Study of human epithelial cell detachment and damage: Effects of proteases and oxidants

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Abstract
Polymorphonuclear leucocyte (PMN) accumulation is associated with damage to airways epithelial cells in bronchitis. bronchiectasis and some forms of asthma. PMNs release several molecules which may mediate this damage, particularly proteases and oxidants. Using an in vitro model of intact human amnionic epithelial ells (EC) attached to native basement membrane (BM). we evaluated the capacity of several proteases and oxidants to induce detachment of EC from the BM. Maximum desquamation was observed with collagenase, elastase and trypsin, with minimum effective concentrations required to produce 50% EC-desquamation (MEC50) for highly purified collagenase, pancreatic elastase, human leucocyte elastase, human leucocyte cathepsin-G (Cath-G), trypsin, and kallikrein being 3616 989 U/mL, 323 147 U/mL, 858267 U/mL, 36020 U/mL, 34049 BAEE U/mL and 30023 U/mL, respectively. Urokinase (20 U/mL) and plasmin (500 U/mL) produced no desquamation in this system. Relatively high concentrations of oxidants also produced detachment (MEC50 for H2O2 and HOCl being 0590006 mol/L and 00150009 ml/L, respectively and pretreatment of EC membranes with non-detaching concentrations of H2O2 rendered them 10 fold more susceptible to protease-induced desquamation, suggesting synergism. Reduced glutathione (GSH), N-acetyl cysteine (NAC), ethylenediamine tetra-acetic acid (EDTA) and 1,10 phenanthroline ablated collagenase induced EC-detachment. Elastase induced detachment was sensitive to inhibition by phenyl methyl sulfonyl fluoride (PMSF) and 1-anti-proteinase (1-AP) and. to a lesser extent by aprotinin; trypsin-induced detachment was ablated by PMSF. 1-AP and soybean trypsin inhibitor (SBTI) but not by 1,10 phenanthroline or EDTA. Cath-G induced detachment was profoundly inhibited by SBTI GSH and NAC. These data demonstrate that human EC can be detached from intact BM by several PMN products, including collagenase, Cath-G and elastase, and that PMN-mediated detachment tan be prevented by Cath-G and collagenase, inhibitors. The data suggest a role for proteases, particularly Cath-G and collagenase, plus oxidants in synergism with proteases, in mediating PMN-induced EC detachment.