Differential effect of soluble fibrinogen as a neutrophil activator V. Vitorino de Almeida ⁎, A. Calado, H.S. Rosário, C. Saldanha Unidade de Biologia Microvascular e Inflamação, Instituto de Medicina Molecular, Instituto de Bioquímica, Faculdade de Medicina, Universidade de Lisboa, Portugal abstract article info Article history: Accepted 1 February 2012 Available online 9 February 2012 A fundamental paradigm involved in acute inflammatory responses to invading pathogens and tissue damage is the migration of specific leukocyte populations to the affected tissues to mount an initial innate response to the aggression. The recruitment of polymorphonuclear neutrophils (PMNs) from the blood is a central event in this respect. The aim of this study was to understand whether fibrinogen is able to modulate the pattern of neutrophil ac- tivation and thus contribute to neutrophil recruitment. We demonstrated that fibrinogen induces free radical production by neutrophils without modifying the activation status of Mac-1 (αMβ2, CD11b/CD18), the pre- viously identified neutrophil receptor for fibrinogen. This data indicates that fibrinogen must have an addi- tional different binding site in the neutrophil membrane. Importantly, we propose that as Mac-1 activation was not affected by the binding of fibrinogen, activated neutrophils can further maintain their ability to mar- ginate, roll and adhere to the endothelial walls. © 2012 Elsevier Inc. All rights reserved. Introduction Neutrophil recruitment is crucial for a successful inflammatory re- sponse. In this process, neutrophils initially establish several contacts with the vascular endothelium leading to rolling and firm adhesion to endothelium. This initial step is determinant for the migration of neu- trophils to the inflammation site. This process is mediated by several integrins, including the β 2 integrin family, such as Mac-1 (α M β 2, CD11b/CD18). Circulating neutrophils maintain their integrin recep- tors in a low affinity, non-adhesive state and, in response to local in- flammatory stimuli, are rapidly activated to bind specific ligands (Flick et al., 2004a, 2004b). Mac-1 is a multiligand receptor that mediates leukocyte adhesion during the inflammatory response. One of its ligands is the plasma protein, fibrinogen, a large multidomain protein consisting of two pairs of α, β and γ polypeptide chains organized into three major structural regions: a central E and two peripheral D regions held together by coiled-coil connectors. This multifunctional glycoprotein participates in haemostasis and has adhesive and inflammatory func- tions through specific interactions with cells. Fibrinogen is synthesized by hepatocytes, and then secreted into the circulating bloodstream and it is the main protein component of blood clots. However fibrinogen can mediate diverse biological responses due to its particular molecular structure that comprises binding sites for receptors expressed on cells that are central mediators of the inflammatory process (Kamath and Lip, 2003). As a ligand for the neutrophil Mac-1 integrin, it has been reported to play a role in neutrophil signalling by modulating the generation of second messengers, production of oxygen free radicals and cell adhe- sion in inflammatory conditions. The concentration of this glycoprotein increases in inflammatory conditions, and for this reason, it is considered a risk factor for diverse pathological conditions such as cardiovascular disease and stroke (Kakafika et al., 2007). Materials and methods Reagents HBSS: Hank's Balance salt solution with NaHCO 3 without phenol red, calcium chloride and magnesium sulphate, Sigma; red blood cell lysing buffer: 155 mM ammonium chloride (NH 4 Cl), 10 mm po- tassium hydrogen carbonate (KHCO 3 ), 0,1 mM EDTA; Ficoll-Paque Plus, Amersham Biosciences; DextranT500 a 6% (p/v), Pharmacosmos; Human Fibrinogen, Sigma; Fibrinogen Alexa 488; anti- total Mac-1 anti- body labelled with Phycoerythrin (PE) referred in this manuscript as anti-Mac-1(total antibody, eBioscience; anti-activated Mac-1 antibody labelled with Allophycocyanin (APC) referred in this manuscript as anti-Mac-1(activated) antibody, eBioscience; fMLP (N-formyl– methionine–leucine–phenylalanine), Calbiochem. Methods Isolation of human neutrophils Human neutrophils were isolated from heparinised peripheral venous blood from healthy donors. After erythrocyte sedimentation Microvascular Research 83 (2012) 332–336 ⁎ Corresponding author. E-mail address: vandaalmeida@fm.ul.pt (V.V. de Almeida). 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2012.02.001 Contents lists available at SciVerse ScienceDirect Microvascular Research journal homepage: www.elsevier.com/locate/ymvre