Chi-Kuang Huang

Neutrophils lacking phosphoinositide 3-kinase γ show loss of directionality during N-formyl-Met-Leu-Phe-induced chemotaxis

Confocal imaging and time-lapsed videomicroscopy were used to study the directionality, motility, rate of cell movement, and morphologies of phosphoinositide 3-kinase γ (PI3K)γ−/− neutrophils undergoing chemotaxis in Zigmond chambers containing N-formyl-Met-Leu-Phe gradients. Most of the PI3Kγ−/− neutrophils failed to translocate up the cheomotactic gradient. A partial reduction in cell motility and abnormal morphologies were also observed. In the wild-type neutrophils, the pleckstrin homology domain-containing protein kinase B (AKT) and F-actin colocalize to the leading edge of polarized neutrophils oriented toward the gradient, which was not observed in PI3Kγ−/− neutrophils. In PI3Kγ−/− neutrophils, AKT staining consistently failed to perfectly overlap with the F-actin. This failure was observed as an F-actin-filled region of 2.3 ± 0.5 μm between AKT and the cell membrane. These data suggest that PI3Kγ regulates neutrophil chemotaxis primarily by controlling the direction of cell migration and the intracellular colocalization of AKT and F-actin to the leading edge.

P47(phox)-deficient NADPH oxidase defect in neutrophils of diabetic mouse strains, C57BL/6J-m db/db and db/+.

Deficiencies in neutrophil NADPH oxidase proteins have been demonstrated in humans with chronic granulomatous disease. However, no spontaneous mutation in murine NADPH oxidase has been reported. In this study we report that neutrophils from the diabetic mouse strains, C57BL/6J‐m heterozygous lean (leprdb/+ ) and homozygous obese (leprdb/db) mice produced no superoxide on stimulation. An absence of intact p47phox but not other oxidase proteins was observed in both mouse strains through the use of immunoblotting. Molecular analysis by reverse transcriptase‐polymerase chain reaction identified three abnormal p47phox mRNA transcripts. Sequencing of genomic DNA of p47phox revealed a point mutation at the –2 position of exon 8, which is consistent with aberrant splicing of the p47phox transcript. These results indicate that the C57BL/6J‐m db/db and db/+ mice are the first spontaneously derived murine model of NADPH oxidase deficiency involving a p47phox mutation. J. Leukoc. Biol. 67: 210–215; 2000.

Serum withdrawal-induced post-transcriptional stabilization of cyclooxygenase-2 mRNA in MDA-MB-231 mammary carcinoma cells requires the activity of the p38 stress-activated protein kinase.

Overexpression of thecyclooxygenase-2 (COX-2) gene is observed in several neoplastic diseases. However, molecular mechanisms involved in the regulation of expression of COX-2 are not well understood. In this report, we describe a unique post-transcriptional regulatory mechanism of COX-2 mRNA stabilization in MDA-MB-231 cells, a highly metastatic cell line derived from a human mammary tumor. High levels of COX-2 mRNA, protein, and enzyme activity were induced by serum withdrawal, which were potently inhibited by the addition of serum or >100-kDa serum factor. Nuclear run-on analysis and actinomycin D chase experiments indicate that regulation is primarily at the level of post-transcriptional mRNA stability. Interestingly, SB203580, an inhibitor of the p38 stress-activated protein kinase (SAPK), and overexpression of the dominant-negative p38α construct potently inhibited the serum withdrawal-induced COX-2 mRNA levels. Indeed, the half-life of COX-2 mRNA decreased from 9 to 4.5 h after SB203580 treatment, suggesting that signal transduction by the p38 SAPK pathway is required for COX-2 mRNA stability.

Abnormal Migration Phenotype of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2−/− Neutrophils in Zigmond Chambers Containing Formyl-Methionyl-Leucyl-Phenylalanine Gradients

Time-lapsed video microscopy and confocal imaging were used to study the migration of wild-type (WT) and mitogen-activated protein kinase-activated protein kinase 2 (MK2)−/− mouse neutrophils in Zigmond chambers containing fMLP gradients. Confocal images of polarized WT neutrophils showed an intracellular gradient of phospho-MK2 from the anterior to the posterior region of the neutrophils. Compared with WT neutrophils, MK2−/− neutrophils showed a partial loss of directionality but higher migration speed. Immunoblotting experiments showed a lower protein level of p38 mitogen-activated protein kinase and a loss of fMLP-induced extracellular signal-related kinase phosphorylation in MK2−/− neutrophils. These results suggest that MK2 plays an important role in the regulation of neutrophil migration and may also affect other signaling molecules.

Leukocyte-specific gene 1 protein (LSP1) is involved in chemokine KC-activated cytoskeletal reorganization in murine neutrophils in vitro

Leukocyte‐specific gene 1 protein (LSP1) is a cytoskeletal‐associated protein of leukocytes that in vitro cross‐links F‐actin into extensively branched bundles of mixed polarity. In this study, we examined chemotaxis and superoxide production in neutrophils prepared from wild‐type (WT) and Lsp1 knockout mice. Compared to WT neutrophils, Lsp1‐/‐ neutrophils showed impairment in both migration speed and chemotaxis direction during chemokine KC‐directed chemotaxis. When examined by confocal microscopy, chemotaxing Lsp1‐/‐ neutrophils showed abnormal morphologies. They had discontinuous primary actin‐rich cortexes and large membrane protrusions. When stimulated by phorbol 12‐myristate 13‐acetate (PMA), Lsp1‐/‐ peritoneal neutrophils produce more superoxide than WT. The data presented suggest that LSP1 plays important roles in the regulation of neutrophil morphology, motility, and superoxide production.

Genetic control of neutrophil superoxide production in diabetes-resistant ALR/Lt mice.

The neutrophil oxidative burst reaction differentiates ALR/Lt mice, known for an unusual systemic elevation of antioxidant defenses, from ALS/Lt mice, a related strain known for reduced ability to withstand oxidative stress. Neutrophils from marrow of ALS mice produced a normal neutrophil oxidative burst following phorbol ester stimulation. In contrast, ALR mice exhibited a markedly suppressed superoxide burst. F1 progeny from reciprocal outcrosses between ALR and ALS mice exhibited an intermediate burst level, higher than ALR but significantly lower than ALS. To elucidate the genetic basis for this strain difference, F1 mice were backcrossed to ALS mice, and marrow neutrophils isolated from the progeny were phenotyped for oxidative burst capacity. A genome-wide sweep using polymorphic markers distinguishing the two parental strains was performed to map the trait. A 1:1 phenotypic distribution was observed, and a locus (Suppressor of superoxide production, Susp) controlling this phenotype was mapped to Chromosome 3 near D3Mit241 at 33.1 cM. This locus probably represents an important regulatory element in the overall ALR strain resistance to oxidative stress, since diminished ability to mount a neutrophil burst in backcross segregants correlated with elevated hepatic superoxide dismutase 1 (SOD1) activity, an ALR strain characteristic.