Michael Hannigan

Directional sensing requires Gβγ-mediated PAK1 and PIXα-dependent activation of Cdc42

Efficient chemotaxis requires directional sensing and cell polarization. We describe a signaling mechanism involving G beta gamma, PAK-associated guanine nucleotide exchange factor (PIX alpha), Cdc42, and p21-activated kinase (PAK) 1. This pathway is utilized by chemoattractants to regulate directional sensing and directional migration of myeloid cells. Our results suggest that G beta gamma binds PAK1 and, via PAK-associated PIX alpha, activates Cdc42, which in turn activates PAK1. Thus, in this pathway, PAK1 is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation. This G beta gamma-PAK1/PIX alpha/Cdc42 pathway is essential for the localization of F-actin formation to the leading edge, the exclusion of PTEN from the leading edge, directional sensing, and the persistent directional migration of chemotactic leukocytes. Although ligand-induced production of PIP(3) is not required for activation of this pathway, PIP(3) appears to localize the activation of Cdc42 by the pathway.

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.

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.

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.

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.

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

Abstract 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 HgCl2 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 HgCl2-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 HgCl2 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.

NOD Mice Having a Lyn Tyrosine Kinase Mutation Exhibit Abnormal Neutrophil Chemotaxis

Neutrophils from NOD (Non‐Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes, and loss of directionality during chemotaxis (compared to wild‐type [WT] C57BL/6 mice). Additionally, F‐actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E393→K) (E372 of LynB), affecting fMLP‐induced tyrosine phosphorylation. These data indicate that the Lyn mutation in NOD neutrophils is likely responsible for dysregulation of neutrophil adhesion and directed migration, implying the role of Lyn in modulating diabetic patients susceptibility to bacterial and fungal infections. J. Cell. Physiol. 232: 1689–1695, 2017.