Antigen-induced airway hyperresponsiveness in absence of broncho-obstruction in sensitized guinea pigs

Álvarez-González

Elizabeth

et al. 2020

Preprint

Background: Airway obstruction in asthma is driven by airway smooth muscle (ASM) contraction. Airway obstruction can be induced extrinsically by direct stimulation of ASM with contractile agonists or by indirect provocation with antigens, while the airway baseline tone is dependent on intrinsic obstruction. The ASM phenotypes involved in all types of obstruction seem to be related.Methods: To determination the associations of the ASM phenotypes involved in different types of airway obstruction, guinea pigs were sensitized to ovalbumin and repetitively challenged with antigen. At the third challenge, histamine provocation was used to evaluate airway responsiveness (AR), and lung samples were obtained to calculate the airway wall area. ASM cells from the trachea were disaggregated to determine 1) the percentage of cells that expressed transforming growth factor-β1 (TGF-β1), interleukin-13 (IL-13) and sarco-endoplasmic Ca2+ ATPase-2b (SERCA2b) by flow cytometry; 2) SERCA2B gene expression by RT-PCR; 3) the level of reduced glutathione (GSH) by ELISA; and 4) the sarcoplasmic reticulum Ca2+ refilling rate by microfluorometry. The control guinea pig group received only saline instead of ovalbumin. Comparisons were made using t-tests, and the associations were determined using Spearman correlation coefficient analysis.Results: Antigenic challenges induced airway obstruction and progressive incremental changes in airway baseline tone. The AR to histamine and the expression of TGF-β1 in ASM cells was increased in the asthma model. The airway wall mass and expression of IL-13 and SERCA2b in ASM cells were similar between groups. SERCA2B gene expression and GSH levels were reduced in the asthma group. The extent of antigen-induced airway obstruction was directly associated with ASM cell TGF-β1 expression and the degree of AR. The magnitude of AR and antigen-induced airway obstruction showed an inverse correlation with GSH levels. The airway baseline tone showed an inverse association with SERCA2b expression. No relationship was observed between direct or indirect airway obstruction and the airway tone. After caffeine withdrawal, the rate of sarcoplasmic reticulum Ca2+ refilling was similar in both groups.Conclusions: Each type of airway obstruction depends on different ASM phenotypes: 1) direct and indirect airway obstruction seems to be sensitive to the level of ASM oxidative stress; 2) indirect obstruction induced by antigen appears to be influenced by the expression of TGF-β1 in ASM; and 3) the SERCA2b expression level in ASM cells plays a role in the intrinsic airway tone.

Section: Discussionmentioning

confidence: 45%

Different airway smooth muscle cells phenotypes are involved in intrinsic and extrinsic obstruction in guinea pig asthma model

Álvarez-González

Elizabeth

et al. 2020

Preprint

“…The guinea pig has served as a helpful model in the study of asthma, with some advantages over other models such as the rat and mouse, because it shares various pharmacological characteristics of human asthma, besides the strong airway obstructive response that guinea pigs exhibit after agonist stimulation [ 32 ]. Responses to antigen challenge in guinea pig asthma models are characterised by a rapid and transient airway obstruction in sensitised animals [ 23 , 27 ]. In the current study, the values of basal airway obstruction index were similar between control and experimental groups (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Guinea pigs were sensitised and challenges were performed according to previously described methods [ 23 , 27 ]. The antigen sensitisation of guinea pigs was performed by intraperitoneal (0.5 mg/ml) and subdermal (0.5 mg/ml) injections with a combination of 60 μg/ml ovalbumin plus 1 mg/ml aluminium hydroxide dispersed in saline solution (Figure 1 ).…”

Section: Methodsmentioning

confidence: 99%

“…In guinea pigs, airway hyperresponsiveness was measured after antigen challenge in sensitised ( n = 18; asthma model) and non-sensitised ( n = 13; control group) animals [ 27 ]. Airway responsiveness was evaluated on day 35 (third ovalbumin challenge) by exposing each animal to increasing non-cumulative doses of histamine aerosols (0.001 to 0.32 mg/ml; Sigma Chemical Co., St. Louis, MO, US) after an initial broncho-obstructive index acquisition before and after ovalbumin administration.…”

Section: Methodsmentioning

confidence: 99%

“…Anti-human interferon-γ (IFN-γ; R&D System, Minneapolis, USA), interleukin-4 (IL-4; R&D System, Minneapolis, USA) and IL-5 (clone TRFK5; BD Pharmingen, USA), antibodies were used in lung homogenates of sensitized and non sensitised guinea pigs ( n = 6, each group) to measure cytokines by ELISA as previously described [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

