Interleukin-1beta causes acute lung injury via alphavbeta5 and alphavbeta6 integrin-dependent mechanisms

Abstract

Interleukin (IL)-1beta has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1beta-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1beta production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta attenuated the IL-1beta-induced ALI. The results of in vitro studies indicated that IL-1beta caused the activation of transforming growth factor-beta via RhoA/alphavbeta6 integrin-dependent mechanisms and the inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta signaling completely blocked the IL-1beta-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1beta increased protein permeability across lung endothelial cell monolayers via RhoA- and alphavbeta5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the alphavbeta5 and alphavbeta6 integrins had an additive protective effect against IL-1beta-induced ALI. In summary, these results demonstrate a critical role for the alphavbeta5/beta6 integrins in mediating the IL-1beta-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.

Figure 1

IL-1β increases mouse lung vascular permeability that is partially mediated via αvβ6 integrin– and TGF-β–dependent mechanisms. A, Levels of hIL-1β were measured in BAL fluid by ELISA. B and C, Extravascular plasma equivalents (EPE) (μL) and excess lung water (ELW) (μL) were measured in mouse lung after recombinant adenovirus (2.5×10⁸ plaque-forming units) expressing human IL-1β (Ad-hIL-1β), control adenovirus, or PBS was instilled intratracheally into wild-type mice (C57BL/6J). D and E, Recombinant adenovirus (2.5×10⁸ plaque-forming units) expressing human IL-1β (Ad-hIL-1β), control adenovirus, or PBS was instilled intratracheally into wild-type mice. Mice were euthanized at day 7. Some of the wild-type mice were treated with blocking or control Ab for αvβ6 integrin (1 mg/kg IP at day 3) and/or soluble chimeric TGF-β type II receptor or vehicle (sTGFβ II Rec; 2 mg/kg IP at day 0, 3, and 5). For all experiments, data are shown as means±SEM (n=8/group). *P<0.05 compared with control mice (Ad-Empty) (A through C) or with Ad-hIL-1β and control Ab or vehicle (D through E).

Figure 2

IL-1β increases protein permeability across rat ATII cell monolayers via αvβ6 integrin or TGF-β–dependent mechanisms. Rat ATII cell monolayers were exposed to IL-1β (10 ng/mL) or its vehicle for 4 hours. Some cell monolayers were pretreated with blocking Ab to the integrin αvβ6 (30 μg/mL) and TGF-β (10 μg/mL) or their respective control isotype Ab. Paracellular protein permeability was measured with ¹²⁵I-albumin. Data are shown as means±SEM. *P≤0.05 from controls.

Figure 3

IL-1β activates TGF-β signaling via a RhoA/αvβ6 integrin–dependent mechanism in rat ATII cell monolayers. A, Rat ATII cell monolayers were stimulated with 1 of the following cytokines/chemokines: IL-1β (10 ng/mL), tumor necrosis factor (TNF)-α (10 ng/mL), interferon (IFN)-γ (10 ng/mL), IL-11 (25 ng/mL), cytokine-induced neutrophil chemoattractant (CINC)-1 (100 ng/mL), KC (500 ng/mL), MCP-1 (100 ng/mL), MIP-2 (100 ng/mL), MIP-3β (250 ng/mL), and exodus-2 (100 ng/mL) before being cocultured with mink lung epithelial reporter cells, as described in the expanded Materials and Methods section in the online data supplement. Cell lysates were assayed for luciferase activity. B, Rat ATII cell monolayers were stimulated with IL-1β (10 ng/mL) for 24 hours, and TGF-β activation was measured by coculture with mink lung epithelial cells as described in the online data supplement. C, Rat ATII cell monolayers were exposed to IL-1β (10 ng/mL) or its vehicle for 4 hours. Some cell monolayers were pretreated with a RhoA kinase inhibitor (Y-27632) (10 μmol/L) or its vehicle before exposure to IL-1β or its vehicle. Active TGF-β was detected using ELISA. D, Rat ATII cell monolayers were stimulated with IL-1β for 30 minutes, cells were lysed, and phospho- and total Smad2 were detected by Western blotting. Some cell monolayers were pretreated with blocking Ab to the integrin αvβ6 (30 μg/mL) and TGF-β (10 μg/mL) or their respective control isotype Ab. E, Rat ATII cell monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 10 minutes. Some cell monolayers were pretreated with blocking Ab to the integrin αvβ6 (30 μg/mL) or its respective control isotype Ab. RhoA activity was measured as described in the online data supplement. All experiments were performed at least in triplicate and repeated 3 times. Data are shown as means±SEM. *P≤0.05 from controls.

Figure 4

IL-1β increases protein permeability across BPAECs. A, BPAEC monolayers were treated with IL-1β (3 to 50 ng/mL) or its vehicle for 1 hour. Some cell monolayers were pretreated with IL-1RA (10 μg/mL) or its vehicle before exposure to IL-1β or its vehicle. Paracellular protein permeability was measured with ¹²⁵I-albumin. B, BPAECs were treated with IL-1β (10 ng/mL) or its vehicle for 1 to 12 hours. Paracellular protein permeability was measured with ¹²⁵I-albumin. Data are shown as percentages of controls; results are shown as means±SEM. *P≤0.05 from controls.

