Materials and MethodsNeuronal Cell Cultures and Cytotoxicity. Primary cortical or hippocampal neuronal cultures were prepared as previously described (7). To induce excitotoxicity, the cells were prewashed with Tris-buffered control salt solution (CSS; 120 mM NaCl͞5.4 mM KCl͞1.8 mMCaCl 2 ͞25 mM Tris⅐HCl, pH 7.4͞15 mM glucose) and treated with CSS containing 300 M NMDA for 5 min. Toxicity was assayed 20-24 h after NMDA exposure by microscopic examination with computer-assisted cell counting. Total and dead cells were determined by nuclei staining with 100 ng͞ml 4Ј,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) (10 M), respectively. After a 10-min incubation, the cells were examined under a fluorescence microscope (Zeiss) with excitation at 360 nm. Cell death was determined as the ratio of dead to total cells and quantified by counting 1,000 cells. For staining of dead cells by terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL) assay, cells were fixed in 4% paraformaldehyde͞PBS and then stained by using a TUNEL Assay Kit (Molecular Probes) following protocols provided by the manufacturer. The cell death inhibitory effect of various agents was examined essentially as described (7,8).Western blotting was performed essentially as described (9).Focal Cerebral Ischemia Model. C57BL͞6 mice weighing 17-25 g were used for transient focal cerebral ischemia. After a midline neck incision, the left common carotid artery was isolated from the vagus nerve and ligated. The external carotid artery also was ligated, and the internal carotid artery was isolated carefully from the surrounding tissue. An 8-0 nylon filament (Ethicon, Somerville, NJ) was inserted into the common carotid artery through a small incision made in the proximity of the carotid bifurcation and advanced to the proximal part of the anterior cerebral artery to compromise the middle cerebral artery (MCA) flow. The filament was fixed in position by ligature. In sham-operated animals, the above procedures were performed except for the insertion of an intraluminal filament. For histological examinations, mice were perfused transcardially with heparinized PBS followed by 4% paraformaldehyde͞PBS for tissue fixation. Brains were removed and postfixed in 4% paraformaldehyde͞PBS at 4°C overnight. Coronal frozen sections (20 m) were prepared on a cryostat and stored at Ϫ80°C until use. The frozen sections were thawed, washed three times in PBS, permeabilized with 0.1% Triton X-100͞PBS at room temperature for 5 min, and then blocked in 5% skim milk͞3% BSA/PBS for 60 min. Subsequently, they were incubated with primary antibodies (1:200) at 4°C overnight and with secondary antibodies at room temperature for 2 h, and immunoreactivity was visualized by the avidin-biotin complex (ABC) method.Cell Lines and Cell Death Assays. HeLa cells, a human cervical carcinoma-derived cell line, were maintained in DMEM with 10% FBS, 2 mM L-glutamine, and 100 units of penicillin͞ streptomycin at 37°C with a 5% CO 2 atmosphere in a humidified incubator. P...

Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

The recruitment and activation of neutrophils at infected tissues is essential for host defense against invading microorganisms. However, excessive neutrophil recruitment or activation can also damage the surrounding tissues and cause unwanted inflammation. Hence, the responsiveness of neutrophils needs to be tightly regulated. In this study, we have investigated the functional role of tumor suppressor PTEN in neutrophils by using a mouse line in which PTEN is disrupted only in myeloid-derived cells. Chemoattractant-stimulated PTEN ؊/؊ neutrophils displayed significantly higher Akt phosphorylation and actin polymerization. A larger fraction of these neutrophils displayed membrane ruffles in response to chemoattractant stimulation. In addition, chemoattractant-induced transwell migration and superoxide production were also augmented. Single-cell chemotaxis assays showed that PTEN ؊/؊ neutrophils have a small (yet statistically significant) defect in directionality. However, these neutrophils also showed an increase in cell speed. As a result, overall chemotaxis, which depends on speed and directionality, was not affected. Consistent with the increased responsivenessof PTEN ؊/؊ neutrophils, the in vivo recruitment of these cells to the inflamed peritoneal cavity was significantly enhanced. Thus, as a physiologic-negative regulator, PTEN should be a promising therapeutic target for modulating neutrophil functions in various infectious and inflammatory dis- IntroductionNeutrophils are the most abundant cell type among circulating white blood cells. The recruitment and activation of neutrophils are important components of the innate immune system. In response to inflammatory stimuli, neutrophils migrate from the blood to infected tissues, where they protect their host by engulfing, killing, and digesting invading bacterial and fungal pathogens. Conversely, excessive neutrophil accumulation or hyperresponsiveness of neutrophils can also be detrimental to the system. The toxic reactive oxygen species and granule enzymes (eg, proteases) released by neutrophils can damage surrounding tissues and cause unwanted and exaggerated tissue inflammation. Hence, the response of neutrophils to inflammatory stimuli needs to be well controlled.Neutrophils get recruited to the site of infection by responding to a variety of chemokines, leukotrienes, complement peptides, and some chemicals released by bacteria directly, such as peptides bearing the N-formyl group (formyl-peptides). 1-3 All these responses are mediated by heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins)-coupled receptors (GPCRs). One essential downstream target of GPCRs is PtdIns(3,4,5)P3, an inositol phospholipid which has been implicated in a variety of neutrophil functions such as polarization, chemotaxis, and superoxide generation. [4][5][6] PtdIns(3,4,5)P3 exerts its function by mediating protein translocation via pleckstrin homology (PH) domains on the protein. 7,8 This membrane translocation is crucial for these proteins to fulfill the...