α4β1 and αMβ2 Integrin Expression and Pro-Proliferative Properties of Eosinophil Subtypes in Asthma

Abstract

Eosinophilic inflammation is one of the main pathophysiological features in asthma. Two subtypes of eosinophils exist in the lung and systemic circulation: lung-resident eosinophils (rEOS) and inflammatory eosinophils (iEOS). We evaluated the expression of α4β1 and αMβ2 integrins of eosinophil subtypes and their influence on airway smooth muscle (ASM) cell proliferation and viability in asthma. We included 16 severe non-allergic eosinophilic asthma (SNEA) patients, 13 steroid-free, non-severe allergic asthma (AA) patients, and 12 healthy control subjects (HS). For AA patients, a bronchial allergen challenge with Dermatophagoides pteronyssinus was performed. The eosinophil subtypes were distinguished using magnetic bead-labeled antibodies against surface CD62L, and individual combined cell cultures were prepared with ASM cells. The integrins gene expression was analyzed by a quantitative real-time polymerase chain reaction. Proliferation was assessed by the Alamar blue assay, and viability by annexin V and propidium iodide staining. rEOS-like cells were characterized by the relatively higher gene expression of the β1 integrin subunit, whereas iEOS-like cells were characterized by the αM and β2 integrin subunits. The inclusion of either eosinophil subtypes in co-culture significantly increased the proliferation of ASM cells, and the effect of rEOS-like cells was stronger than iEOS-like cells (p < 0.05). Furthermore, rEOS-like cells had a more pronounced effect on reducing ASM cell apoptosis compared to that of iEOS-like cells (p < 0.05). Lastly, the bronchial allergen challenge significantly enhanced only the iEOS-like cells’ effect on ASM cell proliferation and viability in AA patients (p < 0.05). These findings highlight the different expression of α4β1 and αMβ2 integrins on distinct eosinophil subtypes in asthma. Therefore, rEOS-like cells have a stronger effect in stimulating ASM cell proliferation and viability; however, contact with specific allergens mainly enhances pro-proliferative iEOS-like cell properties.

IL-5 and GM-CSF, but Not IL-3, Promote the Proliferative Properties of Inflammatory-like and Lung Resident-like Eosinophils in the Blood of Asthma Patients

“…Esnault et al compared the effects of β-chain cytokines on eosinophil biology and demonstrated that IL-3 has a stronger effect than any other βc family cytokines in activating eosinophils [ 14 ]. However, despite the strong effect of IL-3 on eosinophil activity, the effects might be related to the stronger affinity to eosinophil IL-3 receptors rather than concentration in asthmatic patients’ blood [ 22 ].…”

“…The phenotyping of eosinophils was based on magnetic bead-conjugated antibodies against L-selectin. These procedures are described in detail in [ 22 ].…”

“…We used 0.5 × 10 5 viable eosinophils and 2 × 10 5 ASM cells grown for 3 days for all experiments. ASM cell proliferation was assessed using an Alamar blue assay, with details described in [ 22 ].…”

“…The expression levels of IL-3, IL-5, and GM-CSF genes were determined for both eosinophil subtypes with qPCR using the commercial Power SYBR ® Green RNA-to-CT™ 1-Step kit (Applied Biosystems, Foster City, CA, USA). The process is described in detail [ 22 ]. The gene expression changes were evaluated based on a fold change between different eosinophil subtypes in the same group and of the same eosinophil subtypes between different groups.…”