This study tested the hypothesis that human lung tryptase (HLT) would increase interleukin 8 (IL-8) expression and excretion from cell culture isolates of human airway smooth muscle cells (HASMCs). HLT is a serine protease present in human lung mast cells. IL-8 is a cytokine known to be involved in inflammatory processes. It has been shown in prior research that HLT up-regulates IL-8 expression and excretion from human airway fibroblast and epithelial cell lines. We endeavor to see if the same effects are seen in HASMCs. HASMCs for this experiment were obtained from human lung transplants. Cells were grown in 96 well plates until almost confluent. The tryptase was obtained from a commercial source, which was purified from human mast cell leukemia cells. Tryptase catalytic activity was assayed using CPK, a tripeptide containing HLT target sequence. Cell culture incubation with TNF-α, HLT and serum was done at a 10 μM concentration and at time points of 4, 8, and 24 hours. At each time point 200 μL of extracellular fluid was removed from triplicates of each well. IL-8 protein levels in extracellular fluid were determined using a commercial ELISA kit. Samples incubated with a negative control, which consisted of serum free media, showed no increase in IL-8 levels across all time points. Samples incubated with HLT also showed no increase in IL-8 levels across all time points. Samples incubated with TNF-α, an established inducer of IL-8, showed a significant increase in IL-8 levels after being incubated for 8 and 24 hours. The results of the ELISA were performed in duplicate. The ELISA results showed that while HASMC increased IL-8 production and excretion in response to TNF-α, no such response was produced by HLT or the negative control. HLT is known to be unstable with variable activity. We cannot dismiss poor HLT activity and stability in the lack of an HLT-induced increase in IL-8.
Financial support for this research was provided by Western University of Health Sciences Summer Research Fellowship Program.
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