BACKGROUND: Chemokines are inflammatory mediators that activate and recruit specific leukocyte subpopulations. We have recently shown a role for certain chemokines in a warm in situ rat model of lung ischemia reperfusion injury. After hypoxic stress, rat pulmonary artery endothelial cells (RPAECS) potentiate and direct neutrophil sequestration, and, therefore, contribute to the development of tissue injury. The present studies were performed to determine whether RPAECS subjected to in vitro hypoxia and reoxygenation (H&R) secrete chemokines, and, if so, to define the regulatory mechanisms involved. MATERIALS AND METHODS: RPAECS were isolated from 21-day-old Long-Evans rats and were rendered hypoxic (pO(2) 0.5%) for 2 hours and reoxygenated for up to 6 hours. Secreted chemokine content was quantified using sandwich enzyme-linked immunosorbent assay techniques. Mechanistic studies assessed chemokine messenger ribonucleic acid (mRNA) expression by Northern blot, as well as the nuclear translocation of proinflammatory transcription factors nuclear factor kappa beta (NFkappaB), early growth response (EGR), and activator protein-1 (AP-1) by electromobility shift assays. Supershift analysis for EGR-1 was also performed. RESULTS: RPAECS showed a marked increase in the secretion of the chemokines cytokine induced neutrophil chemoattractant and monocyte chemoattractant protein-1 in response to H&R, which was dependent on de novo mRNA transcription and protein translation. Furthermore, in vitro H&R induced the nuclear translocation of the proinflammatory transcription factors NFkappaB and EGR-1 early during reoxygenation. CONCLUSIONS: RPAECS secrete significant amounts of cytokine induced neutrophil chemoattractant and monocyte chemoattractant protein-1 in response to in vitro H&R. The secretion of both chemokines is dependant on de novo mRNA transcription and protein translation, and may be regulated by NFkappaB and EGR-1 activation.
BACKGROUND: Chemokines are inflammatory mediators that activate and recruit specific leukocyte subpopulations. We have recently shown a role for certain chemokines in a warm in situ rat model of lung ischemia reperfusion injury. After hypoxic stress, rat pulmonary artery endothelial cells (RPAECS) potentiate and direct neutrophil sequestration, and, therefore, contribute to the development of tissue injury. The present studies were performed to determine whether RPAECS subjected to in vitro hypoxia and reoxygenation (H&R) secrete chemokines, and, if so, to define the regulatory mechanisms involved. MATERIALS AND METHODS: RPAECS were isolated from 21-day-old Long-Evans rats and were rendered hypoxic (pO(2) 0.5%) for 2 hours and reoxygenated for up to 6 hours. Secreted chemokine content was quantified using sandwich enzyme-linked immunosorbent assay techniques. Mechanistic studies assessed chemokine messenger ribonucleic acid (mRNA) expression by Northern blot, as well as the nuclear translocation of proinflammatory transcription factors nuclear factor kappa beta (NFkappaB), early growth response (EGR), and activator protein-1 (AP-1) by electromobility shift assays. Supershift analysis for EGR-1 was also performed. RESULTS: RPAECS showed a marked increase in the secretion of the chemokines cytokine induced neutrophil chemoattractant and monocyte chemoattractant protein-1 in response to H&R, which was dependent on de novo mRNA transcription and protein translation. Furthermore, in vitro H&R induced the nuclear translocation of the proinflammatory transcription factors NFkappaB and EGR-1 early during reoxygenation. CONCLUSIONS: RPAECS secrete significant amounts of cytokine induced neutrophil chemoattractant and monocyte chemoattractant protein-1 in response to in vitro H&R. The secretion of both chemokines is dependant on de novo mRNA transcription and protein translation, and may be regulated by NFkappaB and EGR-1 activation.
Authors: E Barker; P Murison; P Macchiarini; A Jones; C Otto; H-J Rothkoetter; K Haverson; M Bailey; M Birchall; C Stokes Journal: Clin Exp Immunol Date: 2006-12 Impact factor: 4.330
Authors: Anton S McCourtie; Heather E Merry; Patrick S Wolf; Elizabeth FitzSullivan; John C Keech; Alexander S Farivar; Michael S Mulligan Journal: Ann Thorac Surg Date: 2010-06 Impact factor: 4.330
Authors: Anton S McCourtie; Alexander S Farivar; Steven M Woolley; Heather E Merry; Patrick S Wolf; Brendan Mackinnon-Patterson; John C Keech; Elizabeth Fitzsullivan; Michael S Mulligan Journal: Ann Thorac Surg Date: 2008-12 Impact factor: 4.330