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Study of human epithelial cell detachment and damage: effects of proteases and oxidants
A H Mendis1, T J Venaille, B W Robinson
1University Department of Medicine, Queen Elizabeth II Medical Centre, Sir Charles Gairdner Hospital, Nedlands, Australia.
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 cells (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, 32.3 +/- 14.7 U/mL, 85.8 +/- 26.7 U/mL, 360 +/- 20 U/mL, 340 +/- 49 BAEE U/mL and 300 +/- 23 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 0.59 +/- 0.006 mol/L and 0.015 +/- 0.009 mol/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 alpha 1-anti-proteinase (alpha 1-AP) and, to a lesser extent by aprotinin; trypsin-induced detachment was ablated by PMSF, alpha 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 can 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.
Insights
Polymorphonuclear leucocytes (PMNs) release proteases and oxidants that damage airway epithelial cells. This study shows PMN products like collagenase and cathepsin-G can detach cells from the basement membrane, with potential for synergistic damage with oxidants.
Area of Science:
- Cell Biology
- Biochemistry
- Respiratory Medicine
Background:
- Polymorphonuclear leucocyte (PMN) accumulation is linked to airway epithelial cell damage in inflammatory lung diseases like bronchitis and asthma.
- PMNs release damaging molecules, primarily proteases and oxidants, contributing to airway pathology.
Purpose of the Study:
- To investigate the capacity of various proteases and oxidants released by PMNs to induce detachment of human amnionic epithelial cells (EC) from their native basement membrane (BM) in an in vitro model.
- To identify specific PMN products responsible for epithelial cell desquamation and explore potential synergistic interactions with oxidants.
- To evaluate the efficacy of inhibitors in preventing protease-induced epithelial cell detachment.
Main Methods:
- An in vitro model using intact human amnionic epithelial cells (EC) cultured on native basement membrane (BM) was employed.
- Various purified proteases (collagenase, elastase, cathepsin-G, trypsin, kallikrein, urokinase, plasmin) and oxidants (H2O2, HOCl) were applied to assess their capacity to induce EC detachment (desquamation).
- Minimum effective concentrations (MEC50) for desquamation were determined, and synergistic effects between oxidants and proteases were investigated. Inhibitory effects of various agents (GSH, NAC, EDTA, 1,10 phenanthroline, PMSF, α1-AP, aprotinin, SBTI) were tested.
Main Results:
- Collagenase, elastase, trypsin, and cathepsin-G were potent inducers of EC detachment from BM, with specific MEC50 values determined for each.
- Oxidants like H2O2 and HOCl also caused detachment at high concentrations, and pretreatment with H2O2 enhanced susceptibility to protease-induced detachment, indicating synergism.
- Specific inhibitors effectively blocked protease-induced detachment: collagenase-induced detachment was inhibited by GSH, NAC, EDTA, and 1,10 phenanthroline; elastase detachment by PMSF and α1-AP; trypsin detachment by PMSF, α1-AP, and SBTI; and cathepsin-G detachment by SBTI, GSH, and NAC.
Conclusions:
- Human epithelial cells can be detached from the basement membrane by several PMN-derived products, notably collagenase, cathepsin-G, and elastase.
- Protease-induced epithelial cell detachment can be significantly inhibited by specific agents, suggesting therapeutic potential for protease inhibitors.
- The findings highlight a crucial role for proteases, particularly cathepsin-G and collagenase, and their synergistic interaction with oxidants in mediating PMN-induced epithelial cell detachment in airway inflammatory conditions.