title: Trypsin inhibits lipopolysaccharide signaling in macrophages via toll-like receptor 4 accessory molecules.
abstract: OBJECTIVE
  To examine the role of trypsin in the immune response of macrophages and to determine whether protease-activated receptors (PARs) are involved in the effects of trypsin.
  
  
  METHODS
  We used RAW264.7 cells and peritoneal macrophages isolated from C57BL/6 wild-type mice, PAR2 knockout mice, and ddY mice. Macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of trypsin, thrombin, and PAR subtype-specific agonists (PARs-AP). Activation of macrophages was quantified by nitric oxide production and expression of inflammatory mediators, such as inducible nitric oxide synthase, interleukin-1β, and interleukin-6. To clarify the effect of trypsin on LPS receptors, we also investigated the expression of toll-like receptor 4 (TLR4), soluble MD-2 (sMD-2), membrane-bound MD-2 (mMD-2), soluble CD14 (sCD14), and membrane-bound CD14 (mCD14). To directly investigate the effect of trypsin on CD14 protein, we expressed recombinant CD14 protein.
  
  
  RESULTS
  Trypsin inhibited LPS-induced nitric oxide production and expression of inducible nitric oxide synthase, interleukin-1β, and interleukin-6. The same inhibitory effects of trypsin were observed in wild-type macrophages and in PAR2 knockout macrophages. Furthermore, the other PAR agonists, thrombin, PAR1-AP, PAR2-AP, and PAR4-AP, did not mimic the effect of trypsin. Although trypsin did not affect TLR4 or mMD-2 expression, sCD14, mCD14, and sMD-2 expressions were decreased by trypsin. Furthermore, trypsin also degraded recombinant CD14 protein.
  
  
  CONCLUSIONS
  Trypsin inhibited LPS signaling PAR-independently via degradation of TLR4 accessory molecules. This observation provides a better understanding of the complicated immune response in acute pancreatitis.
authors: Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Japan.
authors: Hiroyuki
authors: H
authors: Komatsu
authors: Akihiro
authors: A
authors: Shimose
authors: Takashi
authors: T
authors: Shimizu
authors: Yu
authors: Y
authors: Mukai
authors: Jun
authors: J
authors: Kobayashi
authors: Takashi
authors: T
authors: Ohama
authors: Koichi
authors: K
authors: Sato
mesh_terms: D000818:Animals; D007249:Inflammation; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D044462:Receptors, Proteinase-Activated / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D044462:Receptors, Proteinase-Activated; D011994:Recombinant Proteins; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D014357:Trypsin; D013917:Thrombin