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-1beta 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.

Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.

1 Recruitment to activated tyrosine kinase growth factor receptors of Grb2 and p21 ras leads to downstream activation of the kinases Raf, MAPK/Erk kinase (Mek) and, subsequently, extracellular signal-regulated kinase (Erk). Activated Erk phosphorylates speci®c serine residues within cytosolic phospholipase A 2 (PLA 2 ), promoting enzyme translocation to membranes and facilitating liberation of arachidonic acid (AA). 2 In the A549 human adenocarcinoma cell line dexamethasone inhibited epidermal growth factor (EGF)-stimulated cytosolic PLA 2 (cPLA 2 ) activation and AA release by blocking the recruitment of Grb2 to the activated EGF receptor (EGF-R) through a glucocorticoid receptor (GR)-dependent (RU486-sensitive), transcription-independent (actinomycin-insensitive), mechanism. 3 The dexamethasone-induced block of Grb2 recruitment was parallelled by changes in phosphorylation status and subcellular localization of lipocortin 1 (LC1) and an increase in the amount of the tyrosine phosphoprotein co-localized with EGF-R. 4 Like dexamethasone, peptides containing E-Q-E-Y-V from the N-terminal domain of LC1 also blocked ligand-induced association of Grb2, p21 ras and Raf. 5 Our results point to an unsuspected rapid eect of glucocorticoids, mediated by occupation of GR but not by changes in gene transcription, which is brought about by competition between LC1 and Grb2 leading to a failure of recruitment o signalling factors to EGF-R

Acute inflammation can be considered in terms of a series of checkpoints where each phase of cellular influx, persistence, and clearance is controlled by endogenous stop and go signals. It is becoming increasingly apparent that in addition to initiating the inflammatory response, eicosanoids may also mediate resolution. This suggests there are two phases of ara‐chidonic acid release: one at onset for the generation of proinflammatory eicosanoids and one at resolution for the synthesis of proresolving eicosanoids. What is unclear is the identity of the phospholipase (PLA2) isoforms involved in this biphasic release of arachidonic acid. We show here that type VI iPLA2 drives the onset of acute pleurisy through the synthesis of PGE2, LTB4, PAF, and IL‐1β. However, during resolution there is a switch to a sequential induction of first sPLA2 (types IIa and V) that mediates the release of PAF and lipoxin A4, which, in turn, are responsible for the subsequent induction of type IV cPLA2 that mediates the release of arachidonic acid for the synthesis of proresolving pros‐taglandins. This study is the first of its kind to address the respective roles of PLA2 isoforms in acute resolving inflammation and to identify type VI iPLA2 as a potentially selective target for the treatment of inflammatory diseases.—Gilroy, D. W., Newson, J., Sawmynaden, P., Willoughby, D. A., Croxtall, J. D. A novel role for phospholipase A2 isoforms in the checkpoint control of acute inflammation.

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall ('30 mmHg) Septic shock is usually caused by infection with Gram-negative bacteria, most commonly Escherichia coli, which release endotoxin (or bacterial lipopolysaccharide; LPS) into the blood stream. Endotoxin induces the expression of a calciumindependent isoform of nitric oxide synthase (iNOS) in various cells in vitro and in a number of tissues in vivo (1-3). Thus, an enhanced formation of nitric oxide by iNOS contributes to several key features of the pathophysiology of septic shock including hypotension (4, 5) and vascular hyporeactivity to vasoconstrictor agents (6-9). In various cells including endothelial cells, vascular smooth muscle cells, macrophages, neutrophils, and hepatocytes, glucocorticoids inhibit the expression of iNOS induced by LPS or cytokines (10-12); pretreatment with dexamethasone prevents the induction of iNOS and the vascular hyporeactivity caused by endotoxin in vitro (13) and in vivo (8,14,15). Thus, glucocorticoids are potent inhibitors of the induction of iNOS caused by LPS in vitro and in vivo.

Pharmacology. In the article "Lipocortin 1 mediates dexamethasone-induced growth arrest of the A459 lung adenocarcinoma cell line" by J. D. Croxtall and R. J. Flower, which appeared in number 8, April 15, 1992, of Proc. Natl. Acad. Sci. USA (89,(3571)(3572)(3573)(3574)(3575)

1 We have examined the e ects of 12 glucocorticoids as inhibitors of A549 cell growth. 2 Other than cortisone and prednisone, all the glucocorticoids inhibited cell growth and this was strongly correlated (r=0.91) with inhibition of prostaglandin (PG)E 2 formation. 3 The molecular mechanism by which the active steroids prevented PGE 2 synthesis was examined and three groups were identi®ed. Group A drugs did not inhibit arachidonic acid release but inhibited the induction of COX2. Group B drugs were not able to inhibit the induction of COX2 but inhibited arachidonic acid release through suppression of cPLA 2 activation. Group C drugs were apparently able to bring about both e ects. 4 The inhibitory actions of all steroids was dependent upon glucocorticoid receptor occupation since RU486 reversed their e ects. However, group A acted through the NF-kB pathway to inhibit COX2 as the response was blocked by the inhibitor geldanamycin which prevents dissociation of GR and the e ect was blocked by APDC, the NF-kB inhibitor. On the other hand, the group B drugs were not inhibited by NF-kB inhibitors or geldanamycin but their e ect was abolished by the src inhibitor PP2. Group C drugs depended on both pathways. 5 In terms of PGE 2 generation, there is clear evidence of two entirely separate mechanisms of glucocorticoid action, one of which correlates with NF-kB mediated genomic actions whilst the other, depends upon rapid e ects on a cell signalling system which does not require dissociation of GR. The implications for these ®ndings are discussed.

The Ca(2+)- and phospholipid-binding protein Anx-A1 (annexin 1; lipocortin 1) has been described both as an inhibitor of phospholipase A(2) (PLA(2)) activity and as a mediator of glucocorticoid-regulated cell growth and eicosanoid generation. Here we show that, when compared with Anx-A1(+/+) cells, lung fibroblast cell lines derived from the Anx-A1(-/-) mouse exhibit an altered morphology characterized by a spindle-shaped appearance and an accumulation of intracellular organelles. Unlike their wild-type counterparts, Anx-A1(-/-) cells also overexpress cyclo-oxygenase 2 (COX 2), cytosolic PLA(2) and secretory PLA(2) and in response to fetal calf serum, exhibit an exaggerated release of eicosanoids, which is insensitive to dexamethasone (10(-8)- 10(-6) M) inhibition. Proliferation and serum-induced progression of Anx-A1(+/+) cells from G(0)/G(1) into S phase, and the associated expression of extracellular signal-regulated kinase 2 (ERK2), cyclin-dependent kinase 4 (cdk4) and COX 2, is strongly inhibited by dexamethasone, whereas Anx-A1(-/-) cells are refractory to the drug. Loss of the response to dexamethasone in Anx-A1(-/-) cells occurs against a background of no apparent change in glucocorticoid receptor expression or sensitivity to non-steroidal anti-inflammatory drugs. Taken together, these observations suggest strongly that Anx-A1 functions as an inhibitor of signal-transduction pathways that lead to cell proliferation and may help to explain how glucocorticoids regulate these processes.