Roles of PI3K in neutrophil function.

Neutrophils are terminally differentiated cells that play a vital role in host defense. It has recently become evident that phospholipid regulation plays an import role in many neutrophil functions. We review the regulation of neutrophil functions such as chemotaxis, superoxide production, and phagocytosis by phosphatidylinositol-3,4,5-trisphosphate (PIP3), which is generated in neutrophils by PI3K(gamma). Several lines of evidence are presented demonstrating the importance of this kinase in regulating chemotaxis, in particular the directionality of chemotactic migration. Evidence suggesting that this kinase is important for phagocytosis, especially during engulfment and the internalization of large particles, is also reviewed. Finally, it is suggested that PI3K is important for superoxide production and neutrophil priming. The common link between these seemingly diverse functions is that PI3K(gamma), via its phospholipid products, appears to be providing spatial-temporal cues for the binding of actin-organizing proteins.

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 (lepr(db/+)) and homozygous obese (lepr(db/db)) mice produced no superoxide on stimulation. An absence of intact p47(phox) 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 p47(phox) revealed a point mutation at the -2 position of exon 8, which is consistent with aberrant splicing of the p47(phox) 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 p47(phox) mutation.

Thymus atrophy and changes in thymocyte subpopulations of BN rats with mercury-induced renal autoimmune disease.

Administration of low doses of mercury induces autoantibodies to laminin and autoimmune glomerulonephropathy in BN, MAXX and DZB rats as well as in (BN x LEW)F1 hybrids. LEW strain rats are resistant to these immunotoxic effects. Susceptible rats also show lymphoid hyperplasia in spleen and lymph nodes and severe thymic atrophy. It is still uncertain whether these mercury-induced changes have any role in the induction of autoimmune responses to laminin. In the present study, we have examined the effects of mercury on the thymus of susceptible and resistant rats. Histological analysis of thymuses from BN rats revealed extensive disorganization within 15 days following mercury treatment, with loss of demarcation between cortex and medulla. Numbers of thymus cells were significantly decreased in both BN and (BN x LEW)F1 hybrid rats injected with HgCl2. There was no apparent increase in apoptotic cells in the thymus of these animals. By flow cytometry we detected a relative and absolute loss of double-positive CD4+ CD8+ thymocytes in BN (but not in LEW rats) within 15 days of mercury treatment. There was a corresponding increase in the relative proportion of single-positive (CD4+ or CD8+) and double-negative CD4- CD8- thymocytes in mercury-treated BN rats. Absolute increases in the number of CD4+ single-positive thymocytes were also observed. In contrast, mercury-treated LEW rats had no changes in thymus architecture or significant decreases in cell numbers. Since the thymus is important in both position and negative selection of developing thymocytes, immunotoxic effects of mercury on its structure and thymocyte subpopulations may have multiple consequences. Alternatively, we suggest the hypothesis that autoimmunity (and in particular autoantibodies to laminin) may be responsible for the changes observed in the thymus.

Lack of graft-versus-host-like pathology in mercury-induced autoimmunity of Brown Norway rats.

The repeated administration of mercury to Brown Norway (BN) rats induces the production of autoantibodies to laminin 1 and other autoantigens, accompanied by renal deposition of immunoglobulins and a membranous glomerulonephropathy. A graft-versus-host-like (GVHL) syndrome, characterized by widespread necrotizing leukocytoclastic vasculitis of the bowel, skin, and other tissues, has also been observed after mercury treatment of BN rats. These findings have suggested that the autoimmunity caused by the administration of mercury to BN rats may result as a xenobiotic-induced GVHL effect under the control of OX22+ T lymphocytes. However, previous studies of mercury-induced autoimmunity have never reported any evidence of GVHL lesions. Therefore, we have carefully examined various tissues from a large group of BN rats injected with HgCl(2) to identify possible areas of inflammatory reactions that may have been unnoticed in previous investigations. In addition, we have determined by flow cytometry whether exposure to mercury results in percentage and numerical alterations of OX22+ or other lymphocyte subpopulations in lymphoid organs of HgCl(2)-treated BN rats. The present article confirms that mercury induces autoimmune responses to laminin 1 but does not corroborate the hypothesis of a GVHL syndrome regulated by OX22+ lymphocytes. First, changes in OX22+ cells during treatment with HgCl(2) were infrequent and had no significant correlation with the kinetics of autoimmune responses to laminin 1. Second, we detected no GVHL lesions in skin and intestine of mercury-treated BN rats.

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.