title: Constitutive and LPS-induced expression of MCP-1 and IL-8 by human uveal melanocytes in vitro and relevant signal pathways.
abstract: OBJECTIVE
  Melanocytes are one of the major cellular components in the uvea. Interleukin-8/CXCL8 and monocyte chemoattractant protein-1 (MCP-1/CCL2) are the two most important proinflammatory chemokines. We studied the constitutive and lipopolysaccharide (LPS)-induced expression of IL-8 and MCP-1 in cultured human uveal melanocytes (UM) and explored the relevant signal pathways.
  
  
  METHODS
  Conditioned media and cells were collected from UM cultured in medium with and without stimulation of LPS. Interleukin-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Nuclear factor (NF)-κB in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases1/2 (ERK1/2), and c-Jun N-terminal kinase1/2 (JNK1/2) in cells cultured with and without LPS were measured by ELISA kits. Inhibitors of p38 (SB203580), ERK1/2 (UO1026), JNK1/2 (SP600125), and NF-κB (BAY11-7082) were added to the cultures to evaluate their effects.
  
  
  RESULTS
  Low levels of IL-8 and MCP-1 proteins were detected in the conditioned media in UM cultured without serum. Lipopolysaccharide (0.01-1 μg/mL) increased IL-8 and MCP-1 mRNAs and proteins levels in a dose- and time-dependent manner, accompanied by a significant increase of phosphorylated JNK1/2 in cell lysates and NF-κB in nuclear extracts. Nuclear factor-κB and JNK1/2 inhibitors significantly blocked LPS-induced expression of IL-8 and MCP-1.
  
  
  CONCLUSIONS
  This is the first report on the expression and secretion of chemokines by UM. The data suggest that UM may play a role in the pathogenesis of ocular inflammatory diseases.
authors.affiliation: Tissue Culture Center, New York Eye and Ear Infirmary at Mount Sinai Health Center, New York, New York, United States.
authors.forename: Dan-Ning
authors.initials: DN
authors.lastname: Hu
authors.affiliation: Tissue Culture Center, New York Eye and Ear Infirmary at Mount Sinai Health Center, New York, New York, United States.
authors.forename: Mingchao
authors.initials: M
authors.lastname: Bi
authors.affiliation: Department of Pathology, Mount Sinai Hospital, New York, New York, United States.
authors.forename: David Y
authors.initials: DY
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Mount Sinai Hospital, New York, New York, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Ye
authors.affiliation: Department of Pathology, New York Eye and Ear Infirmary at Mount Sinai Health Center, New York, New York, United States Department of Ophthalmology, New York Eye and Ear Infirmary at Mount Sinai Health Center, New York, New York, United States; New York Medical College, Valhalla, New York, United States.
authors.forename: Steven A
authors.initials: SA
authors.lastname: McCormick
authors.affiliation: Immunopathology Section, Laboratory of Immunology, National Eye Institute, Bethesda, Maryland, United States.
authors.forename: Chi-Chao
authors.initials: CC
authors.lastname: Chan
mesh_terms: D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008544:Melanocytes / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014602:Uvea / Q000166:cytology* / Q000378:metabolism*
chemical_list: D018932:Chemokine CCL2; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D020929:Mitogen-Activated Protein Kinase Kinases
keywords: IL-8; JNK; LPS; MCP-1; NF-κB; p38 MAPK; uveal melanocyte; uveitis