Functional Analysis of Antimicrobial Peptides in Protection from Bacterial Infection

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Initially, we examined the role of inducible hBD in the acute phase of in vitro M.pneumoniae infection. We observed that chemically synthesized hBD-2 and hBD-3, but not hBD-1, show effective antimicrobial activity against M.pneumoniae. To induce hBD production, a human pulmonary squamous cell line EBC-1 was incubated with a potent stimulant, IL-1β. Culture supernatant and total RNA from the EBC-1 were examined for hBD expression. hBD-2 in the supernatant was detected by western blot analysis. In addition, hBD-2 mRNA was strongly up-regulated by IL-1bβ, but neither hBD-1 nor hBD-3 was apparently up-regulated. To determine whether hBD induces the protection from infection, IL-1β-treated EBC-1 cell were infected with M.pneumoniae. IL-1β-treated EBC-1 significantly inhibited the growth of M.pneumoniae, as judged by colony assay. Thus, our results suggest that hBD-2 produced by IL-1β-treated EBC-1 cells plays a role in the protection of early stage of M.pneumoniae infection.Subsequently, roles of antimicrobial peptides, in particular CRAMP, in in vivo infection model were examined. There seems to be no reports regarding the CRAMP and Mycoplasma infection. CRAMP, a family of cathelicidin, was chemically synthesized. The CRAMP showed an effective antimicrobial activity against M.pneumoniae. To induce CRAMP in vivo, BALB/c mice were intranasally infected with M.pneumoniae, and infected lungs taken from the mice at 6,12,24 and 48 hours postinfection were examined for CRAMP expression. Western blot analysis showed that the homogenate from the lungs at 24 hours after infection contains CRAMP. In addition, CRAMP mRNA from the infected lungs was detected by using RT-PCR. Immunostaining showed that there are many CRAMP-positive neutrophils in the peribronchial area, but no definitive CRAMP-positive epithelial cells. At present underway is investigation on the association between CRAMP-positive neutrophils and protective effect