Induction of nitric oxide synthases in primary human cultured mast cells by IgE and proinflammatory cytokines

The mast cells have been suggested to be a cellular source of nitric oxide (NO) which level is increased in the pathogenesis of asthma. However, isoforms of the NO generating enzyme, nitric oxide synthase (NOS), in primary human mast cells have not been defined due to the lack of a suitable model. We hence examined directly the expression of NOS mRNA and proteins in primary human cultured mast cells (HCMC). Mature HCMC were cultured from CD34+ progenitors isolated from buffy coat preparations and were subjected to IgE sensitisation, IgE receptor mediated activation and cytokines induced stimulation. While expression of NOS mRNA was detected by conventional reverse transcription-polymerase chain reaction (RT-PCR) and quantitatively analyzed with real-time RT-PCR, expression of NOS proteins was detected by immunostaining. In non-stimulated HCMC incubated in medium alone, expressions of NOS were not detected. While overnight incubation of HCMC with IgE significantly increased the expression of NOS2 and NOS3, only NOS2 expression was up-regulated after overnight incubation with a mixture of TNF-α, IFN-γ and IL-1β. Cross-linking of IgE with anti-IgE further increased NOS2 expression with a concomitant decrease in NOS3 expression. NOS1 was not detected in all treatments. In conclusion, we have shown for the first time that NOS2 and NOS3 expressions are induced in primary human mast cells following appropriate stimulations. Comparisons between the differential expressions of NOS isoforms in HCMC to the changes in NOS expressions in asthma models suggest that the mast cell is a source of NO in asthmatic airways.