Inactivation of CD11b in a mouse transgenic model protects against sepsis-induced lung PMN infiltration and vascular injury

Abstract

ϩ/ϩ mice interfered with lung PMN infiltration induced by E. coli and prevented the increase in lung microvessel permeability and edema formation, with most of the protection seen in the 1-h period after the E. coli. Thus our results demonstrate that CD11b plays a crucial role in mediating lung PMN sequestration and vascular injury in the early phase of gram-negative septicemia. The NIF ϩ/ϩ mouse model, in which CD11b is inactivated by binding to NIF, is a potentially useful model for in vivo assessment of the role of PMN CD11b in the mechanism of vascular inflammation. neutrophil inhibitory factor; NIF ϩ/ϩ mice; CD11b/CD18 integrin; neutrophil function; sepsis-induced lung injury; polymorphonuclear leukocyte RECRUITMENT OF polymorphonuclear neutrophils (PMNs) in lungs has been implicated in the development of acute lung injury (ALI) (17, 28). The process of recruitment involves PMN adhesion to endothelial cells via ␤ 2 -integrins and transendothelial migration of PMNs into tissues (33). The PMN adhesion cascade comprises elements of tethering, rolling, activation, and firm adhesion (4, 33). Rolling and capture of PMNs are in large part mediated by endothelial P-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1). Stable adhesion of PMNs is mediated by the binding of PMN ␤ 2 -integrins CD11a/CD18 and CD11b/CD18 to intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell surface (33). The ␤ 2 -integrins, lymphocyte function associated antigen-1 (LFA-1; CD11a/CD18) and Mac-1 (CD11b/CD18), are the major negative regulators of PMN rolling velocity; hence, deletion of ␤ 2 -integrin in mice (CD18 Ϫ/Ϫ mice) was shown to increase the PMN rolling velocity (8, 16). ␤ 2 -Integrins are also capable of binding to multiple ligands; Mac-1 recognizes extracellular matrix proteins (fibronectin, laminin, collagen, and elastin), ICAM-1 and ICAM-2 (11), blood coagulation proteins [fibrinogen (1), factor X (2), and kininogen (15)], neutrophil inhibitory factor (NIF) (25), complement pathway product (iC3b) (3), and nonprotein ligands [e.g., lipopolysaccharide (LPS), ␤-glucan (35), and heparin (6)]. The broad binding specificity of Mac-1 is not shared by LFA-1 even though the two have overlapping functional properties (44). Functional defects have been reported for Mac-1 gene deletion in mice; these include impaired PMN respiratory burst response (5), absence of PMN spreading and adhesion (5,7,21,34), and reduced susceptibility to ischemia/reperfusioninduced tissue injury (32).NIF is a 47-kDa protein isolated from the parasite Ancylostoma caninum that interferes with the host-defense response by apparently inactivating CD11b (25). Recombinant NIF inhibited the adhesion of activated human PMNs to the endothelium and release of PMN H 2 O 2 over a concentration range (IC 50 10-20 nM) (25). NIF binding sites on PMNs showed selective, high-affinity binding to the I-domain of CD11b (25) and low-affinity binding to CD11a in phorbol ester-stimulated PMNs (20). NIF protein has 257 amino acids preceded by a 17-amino acid lea...

Blockade of Class IA Phosphoinositide 3-Kinase in Neutrophils Prevents NADPH Oxidase Activation- and Adhesion-dependent Inflammation

“…Mac-1-deficient mice (Mac-1 Ϫ/Ϫ ) were obtained from Dr. C. Ballantyne, Baylor College of Medicine, Houston, TX (24). We generated NIF transgenic mice (NIF ϩ/ϩ ) as described (25). All mice were housed under specific pathogen-free conditions with access to food and water ad libitum in the Animal Care Facility and all studies were made in accordance with institutional guidelines.…”

“…Isolation of Mouse PMNs-Neutrophils were purified from mouse bone marrow and peripheral venous blood using a discontinuous Percoll gradient as described with modifications (25,26). Purity of PMN preparations as assessed by examination of HEMA3 (Fisher)-stained cytospin (Shandon, Pittsburgh, PA) preparations (27) was ϳ90 -95% and viability assessed by Trypan blue exclusion was Ͼ95%.…”

“…Mouse PMNs loaded with calcein-AM (2 g/ml) (Molecular Probes, Eugene, OR) for 30 min at 37°C were added to fibrinogen-coated plates. The assay for PMN adhesion was performed as described by us (25).…”

Group V Phospholipase A2 in Bone Marrow-derived Myeloid Cells and Bronchial Epithelial Cells Promotes Bacterial Clearance after Escherichia coli Pneumonia

“…Although significantly less LTB 4 and PGE 2 is produced by macrophages from GV Ϫ/Ϫ than GV ϩ/ϩ mice (16) and GV Ϫ/Ϫ mice have lower levels of LTE 4 in the peritoneal cavity after injection of zymosan than GV ϩ/ϩ mice (16), we failed to identify differences in the levels of any eicosanoids in BAL fluid, measured independently by LC/MS/MS and ELISA, between GV ϩ/ϩ and GV Ϫ/Ϫ mice after lung infection with E. coli. PMN migration into the alveolar space after pulmonary E. coli infection is dependent on CD11b/ CD18 on the surface of PMN (30). Although exogenous human GV sPLA 2 can increase surface expression of CD11b and focal clustering of this integrin on eosinophils (34), we also failed to identify differences in CD11b/CD18 expression on the surface of PMN in the alveolar space of GV ϩ/ϩ and GV Ϫ/Ϫ mice after lung E. coli infection.…”

“…Impaired accumulation of PMN in the lungs of GV Ϫ/Ϫ mice does not appear to be due to impaired expression of activated surface CD11b/CD18 on PMN (Fig. 4J), which is necessary for PMNs to migrate from blood into the alveolar space after lung infection with E. coli (30) or to accelerated apoptosis of PMN from GV Ϫ/Ϫ in comparison with GV ϩ/ϩ mice (Fig. 4K).…”