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Antigen‐induced airway hyperresponsiveness and obstruction is related to caveolin‐1 expression in airway smooth muscle in a guinea pig asthma model

Clinical & Translational All

Ramos‐Ramírez

Gutierrez-Aguilar

et al. 2015

BackgroundCaveolin-1 is a fundamental signalling scaffold protein involved in contraction; however, the role of caveolin-1 in airway responsiveness remains unclear. We evaluated the relationship between caveolin-1 expression in airway smooth muscle (ASM) and antigen-induced airway responsiveness and obstruction in a guinea pig asthma model.MethodsAirway obstruction in sensitised guinea pigs, induced by antigenic (ovalbumin) challenges administered every 10 days, was measured. Antigen-induced responsiveness to histamine and the expression of caveolin-1 and cavin 1, 2 and 3 were evaluated at the third ovalbumin challenge. The control group received saline solution instead of ovalbumin.ResultsAfter the first challenge, antigen exposure induced a transient airway obstruction and airway hyperresponsiveness, high levels of IL-4 and IL-5 in lung and airway globet cells proliferation at the third antigenic challenge. Caveolin-1 mRNA levels in total lung decreased in the experimental group compared with controls. Flow cytometric analysis of ASM from the experimental group showed a high number of cells expressing caveolin-1 compared with controls. This increase was confirmed by western blot. Airway obstruction and hyperresponsiveness correlated with the degree of increased caveolin-1 expression in ASM cells (P < 0.05; r = 0.69 and −0.52, respectively). The expression of cavins 1, 2 and 3 in ASM also increased in the experimental group compared to controls. Immunohistochemical findings reveal that differences in ASM caveolin-1 were not evident between groups. Nevertheless, a marked decrease in caveolin-1 and caspase 3 was observed in the pulmonary vascular smooth muscle of asthma model compared with controls. Histological analysis did not reveal differences in smooth muscles mass or subepithelial fibrosis levels in airways between groups. However, an enlargement of smooth muscle mass was observed in the pulmonary microvessels of experimental animals. This enlargement did not induce changes in pulmonary or systemic arterial pressures.ConclusionsOur data suggest that caveolin-1 expression in ASM has a crucial role in the development of antigen-induced airway obstruction and hyperresponsiveness in a guinea pig asthma model. In addition, the asthma model in guinea pigs appears to induce a contractile smooth muscle phenotype in the airways and a proliferative smooth muscle phenotype in pulmonary vessels.

Airway Hyperresponsiveness in Asthma Model Occurs Independently of Secretion of β1 Integrins in Airway Wall and Focal Adhesions Proteins Down Regulation

J of Cellular Biochemistry

Carbajal

Tellez-Jiménez

et al. 2016

Self Cite

The extracellular domains of some membrane proteins can be shed from the cell. A similar phenomenon occurs with β1 integrins (α1β1 and α2β1) in guinea pig. The putative role of β1 integrin subunit alterations due to shedding in airway smooth muscle (ASM) in an allergic asthma model was evaluated. Guinea pigs were sensitized and challenged with antigen. Antigenic challenges induced bronchoobstruction and hyperresponsiveness at the third antigenic challenge. Immunohistochemistry and immunoelectronmicroscopy studies showed that the cytosolic and extracellular domains of the β1 integrin subunit shared the same distribution in airway structures in both groups. Various polypeptides with similar molecular weights were detected with both the cytosolic and extracellular β1 integrin subunit antibodies in isolated airway myocytes and the connective tissue that surrounds the ASM bundle. Flow cytometry and Western blot studies showed that the expression of cytosolic and extracellular β1 integrin subunit domains in ASM was similar between groups. An increment of ITGB1 mRNA in ASM was observed in the asthma model group. RACE-PCR of ITGB1 in ASM did not show splicing variants. The expression levels of integrin-linked kinase (ILK) and paxillin diminished in the asthma model, but not talin. The levels of phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr(696) increased in asthma model. Our work suggests that β1 integrin is secreted in guinea pig airway wall. This secretion is not altered in asthma model; nevertheless, β1 integrin cytodomain assembly proteins in focal cell adhesions in which ILK and paxillin are involved are altered in asthma model. J. Cell. Biochem. 117: 2385-2396, 2016. © 2016 Wiley Periodicals, Inc.