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Neutrophils are an important component of airway inflammation and may interact with human airway smooth muscle cells (HASMC). We investigated the effect of neutrophils and of neutrophil-derived proteases on HASMC survival. When co-incubated with neutrophils (0.1–1 × 106 cells/ml), attachment of human ASMC was reduced to 12.3 ± 4.3% compared with untreated controls after 72 h. HASMC showed nuclear condensation and fragmentation (41.6 ± 8.1% compared with baseline of 3.1 ± 0.4%), and the biochemical markers of apoptosis, annexin V binding (9.7 ± 0.7%; baseline 1.1 ± 0.3%) and cleaved caspase-3 expression, were observed. The proteolytic activity released by neutrophils was essential for the proapoptotic effect because inhibition of elastase activity by α1-antitrypsin and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) reduced HASMC apoptosis. Human neutrophil elastase (0.1–3 μg/ml) induced apoptosis of HASMC, as well as other neutrophil serine proteases, cathepsin G, and proteinase 3. Fibronectin degradation products were present in HASMC supernatants exposed to neutrophil-conditioned media and to neutrophil elastase. The local release of proteases from neutrophils present in airway smooth muscle cells may lead to HASMC apoptosis as a result of matrix degradation and loss of cell attachment. This may limit pathologic changes such as ASMC hyperplasia and extracellular matrix deposition seen in airway remodeling.