title: LPS-stimulated PMN activation and proinflammatory mediator synthesis is downregulated by phosphodiesterase inhibition: role of pentoxifylline.
abstract: BACKGROUND
  Excessive production of reactive oxygen species by PMN is associated with tissue damage during inflammation. LPS interacts with the cell surface receptor CD14, which generates transmembrane signals through Toll-like protein 4 leading to mitogen activated protein kinase (MAPK) p38 activation, cytokine synthesis, PMN beta2-integrin expression and oxidative burst. Phosphodiesterase inhibition decreases proinflammatory cytokine production and tissue injury after LPS challenge. Its effects on PMN function after LPS stimulation, however, have not been fully investigated. We hypothesized that LPS-induced TNF-alpha synthesis and subsequent PMN beta2-integrin expression and oxidative burst are downregulated by concomitant treatment with the non-specific phosphodiesterase inhibitor pentoxifylline (PTX).
  
  
  METHODS
  Whole blood was incubated with HBSS (control), LPS (100 microg/mL), fMLP (1 micromol/L), LPS+PTX (2 mmol/L) and fMLP+PTX for different time intervals at 37C. Oxidative burst, CD14, and CD-11b expression were measured by flow cytometry. Serum TNF-alpha levels were measured by ELISA. In an attempt to localize the site of action of PTX (proximal or distal to PKC) cell surface receptors were bypassed by PMA stimulation (1 microg/mL) and oxidative burst was measured with and without PTX.
  
  
  RESULTS
  Up-regulation of CD14 expression was similar in LPS and LPS+PTX groups. LPS stimulation caused a significant increase in PMN oxidative burst, CD11b expression, and TNF-alpha serum levels. In addition, PMA and fMLP stimulation also caused significant increase in oxidative burst compared with controls. Concomitant addition of PTX to LPS led to a significant decrease in PMN oxidative burst (65%; p < 0.0001), PMN CD11b expression (20%; p = 0.012), and TNF-alpha levels (93%; p < 0.0001). Also, PMA- and fMLP-induced PMN oxidative burst were significantly decreased by PTX [77.5% (p < 0.0001) and 50% (p < 0.01), respectively].
  
  
  CONCLUSIONS
  These results suggest that PTX-inhibition of oxidative burst occurs distal to PKC and may be either due to direct inhibition of NADPH oxidase or inhibition of MAPK phosphorylation, leading to decreased adhesion molecule expression and TNF-alpha synthesis. Its use in clinical scenarios in which PMN are primed may be of clinical relevance.
authors.affiliation: Division of Trauma, Department of Surgery, University of California San Diego School of Medicine, San Diego, California, USA. rcoimbra@ucsd.edu
authors.forename: Raul
authors.initials: R
authors.lastname: Coimbra
authors.forename: William
authors.initials: W
authors.lastname: Loomis
authors.forename: Heidi
authors.initials: H
authors.lastname: Melbostad
authors.forename: Maria
authors.initials: M
authors.lastname: Tobar
authors.forename: Rafael D
authors.initials: RD
authors.lastname: Porcides
authors.forename: David B
authors.initials: DB
authors.lastname: Hoyt
mesh_terms: D000704:Analysis of Variance; D039481:CD11b Antigen / Q000096:biosynthesis / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000187:drug effects; D015536:Down-Regulation; D006801:Humans; D066298:In Vitro Techniques; D018836:Inflammation Mediators / Q000097:blood*; D008070:Lipopolysaccharides; D018375:Neutrophil Activation / Q000187:drug effects*; D010431:Pentoxifylline / Q000494:pharmacology* / Q000627:therapeutic use; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D016897:Respiratory Burst / Q000187:drug effects*; D018805:Sepsis / Q000188:drug therapy* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000187:drug effects
chemical_list: D039481:CD11b Antigen; D015815:Cell Adhesion Molecules; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D010726:Phosphodiesterase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D010431:Pentoxifylline