for 48 h, and after several time points of IL-19 pretreatment, VSMC were stimulated with PDGF, and lysates were examined for phosphorylation of cofilin. B: inhibition of dephosphorylation was further investigated focusing on 4 h of IL-19 pretreatment and two different times of PDGF stimulation. Combined statistical analysis; values and means from four experiments using independently treated groups of VSMC (*P < 0.05 compared with non-IL-19-treated control).
Fig. 6.
Fig. 6.
IL-19 inhibits phosphorylation of heat shock protein (HSP)27. A: representative immunoblot of time course of IL-19 pretreatment. To determine whetehr IL-19 inhibited or decreased HSP activation, VSMC were serum starved for 48 h, and after several different time points of IL-19 pretreatment, VSMC were stimulated with PDGF, and lysates were examined for phosphorylation state of HSP27. B: inhibition of HSP27 phosphorylation was further investigated focusing on 4 h of IL-19 pretreatment and two different times of PDGF stimulation. Combined statistical analysis; values and means from four experiments using independently treated groups of VSMC (*P < 0.05, **P < 0.01 compared with non-IL-19-treated control).
Fig. 7.
Fig. 7.
IL-19 inhibits Rac1 activity. A: representative immunoblot of VSMC serum starved for 48 h, and after 4 h IL-19 pretreatment, stimulated with 40 ng/ml PDGF for the times indicated. PAK-bound Rac1 was identified by Western blot analysis and quantitated by densitometry of the corresponding bands. One-tenth the amount of extract was also blotted with Rac1 antibody to document equal amounts of Rac1 in lysates. Gel shown is representative of three experiments performed on three independently derived VSMC. B: scanning densitometry of three experiments from independently treated VSMC (*P < 0.05 compared with non-IL-19-treated control).
Fig. 8.
Fig. 8.
IL-19 inhibits RhoA activity. A: representative immunoblot of VSMC serum starved for 48 h, and after 4 h IL-19 pretreatment, stimulated with 40 ng/ml PDGF for the times indicated. Rhoteckin-bound RhoA was identified by Western blot analysis and quantitated by densitometry of the corresponding bands. One-tenth the amount of extract was also blotted with RhoA antibody to document equal amounts of RhoA in lysates. Gel shown is representative of three experiments performed on three independently derived VSMC. B: scanning densitometry of three experiments from independently treated VSMC (*P < 0.05, compared with non-IL-19-treated control).
Fig. 9.
Fig. 9.
IL-19 inhibitory effects migration are proximal to Rac1 activity. A: migration by Scratch wound. VSMC were infected with 30 MOI of AdRacV14, and after 48 h, migration by the scratch wound assay was performed as described for Fig. 2. Images are representative of at least 3 slides. B: quantitative analysis of scratch wound area from three different groups of treated VSMC. *Significant difference from 3 different slides of IL-19-treated versus untreated control (P < 0.05). IL-19-treated wound was significantly larger than control, and both RacV14 and RacV14 + IL-19-treated wound sizes were significantly smaller than control (P < 0.05). n.s., not significant.
Fig. 10.
Fig. 10.
IL-19 inhibitory effects on activation of cytoskeletal regulatory proteins are proximal to Rac1 activity. A: representative immunoblots of VSMC infected with 30 MOI of AdRacV14. After 48 h serum starvation, VSMC were pretreated for 4 h with IL-19 and then stimulated 30 min with PDGF. Lysates were examined for phosphorylation state of indicated phosphoproteins as previously described. Gels shown are representative of three experiments performed on three independently derived VSMC. B: quantitative scanning densitometry of three experiments from independently treated VSMC. In all cases the RacV14 and RacV14 + IL-19-treated samples were significantly different from unstimulated controls (*P < 0.05 compared with non-IL-19-treated control), and the RacV14 and RacV14 + IL-19 samples were not significantly different from each other.
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