Induction of inducible nitric oxide synthase mRNA expression and nitric oxide production from macrophages stimulated with high‐molecular size mite antigen HM1

ABSTRACT Background High-molecular size mite antigen fraction (HM1) induced macrophage activation and prolonged airway inflammation from mite-sensitized mice. In the present study, we investigated the inflammatory factors in the HM1-activated macrophage in both non-immunized splenocytes and bronchoalveolar lavage (BAL) from mite-immunized and HM1-exposed mice. Methods Dermatophagoides farinae feces (Dff) extract was divided into HM1 and HM1-depleted fraction (DH) by size-exclusion chromatography. Transcriptional gene induction of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-12 and cytokine-inducible nitric oxide synthase (iNOS) in splenic macrophages stimulated with HM1 or DH was analyzed by using semiquantitative reverse transcription–polymerase chain reaction. Nitric oxide (NO) production was measured by using diaminofluoresceins (DAF), fluorescence indicators. Results Gene expression of TNF-α, IL-12 p40 and iNOS was observed with HM1 stimulation of splenic macrophages from non-immunized mice. In addition, the release of NO induced by HM1-stimulated splenic macrophages increased in a dose-dependent manner. However, splenic macrophages stimulated with DH induced TNF-α and IL-12 p40, but not iNOS, gene expression. Similarly, significant iNOS mRNA expression was detected in alveolar macrophages recovered from HM1-exposed mice, but not from DH-exposed mice. Conclusion In the present study, HM1 induced iNOS mRNA expression and NO production both in vitro and in vivo . The present results suggest that the ability of HM1 to induce NO production may be one of the causes aggravating airway inflammation in HM1-exposed mice.