title: Acanthamoeba-cytopathic protein induces apoptosis and proinflammatory cytokines in human corneal epithelial cells by cPLA2α activation.
abstract: OBJECTIVE
  We have shown that Acanthamoeba interacts with a mannosylated protein on corneal epithelial cells and stimulates trophozoites to secrete a mannose-induced 133 kDa protease (MIP-133), which facilitates corneal invasion and induces apoptosis. The mechanism of MIP-133-induced apoptosis is unknown. The aim of this study was to determine if MIP-133 induces apoptosis and proinflammatory cytokines/chemokines in human corneal epithelial (HCE) cells via the cytosolic phospholipase A(2α) (cPLA(2α)) pathway.
  
  
  METHODS
  HCE cells were incubated with or without MIP-133 at doses of 7.5, 15, and 50 μg/mL for 6, 12, and 24 hours. The effects of cPLA(2α) inhibitors on cPLA(2α), arachidonic acid (AA) release, and apoptosis were tested in vitro. Inhibition of cPLA(2α) involved preincubating HCE cells for 1 hour with cPLA(2α) inhibitors (10 μM methyl-arachidonyl fluorophosphonate [MAFP] or 20 μM arachidonyl trifluoromethyl ketone [AACOCF3]) with or without MIP-133 for 24 hours. Expression of cPLA(2α) mRNA and enzyme was examined by RT-PCR and cPLA(2) activity assays, respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 and DNA fragmentation assays. Expression of IL-8, IL-6, IL-1β, and IFN-γ was examined by RT-PCR and ELISA.
  
  
  RESULTS
  MIP-133 induced significant cPLA(2α) (approximately two to four times) and AA release (approximately six times) from corneal cells while cPLA(2α) inhibitors significantly reduced cPLA(2α) (approximately two to four times) and AA release (approximately three times) (P < 0.05). cPLA(2α) inhibitors significantly inhibited MIP-133-induced DNA fragmentation approximately 7 to 12 times in HCE cells (P < 0.05). MIP-133 specifically activates cPLA(2α) enzyme activity in HCE cells, which is blocked by preincubation with anti-MIP-133 antibody. In addition, MIP-133 induced significant IL-8, IL-6, IL-1β, and IFN-γ production, approximately two to three times (P < 0.05).
  
  
  CONCLUSIONS
  MIP-133 interacts with phospholipids on plasma membrane of HCE cells and activates cPLA(2α). cPLA(2α) is involved in apoptosis, AA release, and activation of proinflammatory cytokines/chemokines from HCE cells. cPLA(2α) inhibitors may be a therapeutic target in Acanthamoeba keratitis.
authors.affiliation: Department of Cell Biology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Trivendra
authors.initials: T
authors.lastname: Tripathi
authors.forename: Ashley Dawn
authors.initials: AD
authors.lastname: Smith
authors.forename: Mahshid
authors.initials: M
authors.lastname: Abdi
authors.forename: Hassan
authors.initials: H
authors.lastname: Alizadeh
mesh_terms: D000048:Acanthamoeba / Q000472:pathogenicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D016718:Arachidonic Acid / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000201:enzymology* / Q000469:parasitology / Q000473:pathology*; D054513:Group IV Phospholipases A2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D015800:Protozoan Proteins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016207:Cytokines; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D012333:RNA, Messenger; C534367:mannose-induced cytopathic protein 133, Acanthamoeba; D016718:Arachidonic Acid; D054513:Group IV Phospholipases A2; C516389:PLA2G4A protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3