Suppression of Eosinophil Integrins Prevents Remodeling of Airway Smooth Muscle in Asthma

Andrius Januskevicius, Reinoud Gosens, Raimundas Sakalauskas, Simona Vaitkiene, Ieva Janulaityte, Andrew J Halayko, Deimante Hoppenot, Kestutis Malakauskas, Andrius Januskevicius, Reinoud Gosens, Raimundas Sakalauskas, Simona Vaitkiene, Ieva Janulaityte, Andrew J Halayko, Deimante Hoppenot, Kestutis Malakauskas

Abstract

Background: Airway smooth muscle (ASM) remodeling is an important component of the structural changes to airways seen in asthma. Eosinophils are the prominent inflammatory cells in asthma, and there is some evidence that they contribute to ASM remodeling via released mediators and direct contact through integrin-ligand interactions. Eosinophils express several types of outer membrane integrin, which are responsible for cell-cell and cell-extracellular matrix interactions. In our previous study we demonstrated that asthmatic eosinophils show increased adhesion to ASM cells and it may be important factor contributing to ASM remodeling in asthma. According to these findings, in the present study we investigated the effects of suppression of eosinophil integrin on eosinophil-induced ASM remodeling in asthma. Materials and Methods: Individual combined cell cultures of immortalized human ASM cells and eosinophils from peripheral blood of 22 asthmatic patients and 17 healthy controls were prepared. Eosinophil adhesion was evaluated using eosinophil peroxidase activity assay. Genes expression levels in ASM cells and eosinophils were measured using quantitative real-time PCR. ASM cell proliferation was measured using alamarBlue® solution. Eosinophil integrins were blocked by incubating with Arg-Gly-Asp-Ser peptide. Results: Eosinophils from the asthma group showed increased outer membrane α4β1 and αMβ2 integrin expression, increased adhesion to ASM cells, and overexpression of TGF-β1 compared with eosinophils from the healthy control group. Blockade of eosinophil RGD-binding integrins by Arg-Gly-Asp-Ser peptide significantly reduced adhesion of eosinophils to ASM cells in both groups. Integrin-blocking decreased the effects of eosinophils on TGF-β1, WNT-5a, and extracellular matrix protein gene expression in ASM cells and ASM cell proliferation in both groups. These effects were more pronounced in the asthma group compared with the control group. Conclusion: Suppression of eosinophil-ASM interaction via RGD-binding integrins attenuates eosinophil-induced ASM remodeling in asthma. Trial Registration: ClinicalTrials.gov Identifier: NCT02648074.

Keywords: TGF-β1; adhesion; airway smooth muscle; asthma; eosinophils; integrins.

Figures

Figure 1
Figure 1
Gene expression of eosinophil integrin. (A) Integrin gene expression differences between eosinophils isolated from asthmatic patients compared with eosinophils from the healthy control group. (B) Influence of a 24-h incubation with airway smooth muscle (ASM) cells on gene expression of eosinophil integrin. Results are mean ± SEM. Asthma group n = 22; control group n = 17. *p < 0.05 compared with healthy eosinophils; #p < 0.05 compared with control group (eosinophils that had not been incubated with ASM cells).
Figure 2
Figure 2
Measurement of eosinophil adhesion. (A) Calibration curve of eosinophil peroxidase (EPO) substrate oxidation dependency by eosinophil count. (B) Eosinophil integrin-suppression efficiency at different Arg-Gly-Asp-Ser (RGDS)/Gly-Arg-Ala-Asp-Ser-Pro (GRADSP) concentrations. (C) Effects of blocking integrin adhesion to airway smooth muscle (ASM) cells in the control group (healthy participants) at various periods of incubation. (D) Effects of blocking integrin adhesion to ASM cells in the asthma group at various periods of incubation. Results are mean ± SEM. Asthma group n = 22; control group n = 17. Control: ASM cells that had not been co-cultured with eosinophils; #p < 0.05 compared with eosinophils that had not been subject to integrin-blocking. Integrin-blocking: 1-h incubation with RGDS/GRADSP in a 0.125 mg/mL final concentration.
Figure 3
Figure 3
Production of transforming growth factor-β1 (TGF-β1). (A) TGF-β1 gene expression differences between eosinophils isolated from asthmatic patients compared with eosinophils from the healthy control group. (B) Changes in TGF-β1 gene expression in ASM cells after exposure to either eosinophils that had or had not been subject to integrin-blocking. (C) Protein levels of free TGF-β1 in growth medium of eosinophil and ASM cell co-cultures. Results are mean ± SEM. Asthma group n = 22; control group n = 17. #p < 0.05 compared with eosinophils that had not been subject to integrin-blocking; *p < 0.05 compared with eosinophils from the control group. Control, ASM cells that had not been co-cultured with eosinophils; EOS control, non-treated eosinophils; integrin-blocking, 1-h incubation with Arg-Gly-Asp-Ser (RGDS)/Gly-Arg-Ala-Asp-Ser-Pro (GRADSP) in a 0.125 mg/mL final concentration.
Figure 4
Figure 4
Eosinophil cationic protein concentration in co-culture growth medium. Results are mean ± SEM. Asthma group n = 22; control group n = 17. Negative control, ASM cells that had not been subject to co-culture with eosinophils; EOS control, non-treated eosinophils; integrin-blocking, 1-h incubation with RGDS/GRADSP in a 0.125 mg/mL final concentration.
Figure 5
Figure 5
Effect of eosinophil integrin-blocking on WNT-5a and extracellular matrix protein production in airway smooth muscle (ASM) cells. (A) Influence of eosinophil integrin suppression on WNT-5a gene expression in ASM cells. (B) Influence of eosinophil integrin suppression on fibronectin gene expression in ASM cells. (C) Influence of eosinophil integrin suppression on collagen gene expression in ASM cells. Results are mean ± SEM. Asthma group n = 22; control group n = 17. Negative control, ASM cells that had not been subject to co-culture with eosinophils; EOS control, non-treated eosinophils: 1-h incubation with Arg-Gly-Asp-Ser (RGDS)/Gly-Arg-Ala-Asp-Ser-Pro (GRADSP) in a 0.125 mg/mL final concentration. Statistical significance, p < 0.05.
Figure 6
Figure 6
Airway smooth muscle (ASM) cell proliferation after incubation with eosinophils. Results are mean ± SEM. Asthma group n = 22; control group n = 17. Control, ASM cells that had not been subject to co-culture with eosinophils; integrin-blocking, 1-h incubation with Arg-Gly-Asp-Ser (RGDS)/Gly-Arg-Ala-Asp-Ser-Pro (GRADSP) in a 0.125 mg/mL final concentration. Statistical significance, p < 0.05.

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