Verena Niggli

Signaling to migration in neutrophils: importance of localized pathways

Neutrophils, a major type of blood leukocytes, are indispensable for host defense of bacterial infections. Directed migration in a gradient of chemotactic stimuli enables these cells to rapidly find the site of infection and destroy the invading pathogens. Chemotactic factors bind to seven-transmembrane-domain receptors and activate heterotrimeric G-proteins. Downstream of these proteins a complex interrelated signaling network is activated in human neutrophils. Stimulation of phospholipase C beta results in activation of protein kinase C isoforms and increases in cytosolic calcium. Activation of the enzyme phosphoinositide 3-kinase results in increased production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidyl 3,4-bisphosphate. In addition, small GTP-binding proteins of the Rho family, the mitogen-activated protein kinase cascade, tyrosine kinases and protein phosphatases are activated. The enzyme phosphoinositide 3-kinase and the small cytosolic GTP-binding proteins Rho and Rac emerge as key regulators of neutrophil migration. A steep internal gradient of phosphatidylinositol 3,4,5-trisphosphate, with a high concentration in the leading lamellae, is thought to regulate polarized actin polymerization and formation of protrusions, together with Rac which may be more directly involved in initiating actin reorganization. Rho may regulate localized myosin activation, tail retraction, cell body traction and dynamics of adhesion. The impact of these different signaling pathways on reversible actin polymerization, development of polarity, reversible adhesion and migration, and the putative targets of these pathways in neutrophils, are reviewed in this article. Insight into mechanisms regulating migration of neutrophils could potentially lead to novel therapeutic strategies for counteracting chronic activation of neutrophils which leads to tissue damage.

IDENTIFICATION OF A PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE-BINDING DOMAIN IN THE N-TERMINAL REGION OF EZRIN

Purified human recombinant ezrin cosediments with large liposomes containing phosphatidylserine (PS). This interaction is optimal at low ionic strength. At physiological ionic strength (130 mM KCl) ezrin interacts strongly with liposomes containing ≥5% phosphatidylinositol‐4,5‐bisphosphate (PIP2), the residual being phosphatidylcholine (PC). When PIP2 is replaced by phosphatidylinositol‐4‐monophosphate (PIP), phosphatidylinositol (PI) or PS, the interaction is markedly reduced. Furthermore we show, that a purified N‐terminal glutathione S‐transferase (GST) fusion protein of ezrin (1–309) still has retained the capacity to interact with PIP2‐containing liposomes, whereas a C‐terminal fusion protein (310–586) has lost this ability.

RHO-KINASE IN HUMAN NEUTROPHILS : A ROLE IN SIGNALLING FOR MYOSIN LIGHT CHAIN PHOSPHORYLATION AND CELL MIGRATION

The role of a Rho‐associated coiled‐coil forming kinase in migration of neutrophils has been investigated. Rho‐associated coiled‐coil forming kinase I was expressed in human neutrophils. Chemotactic peptide led to a Rho‐associated coiled‐coil forming kinase‐dependent increase in phosphorylation of myosin light chain. This was determined with the help of an antibody directed against serine 19‐phosphorylated myosin light chain and an inhibitor of Rho‐associated coiled‐coil forming kinase (Y‐27632). Y‐27632 suppressed myosin light chain phosphorylation and chemotactic peptide‐induced development of cell polarity and locomotion with similar potency (ED50 0.5–1.1 μM). The data strongly suggest that a Rho‐associated coiled‐coil forming kinase isoform, activated in human neutrophils exposed to chemotactic peptide, is important for motile functions of these cells.

Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis.

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting. Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils. An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor.In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.

Microtubule-disruption-induced and chemotactic-peptide-induced migration of human neutrophils: implications for differential sets of signalling pathways

Neutrophil granulocytes rely on a functional actin network for directed migration. Microtubule disassembly does not impair receptor-linked chemotaxis, instead it induces development of polarity and chemokinesis in neutrophils concomitant with polarized distribution of α-actinin and F-actin. Cells stimulated with colchicine, which disassembles microtubules, migrate with a speed comparable to cells exposed to chemotactic peptide. We investigated signalling pathways involved in colchicine-induced neutrophil polarization and migration. Colchicine-induced development of polarity was insensitive to treatment with pertussis toxin, in contrast to chemotactic-peptide-induced shape changes, which were completely abolished by this treatment. Thus, colchicine does not appear to act via activating heterotrimeric Gi proteins. Colchicine does also not seem to act via phosphatidylinositol 3-kinase, as it failed to induce phosphorylation of its downstream target Akt and the potent phosphatidylinositol 3-kinase inhibitor wortmannin failed to inhibit colchicine-induced shape changes. By contrast, wortmannin significantly reduced chemotactic-peptide-induced shape changes. However, the Rho-kinase inhibitor Y-27632 (10 μM) inhibited colchicine-induced development of polarity by 95±3% (n=5) and chemokinesis by 76±9% (n=3), which suggests that the Rho-Rho-kinase pathway has a crucial role in polarity and migration. Indeed, treatment of cells with colchicine induced a significant increase in membrane-bound Rho-kinase II, which is indicative of activation of this protein. This membrane translocation could be prevented by taxol, which stabilizes microtubules. Colchicine also induced a marked increase in myosin light chain phosphorylation, which could be suppressed by Y-27632 and by taxol. In summary, we provide evidence that microtubule disassembly induces in neutrophils a selective activation of Rho-kinase, bypassing activation of heterotrimeric Gi proteins and phosphatidylinositol 3-kinase. This process is sufficient for induction of chemokinesis and mediates increased phosphorylation of myosin light chain and accumulation of F-actin and α-actinin in the leading edge.

