title: Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes.
abstract: OBJECTIVE
  To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β (pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.
  
  
  METHODS
  All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 μg/mL), and/or nigericin (Nig, 30 μmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.
  
  
  RESULTS
  We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1β, but not the processing of pro-IL-1β to IL-1β, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1β, but not the processing of IL-1β, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1β processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments.
  
  
  CONCLUSIONS
  These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.
authors.affiliation: Amy J Galliher-Beckley, Li-Qiong Lan, Lei Wang, Jishu Shi, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Galliher-Beckley
authors.forename: Li-Qiong
authors.initials: LQ
authors.lastname: Lan
authors.forename: Shelly
authors.initials: S
authors.lastname: Aono
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Jishu
authors.initials: J
authors.lastname: Shi
keywords: Caspase-1; Human defensin; Inflammasome; Interleukin-1β processing and release; Monocytes