Figure 5

IL-1β–mediated increase in lung endothelial permeability is αvβ5 integrin–dependent. A, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 1 hour. Some cell monolayers were pretreated with blocking Ab to the αvβ5 integrin or isotype control Ab before exposure to IL-1β or its vehicle. Paracellular protein permeability was measured with ¹²⁵I-albumin. Data are shown as percentages of controls; results are shown as means±SEM. *P≤0.05 from controls, **P≤0.05 from cell monolayers treated with IL-1β alone. B, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 10 minutes. Cell monolayers were either pretreated with blocking Ab to αvβ5 integrin or isotype control Ab before exposure to IL-1β or its vehicle. Cells were then fixed, permeabilized, and incubated with rhodamine–phalloidin. C and D, Recombinant adenovirus (2.5×10⁸ plaque-forming units) expressing human IL-1β (Ad-hIL-1β) or control adenovirus was instilled intratracheally into wild-type mice (C57BL/6J). The animals were euthanized at day 7, and the extravascular plasma equivalents (EPE) (μL) and excess lung water (ELW) (μL) were measured as described in the online data supplement. Mice were also treated with either a specific blocking Ab to αvβ5 integrin or specific blocking Abs to αvβ5 and αvβ6 integrins or their isotype control Abs (1 mg/kg IP at days 1, 3, and 5 after intratracheal instillation of adenoviruses). Data are shown as means±SEM (n=8/group). *P<0.05 compared with mice treated with Ad-hIL-1β and control Ab, **P<0.05 compared with mice treated with Ad-hIL-1β and blocking Ab to αvβ5 integrin.

Figure 6

IL-1β–mediated increase in protein permeability across BPAEC monolayers is RhoA-dependent. A, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 10 minutes. Some cell monolayers were pretreated with blocking Ab to the αvβ5 integrin or isotype control Ab before exposure to IL-1β or its vehicle. RhoA activity was measured as described in the online data supplement. Data are shown as percentages of controls; results are shown as means±SEM. *P≤0.05 from controls. B, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 1 hour. Some cell monolayers were pretreated with a RhoA kinase inhibitor (Y-27632) (10 μmol/L) or its vehicle before exposure to IL-1β or its vehicle. Paracellular protein permeability was measured with ¹²⁵I-albumin. Data are shown as percentages of controls; results are shown as means±SEM. *P≤0.05 from controls, **P≤0.05 from cell monolayers treated with IL-1β alone. C, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 10 minutes. Some cell monolayers were pretreated with a RhoA kinase inhibitor (Y-27632) (10 μmol/L) or its vehicle before exposure to IL-1β or its vehicle. Cells were then fixed, permeabilized, and stained with rhodamine–phalloidin and Ab to the αvβ5 integrin or isotype control Ab.

Figure 7

IL-1β causes adherens junction disassembly and formation of paracellular gaps in BPAEC monolayers. A, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 10 minutes. Some cell monolayers were pretreated with blocking Ab to the αvβ5 integrin or isotype control Ab before exposure to IL-1β or its vehicle. Cell lysates were subjected to immunoprecipitation (IP) with an Ab against β-catenin and immunoblotted (IB) with an Ab to phospho-tyrosine. The same blots were then reprobed with an Ab to β-catenin. B, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 4 hours. Some cell monolayers were pretreated with blocking Ab to the αvβ5 integrin or isotype control Ab before exposure to IL-1β or its vehicle. Cells were then fixed, permeabilized, and incubated with a primary Ab against β-catenin and a fluorescein isothiocyanate–conjugated secondary Ab. C, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 10 minutes. Some cell monolayers were pretreated with a RhoA kinase inhibitor (Y-27632) (10 μmol/L) or its vehicle before exposure to IL-1β or its vehicle. Cell lysates were subjected to immunoprecipitation with an Ab against β-catenin and immunoblotted with an Ab to phospho-tyrosine. The same blots were then reprobed with an Ab to β-catenin. D, BPAEC monolayers were treated with IL-1β (10 ng/mL) or its vehicle for 4 hours. Some cell monolayers were pretreated with a RhoA kinase inhibitor (Y-27632) (10 μmol/L) or its vehicle before exposure to IL-1β or its vehicle. Cells were then fixed, permeabilized, and incubated with a primary Ab against β-catenin and a fluorescein isothiocyanate–conjugated secondary Ab.

Figure 8

Schematics of the effect IL-1β on the alveolar capillary barrier. Our model diagrams the IL-1β signaling pathway that leads to an increase in lung epithelial and endothelial permeability. IL-1β causes an increase in RhoA activity (a), leading to TGF-β activation via the αvβ6 integrin (b). TGF-β induces an increase in permeability (c) and an inhibition of the Na⁺-driven fluid transport (d) in alveolar epithelial cells. Subsequent binding of TGF-β on its receptor on endothelial cells induces an increase in lung endothelial permeability via a RhoA-dependent phosphorylation of the VE-cadherin (f) and a formation of actin stress fibers (i). We further show that IL-1β activates RhoA (g) and increases lung endothelial permeability via the phosphorylation and endocytosis of β-catenin (h) and αvβ5 integrin–dependent stress fiber formation in lung endothelial cells (i).