LPS-Stimulated PMN Activation and Proinflammatory Mediator Synthesis is Downregulated by Phosphodiesterase Inhibition: Role of Pentoxifylline Raul Coimbra, MD, PhD, William Loomis, BS, Heidi Melbostad, BS, Maria Tobar, Rafael D. Porcides, MD, and David B. Hoyt, MD Background: Excessive production of reactive oxygen species by PMN is as- sociated with tissue damage during in- flammation. 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 2-integrin ex- pression and oxidative burst. Phospho- diesterase inhibition decreases proin- flammatory 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- synthesis and sub- sequent PMN 2-integrin expression and oxidative burst are downregulated by concomitant treatment with the non- specific phosphodiesterase inhibitor pentoxifylline (PTX). Methods: Whole blood was incu- bated with HBSS (control), LPS (100 g/ mL), fMLP (1 mol/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- levels were measured by ELISA. In an attempt to localize the site of ac- tion of PTX (proximal or distal to PKC) cell surface receptors were bypassed by PMA stimulation (1 g/mL) and oxida- tive burst was measured with and with- out PTX. Results: Up-regulation of CD14 ex- pression was similar in LPS and LPSPTX groups. LPS stimulation caused a significant increase in PMN oxi- dative burst, CD11b expression, and TNF- serum levels. In addition, PMA and fMLP stimulation also caused signif- icant 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 ox- idative 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 oc- curs 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. Keywords: Pentoxifylline, LPS, Sep- sis, Inflammatory response, Neutrophil, PMN, Oxidative burst, Adhesion mole- cule, TNF-. J Trauma. 2004;57:1157–1163. T he polymorphonuclear neutrophil (PMN) plays a central role in LPS-induced inflammatory response. Activated PMN are recruited to inflammatory sites and constitute the first line of host defense. Following activation, PMN express surface adhesion molecules (2-integrins) that couple with intracellular adhesion molecule-1 (ICAM-1) expressed by endothelial cells and migrate into the tissues. A number of effector functions then develop, including phagocytosis, ox- idative burst, degranulation, and bacterial killing. 1,2 Genera- tion of oxygen reactive species (oxidative burst) by PMN is critical for successful bacterial killing following phagocyto- sis. However, an exaggerated inflammatory response, char- acterized by uncontrolled production of toxic radicals and enzyme release may be injurious to the tissues of the host. A number of respiratory burst agonists, including but not limited to LPS, the chemotactic peptide fMLP, and phorbol myristate acetate (PMA), are capable, by activating distinct intracellular signaling pathways, of inducing marked oxida- tive burst in PMN. Pentoxifylline (PTX), a methylxanthine derivative and non-specific phosphodiesterase inhibitor is known to increase intracellular cyclic AMP (cAMP) and decrease proinflamma- tory mediator synthesis. 3–5 cAMP, an intracellular second messenger, is known to modulate PMN superoxide produc- tion by increasing PKA activity. 6–9 However, most studies demonstrating downregulation of oxidative burst by PTX have been performed using isolated PMN following fMLP and PMA stimulation but not LPS. We have recently shown that PTX attenuates proinflamma- tory mediator production, ICAM-1 expression, and lung injury in animal models of acute and chronic endotoxemia. 10,11 There- fore, we postulated that PTX attenuates PMN oxidative burst in Submitted for July 22, 2004. Accepted for publication September 20, 2004 Copyright © 2004 by Lippincott Williams & Wilkins, Inc. From the Division of Trauma, Department of Surgery, University of California San Diego School of Medicine, San Diego, California. Paper presented at the 34th Annual Scientific Meeting of the Western Trauma Association, February 22–27, 2004, Steamboat Springs, Colorado. Address for reprints: Raul Coimbra, MD, PhD, FACS, Division of Trauma, Department of Surgery, University of California San Diego, School of Medicine, 200 W. Arbor Drive, Campus Box 8896, San Diego, CA 92103– 8896; email: rcoimbra@ucsd.edu. DOI: 10.1097/01.TA.0000151261.28640.F7 The Journal of TRAUMA  Injury, Infection, and Critical Care Volume 57 • Number 6 1157