Researchers: D Gautrin, PhD, K Maghni, DSc, PhD, J-L Malo, MD. Hôpital du Sacré-Coeur de Montréal. Student: Laetitia Lam Shang Leen, 1st year medical student, UdM, summer studentship COPSE.
Introduction: There is evidence that airway obstruction in asthma is not completely reversible and is accompanied by structural changes in the airways (remodelling). Matrix metalloproteinases (MMPs) are extracellular matrix degrading enzymes that play a critical role in normal development and physiological tissue remodelling and repair37. MMPs are synthesized and released as latent (inactive) pro-enzymes, which are activated by a variety of enzymes at the site of inflammation1. Tissue repair and remodelling depend on the balance between the expression of MMPs and their inhibitor, tissue inhibitors of metalloproteinases (TIMPs). TIMPs bind MMPs in a 1:1 stoichiometric fashion. Thus, an increase in the molar ratio of MMP/TIMP may favour tissue injury. Amongst the MMPs family, MMP-9 is the predominant MMP in allergic asthma1. Park and colleagues2 studied the relationship between MMPs (MMP-9 and MMP-2) and their inhibitor with TDI-induced OA to investigate whether their levels were associated with persistence of asthma symptoms. Three groups of subjects were compared: patients with newly diagnosed occupational asthma, patients with persistent symptoms for ≥ 5 years after diagnosis and non-exposed healthy controls. MMP-9 and MMP-9/TIMP-1 levels were significantly greater in patients diagnosed five years earlier than in newly diagnosed patients. Moreover, significant increases in MMP-9 and MMP-9/TIMP-1 levels were found in newly diagnosed patients after a TDI inhalation challenge and active forms of MMP-9 were detected by zymography after the challenge.
To our knowledge, whether hexamethyldiisocyanates (HDI)-induced OA in patients with a confirmed diagnosis or in the pre-clinical stage of the disease is associated with airway remodelling, and potential imbalance in MMP-9/TIMP-1 is unknown. This interesting issue will be addressed in this proposal for a better understanding of HDI-induced OA.
Objective: To determine if markers of airway inflammation (eosinophils, neutrophils, neutrophil-derived myeloperoxidase: MPO) and of remodelling detected in induced sputum are associated with the development of work-related respiratory symptoms suggestive of newly acquired probable work-related asthma.
Hypothesis and Rationale: We hypothesize that the ratio of MMP-9 and its inhibitor TIMP-1 will be greater in individuals who develop work-related symptoms (early stage of work-related asthma) compared to non-symptomatic individuals, and that MMP-9 may derive from infiltrated activated neutrophils (measurements of MPO levels).
Methods: Subjects – Sputum specimens are available from participants of a prospective cohort of apprentices in car-painting exposed to HDI who completed a training program between 2001 and 2004. The specimens were collected in 2007-09 in the course of a seven-year follow-up study from individuals who were still exposed to HDI at work. A symptom questionnaire and methacholine bronchial challenge tests were administered both during apprenticeship and at follow-up; induced sputum was obtained at follow-up. The participants were categorized into symptomatic and asymptomatic at work on the basis of reported respiratory symptoms suggestive of work-related asthma. The proposed study will include the first 15 symptomatic and 15 asymptomatic participants who provided valid sputum specimens. Determination of inflammatory and remodelling markers. Sputum specimens were processed as described by Pizzichini et al4, and will be examined for non-squamous cell counts; the sputum supernatants were stored at -80°C for the proposed determination of markers of inflammation (neutrophil-derived MPO) and of airway remodelling (MMP-9 and its inhibitor TIMP-1). MPO levels will be determined by ELISA5. MMP-9 forms will be determined using two different techniques. Quantitative gelatin zymography (standard curves drawn with human purified Pro-MMP-9, active MMP-9, MMP-9 dimer and MMP-9/TIMP-1 complex) will be used because this technique allows to detect (and activate) the latent pro-MMP9, activated MMP-9 and MMP-9 complexes whereas ELISA (commercial kits) will allow to quantify separately the levels of MMP-9 and TIMP-1, and then to calculate the ratio MMP-9/TIMP-1.
Analysis: Sputum cell counts (eosinophils, neutrophils) and levels of MPO, MMP-9, TIMP-1 and the ratio MMP-9/TIMP-1 will be compared between 15 exposed individuals with symptoms compatible with probable work-related asthma versus 15 asymptomatic exposed controls using nonparametric tests. Correlations between MMP-9 and MPO levels will be also performed using the Pearson correlation test after logarithmic transformation of data.
Anticipated results: If our data indicate that levels of MMP-9 and the ratio MMP-9/TIMP-1 are increased in symptomatic vs asymptomatic workers exposed to HDI, it would be very interesting to follow these individuals to see if early signs of remodelling increase the risk of HDI-induced OA.
Budget: $20,000 is requested for part-time research assistants and for laboratory material needed for analysis of inflammatory mediators and markers of remodelling (purified MMP-9 and complexes, zymography and ELISA kits).
References:
1. Atkinson JJ et al. Am J Respir Cell Mol Biol 2003;28(1):12-24 2. Park HS et al. Clin Exp Allergy 2003;33(1):113-118. 3. Lee KS etal. Clin Exp Allergy 2004;34(2):276-284. 4. Pizzichini E et al. Am J Respir Crit Care Med 1996;154(2 Pt 1):308-317. 5. Maghni K et al. Am J Respir Crit Care Med 2004;169(3):367-372.