title: Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.
abstract: BACKGROUND
  The mucosal mast cell (MMC) granule-specific beta-chymase, mouse mast cell protease-1 (mMCP-1), is released systemically into the bloodstream early in nematode infection before parasite-specific IgE responses develop and TGF-beta1 induces constitutive release of mMCP-1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein-1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated.
  
  
  OBJECTIVE
  To investigate the expression and to compare the mechanisms of constitutive release of the chymase, mMCP-1, and the chemokine, CCL2.
  
  
  METHODS
  MMC homologues were generated by culturing bone marrow cells in the presence of TGF-beta1, IL-3, IL-9 and stem cell factor (SCF). The intracellular distribution of mMCP-1 and CCL2 was examined by confocal microscopy. The involvement of the Golgi complex and of protein synthesis in the constitutive release of mMCP-1 and CCL2 was investigated using the Golgi-disrupting agent brefeldin A and cycloheximide to block protein synthesis. Secreted analytes were quantified by ELISA.
  
  
  RESULTS
  mMCP-1 colocalized with Golgi matrix protein 130 but was most abundant in the granules, whereas CCL2 was not found in the granules but appeared to be located uniquely in the Golgi complex. Extracellular release of mMCP-1 was significantly inhibited ( approximately 40%) by cycloheximide and by the Golgi-disrupting agent brefeldin A, indicating both continuous protein synthesis and transportation via the Golgi complex are required for optimal mMCP-1 secretion. A similar but more marked inhibitory effect with both compounds was demonstrated on the constitutive secretion of CCL2.
  
  
  CONCLUSIONS
  The culture conditions that promote mMCP-1 expression and release by MMC homologues also promote the expression and release of CCL2. Constitutive release involves de novo protein synthesis and requires a functional Golgi complex, suggesting that similar mechanisms of extracellular secretion operate for both mediators.
authors.affiliation: Department of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush Veterinary Centre, Easter Bush, Roslin, Midlothian, UK.
authors.forename: J K
authors.initials: JK
authors.lastname: Brown
authors.forename: P A
authors.initials: PA
authors.lastname: Knight
authors.forename: S H
authors.initials: SH
authors.lastname: Wright
authors.forename: E M
authors.initials: EM
authors.lastname: Thornton
authors.forename: H R P
authors.initials: HR
authors.lastname: Miller
mesh_terms: D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism*; D001854:Bone Marrow Cells; D020126:Brefeldin A / Q000494:pharmacology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D053818:Chymases; D003513:Cycloheximide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000737:chemistry* / Q000187:drug effects; D008297:Male; D008407:Mast Cells / Q000201:enzymology* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D020545:Respiratory Mucosa / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D020126:Brefeldin A; D003513:Cycloheximide; D012697:Serine Endopeptidases; D053818:Chymases; C509230:Mcpt1 protein, mouse