Robert Hannon

Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse

The 37‐kDa protein annexin 1 (Anx‐1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx‐1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX‐2 and cPLA2. In carrageenin‐ or zymosan‐induced inflammation, Anx‐1−/− mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL‐1β generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx‐1−/− polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx‐1−/− mice had reduced cell surface CD 11b (MAC‐1) but enhanced CD62L (L‐selectin) expression and Anx‐1−/− macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx‐1−/−mice that are not seen in the wild‐type animals, suggesting an interaction between sex hormones and inflammation in Anx‐1−/− animals.

Anti-Inflammatory Role of the Murine Formyl-Peptide Receptor 2: Ligand-Specific Effects on Leukocyte Responses and Experimental Inflammation

The human formyl-peptide receptor (FPR)-2 is a G protein-coupled receptor that transduces signals from lipoxin A4, annexin A1, and serum amyloid A (SAA) to regulate inflammation. In this study, we report the creation of a novel mouse colony in which the murine FprL1 FPR2 homologue, Fpr2, has been deleted and describe its use to explore the biology of this receptor. Deletion of murine fpr2 was verified by Southern blot analysis and PCR, and the functional absence of the G protein-coupled receptor was confirmed by radioligand binding assays. In vitro, Fpr2−/− macrophages had a diminished response to formyl-Met-Leu-Phe itself and did not respond to SAA-induced chemotaxis. ERK phosphorylation triggered by SAA was unchanged, but that induced by the annexin A1-derived peptide Ac2–26 or other Fpr2 ligands, such as W-peptide and compound 43, was attenuated markedly. In vivo, the antimigratory properties of compound 43, lipoxin A4, annexin A1, and dexamethasone were reduced notably in Fpr2−/− mice compared with those in wild-type littermates. In contrast, SAA stimulated neutrophil recruitment, but the promigratory effect was lost following Fpr2 deletion. Inflammation was more marked in Fpr2−/− mice, with a pronounced increase in cell adherence and emigration in the mesenteric microcirculation after an ischemia–reperfusion insult and an augmented acute response to carrageenan-induced paw edema, compared with that in wild-type controls. Finally, Fpr2−/− mice exhibited higher sensitivity to arthrogenic serum and were completely unable to resolve this chronic pathology. We conclude that Fpr2 is an anti-inflammatory receptor that serves varied regulatory functions during the host defense response. These data support the development of Fpr2 agonists as novel anti-inflammatory therapeutics.

ENDOGENOUS MONOCYTE CHEMOATTRACTANT PROTEIN-1 RECRUITS MONOCYTES IN THE ZYMOSAN PERITONITIS MODEL

The role of monocyte chemoattractant protein‐1 (MCP‐1) in the recruitment of blood‐derived monocytes in a model of zymosan peritoneal inflammation was investigated. After zymosan injection (1 mg) a rapid influx of polymorphonuclear leukocytes (PMN) and monocytes into the peritoneal cavity associated with mouse MCP‐1 (JE) gene activation and protein secretion in the exudates occurred. MCP‐1 production (maximal at 4 h) preceded the accumulation of monocytes (F4/80‐positive cells, maximally recovered between 16 and 24 h). Treatment of mice with a single injection of anti‐mouse MCP‐1 antibody inhibited 16‐h monocyte accumulation by ~40%, however, a significant decrease in the number of PMN was also measured. Finally, intraperitoneal injection of murine recombinant MCP‐1 (1 μg) produced a selective accumulation of monocytes (F4/80‐positive cells) into the peritoneal cavity. In conclusion, we show the novel existence of a strict relationship between MCP‐1 production and leukocyte accumulation in this model of acute inflammation. J. Leukoc. Biol. 63: 108–116; 1998.

The Annexin Protein Lipocortin 1 Regulates the MAPK/ERK Pathway

Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages.

How to run far: multiple solutions and sex-specific responses to selective breeding for high voluntary activity levels

The response to uniform selection may occur in alternate ways that result in similar performance. We tested for multiple adaptive solutions during artificial selection for high voluntary wheel running in laboratory mice. At generation 43, the four replicate high runner (HR) lines averaged 2.85-fold more revolutions per day as compared with four non-selected control (C) lines, and females ran 1.11-fold more than males, with no sex-by-linetype interaction. Analysis of variance indicated significant differences among C lines but not among HR for revolutions per day. By contrast, average speed varied significantly among HR lines, but not among C, and showed a sex-by-linetype interaction, with the HR/C ratio being 2.02 for males and 2.45 for females. Time spent running varied among both HR and C lines, and showed a sex-by-linetype interaction, with the HR/C ratio being 1.52 for males but only 1.17 for females. Thus, females (speed) and males (speed, but also time) evolved differently, as did the replicate selected lines. Speed and time showed a trade-off among HR but not among C lines. These results demonstrate that uniform selection on a complex trait can cause consistent responses in the trait under direct selection while promoting divergence in the lower-level components of that trait.

Quantitative Analysis of Promoter Activity by Green Fluorescent Protein (GFP) Target/Reporter Strategy in a Novel Transgenic alx/fpr-rs2 Null Mouse

Objective and design: A dual-purpose targeting/reporter vector, incorporating GFP, was utilised to monitor promoter activity in transgenic alx/fpr-rs2 null mice. Materials and Methods:alx/fpr-rs2 null mice were generated by homologous recombination in embryonic stem (ES) cells. A GFP construct was fused inframe within the promoter region allowing representative promoter activity to be monitored by flow cytometry. TNF-α secretion was measured by ELISA. Results: Germline transmission of the alx/fpr-rs2 was confirmed by southern blotting. PCR primer pairs were designed to recognise GFP allowing genotyping. Inducible GFP expression was observed during bone marrow derived macrophage (BMM) differentiation (P<0.01) and following subsequent treatment with LPS (P<0.05), TNF-a secretion mirrored GFP induction. Furthermore GFP positive cell populations were observed in vivo within nai’ve and inflammatory environments. Conclusions: A dual targeting/reporter strategy utilising GFP is a highly effective way of identifying germline transmission in a transgenic colony. In addition the insertion into the promoter region allows a quantitative measure of alx/fpr-rs2 promoter activity and acts as a phenotypic marker in vivo.