A membrane-permeant ester of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is an activator of human neutrophil migration

Activity of phosphatidylinositol (PI) 3‐kinase is required for optimal migration of human neutrophils [Niggli and Keller (1999) Eur. J. Pharmacol. 335, 43–52]. We have tested the direct effect of a product of PI 3‐kinase, phosphatidylinositol 3,4,5‐trisphosphate (PIP3), on neutrophil migration. To this end, a membrane‐permeant ester of PIP3, dilauroyl phosphatidylinositol 3,4,5‐trisphosphate‐heptakis‐(acetooxymethyl)ester (PIP3/AM) was used. PIP3/AM (ED50: 10–17 μM) induced development of polarity and accumulation of F‐actin in the leading lamellae in up to 70% of the cells. These cells exhibited stimulated random migration, comparable to that observed in uniform concentrations of chemotactic peptide. Evidence is provided for a role of Rho‐kinase and for activation of PI 3‐kinase in a positive feedback loop in PIP3/AM‐induced motility.

The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptide-induced locomotion and increases in cytoskeletal actin in human neutrophils

To define a possible role of the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) in motile functions of neutrophils, we have used a potent inhibitor of this enzyme, [1S-(1alpha,6b alpha,9a beta,11alpha,11bbeta)]-1-(acetyloxy)-1,6b,7,8,9a,10,11 ,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno [4,5-h]-2-benzopyran-3,6,9-trione (wortmannin). Wortmannin markedly attenuated chemotactic peptide-induced development of polarity, locomotion and increases in cytoskeletal actin and alpha-actinin in human neutrophils at low, nM, concentrations (ED50 = 4-40 nM; 0.4-3 pmol/10(6) cells). The increase in cytoskeletal actin induced by phorbol-12-myristate-13-acetate in contrast was not affected by wortmannin (18 pmol/10[6] cells). Moreover, the increase in total F-actin induced by an incubation for 1 min with chemotactic peptide was much less sensitive to wortmannin than increases in cytoskeletal actin; 80 pmol/10(6) cells were necessary for half-maximal inhibition. Wortmannin thus appears to primarily affect F-actin organization, rather than polymerization. Inhibition of development of polarity by wortmannin correlated with inhibition of production of phosphatidylinositol 3,4,5-trisphosphate. According to our findings, activation of a wortmannin-sensitive target, very likely PI 3-kinase, is required for optimal chemotactic peptide-induced neutrophil motility.

Structural properties of lipid-binding sites in cytoskeletal proteins

Several cytoskeletal proteins have been shown to interact in vitro with, and in some cases are regulated by, specific membrane lipids. In some cases, evidence for in situ interactions has been provided. The molecular basis for such interactions is now being unravelled. At least five structurally distinct types of lipid-binding sites in cytoskeletal proteins have been identified. However, our understanding of the physiological role of such interactions is still limited. Precise knowledge about the binding-site structures and the actual amino acid residues involved should now enable the expression of mutant proteins that specifically lack the ability to interact with lipids. The impact of these mutations on protein location and function can then be assessed.

Rho kinase is required for CCR7-mediated polarization and chemotaxis of T lymphocytes

We studied the role of Rho kinase and extracellular signal‐regulated kinase (ERK)‐2 in the polarization and migration of T lymphocytes in response to the CCR7 ligands EBI1 ligand chemokine (ELC; CCL19) and secondary lymphoid‐tissue chemokine (SLC; CCL21). Both Rho kinase protein isoforms are expressed in T lymphocytes. Inhibition of the Rho kinases with Y‐27632 strongly inhibited SLC‐ and ELC‐induced polarized morphology and chemotaxis of T lymphocytes. Although the chemokines induced ERK‐2 activation, the blockade of this signaling pathway showed no effect on polarization and migration. This study indicates an important role of Rho kinase in CCR7‐mediated polarization and migration of T lymphocytes, whereas ERK‐2 is not involved in these processes.

Flotillins Interact with PSGL-1 in Neutrophils and, upon Stimulation, Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

Background Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. Methodology/Principal Findings We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. Conclusions/Significance These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.