University of Helsinki, Helsinki 2006
Gelatinases MMP-9 and MMP-2 as indicators of airway inflammation in horses and calves
Doctoral dissertation, June 2006.
Infective respiratory diseases are considered important in both horses and calves, but allergic-type diseases are historically more relevant in horses. Respiratory air quality unquestionably also exerts an impact on food production animals. The aim of this thesis was to investigate environmental dust-induced elevation in gelatinolytic matrix metalloproteinase (MMP) levels in the equine airways and in calves with pulmonary microbial infection. Proteolytic degradation of airway tissue by MMPs has previously been found to correlate with severity of equine recurrent airway obstruction (RAO).
Bronchoalveolar lavage fluid (BALF) of control and heaves-susceptible horses and tracheobronchial lavage fluid (TBLF) of calves with microbial respiratory infection and control calves were analysed for gelatinolytic MMPs (MMP-2 and MMP-9) using zymography. The same method was employed in an in vitro study of proMMP-9 activation capacity of allergens. Gelatinolytic bands were identified by molecular weight, Western immunoblot and/or inhibition by ethylenediaminetetraacetic acid (EDTA). Thus, total gelatinolytic activity, complex forms, proMMP-9, active MMP-9, activation percentage of MMP-9, proMMP-2 and active MMP-2 were analysed.
Consistent with previous studies, 92-kDa MMP-9 was the major gelatinolytic enzyme detected in equine BALF. MMP-2 was elevated in equine BALF following mould inhalation. BALF MMP-9 elevation was dose-dependent in both heaves and control horses following inhalation challenges with hay dust suspension (HDS), endotoxin (LPS) and Aspergillus fumigatus extract (AFE) (the last challenge was conducted with heaves-susceptible horses only). HDSs with different glucan and endotoxin contents caused varying increases in BALF gelatinolytic activities. Mould contamination of HDS, reflected by its glucan concentration, seems to determine the type of gelatinolytic profile detectable in BALF. Soluble and particulate fractions of HDS failed to significantly increase gelatinolytic activity when assessed separately, but in combination the elevations were comparable with those obtained by inhalation of whole HDS, indicating a synergistic effect of glucan and endotoxin as tissue-destruction inducers. Activation of MMP-9 was more enhanced following mould-containing challenges (AFE and HDS with higher glucan concentration). This was further confirmed and extended by in vitro studies using AFE-1 and AFE-2, washed particles of HDS (WP), wash fluid of WP (WF), supernatant of HDS (SUP), HDS and mite extract (MITE) as direct activators of 92-kDa proMMP-9. The two AFEs and MITE were shown to be the most potent activators of 92-kDa proMMP-9 in vitro.
TBLF from calves with clinical symptoms of respiratory disease and positive Pasteurella and Mycoplasma sp. cultures of TBLF samples as well as six healthy controls were analysed for gelatinolytic MMPs. Active MMP-2 and active MMP-9 levels were higher in TBLF of calves with microbial respiratory infection than in controls. The involvement of MMP-2 and MMP-9 in airways of calves with microbial respiratory infection may be important in development of lung lesions, and thus, inhibition of these enzymes may offer a new therapeutic means of treating respiratory infections in calves.
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© University of Helsinki 2006
Last updated 12.05.2006