Regulation of neutrophil apoptosis: A role for protein kinase C and phosphatidylinositol-3-kinase

Neutrophils play a central role in host defense and are recruited in vast numbers to sites of infection where they phagocytose and kill invading bacterial pathogens. Neutrophils have a short half-life that is extended at the inflamed site by pro-inflammatory cytokines and contact with bacterial cell walls. Normal resolution of inflammation involves the removal of neutrophils and other inflammatory cells by the induction of apoptosis. Spontaneous neutrophil apoptosis does not require Fas ligation, but is mediated by caspases 3, 8 and possibly caspase 9 and also involves activation of protein kinase C-. With chronic inflammatory disease, neutrophil apoptosis is delayed by pro-inflammatory cytokines, leading to persistence of neutrophils at the inflamed site and non-specific tissue damage. Here we discuss the evidence for inhibition of neutrophil apoptosis via signaling though PI-3-kinase and downstream pathways, including PDK-1 and PKB. Therapeutic strategies to resolve chronic inflammation could therefore usefully target neutrophil apoptosis and the PI-3-kinase or PKC- signaling pathways.     

Postsynaptic scaffold protein Homer 1a protects against traumatic brain injury via regulating group I metabotropic glutamate receptors

Traumatic brain injury (TBI) produces excessive glutamate, leading to excitotoxicity via the activation of glutamate receptors. Postsynaptic density scaffold proteins have crucial roles in mediating signal transduction from glutamate receptors to their downstream mediators. Therefore, studies on the mechanisms underlying regulation of excitotoxicity by scaffold proteins can uncover new treatments for TBI. Here, we demonstrated that the postsynaptic scaffold protein Homer 1a was neuroprotective against TBI in vitro and in vivo, and this neuroprotection was associated with its effects on group I metabotropic glutamate receptors (mGluRs). Upon further study, we found that Homer 1a mainly affected neuronal injury induced by mGluR1 activation after TBI and also influenced mGluR5 function when its activity was restored. The ability of Homer 1a to disrupt mGluR-ERK signaling contributed to its ability to regulate the functions of mGluR1 and mGluR5 after traumatic injury. Intracellular Ca²⁺ and PKC were two important factors involved in the mediation of mGluR-ERK signaling by Homer 1a. These results define Homer 1a as a novel endogenous neuroprotective agent against TBI.     

Rho signaling inhibition mitigates lung injury via targeting neutrophil recruitment and selectin-AKT signaling

Neutrophils, the early responders of the immune system, eliminate intruders, but their over-activation can also instigate tissue damage leading to various autoimmune and inflammatory disease conditions. As approaches causing neutropenia are associated with immunodeficiency, targeting aberrant neutrophil infiltration offers an attractive strategy in neutrophil-centered diseases including acute lung injury. Rho GTPase family proteins Rho, Rac and Cdc42 play important role as regulators of chemotaxis in diverse systems. Rho inhibitors protected against lung injuries, while genetic Rho-deficiency exhibited neutrophil hyperactivity and exacerbated lung injury. These differential outcomes might be due to distinct effects on different cell types or activation/ inhibition of specific signaling pathways responsible for neutrophil polarity, migration and functions. In this study, we explored neutrophil centric effects of Rho signaling mitigation. Consistent with previous reports, Rho signaling inhibitor Y-27632 provided protection against acute lung injury, but without regulating LPS mediated systemic increase of neutrophils in the circulation. Interestingly, the adoptive transfer approach identified a specific defect in neutrophil migration capacity after Rho signaling mitigation. These defects were associated with loss of polarity and altered actin dynamics identified using time-lapse in vitro studies. Further analysis revealed a rescue of stimulation-dependent L-selectin shedding on neutrophils with Rho signaling inhibitor. Surprisingly, functional blocking of L-selectin (CD62L) led to defective recruitment of neutrophils into inflamed lungs. Further, single-cell level analyses identified MAPK signaling as downstream mechanism of Rho signaling and L-selectin mediated effects. p-AKT levels were diminished in detergent resistance membrane-associated signalosome upon Rho signaling inhibition and blockade of selectin. Moreover, inhibition of AKT signaling as well as selectin blocking led to defects in neutrophil polarity. Together, this study identified Rho-dependent distinct L-selectin and AKT signaling mediated regulation of neutrophil recruitment to inflamed lung tissue.     

A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.     

Neutrophil integrin affinity regulation in adhesion,migration, and bacterial clearance

During an infection, neutrophils are the first immune cells to arrive armed to clear the invading pathogen. In order to do so, neutrophils need to transmigrate from the peripheral blood through the endothelial layer toward the site of inflammation. This process is in most cases dependent on integrins, adhesion molecules present on all immune cells. These molecules are functionally regulated by “inside-out” signaling, where stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs and many factors have been linked to regulation of integrins on neutrophils. Control of integrin conformation and clustering is of pivotal importance for proper cell adhesion, migration, and bacterial clearance. Recently, gelsolin was found to be involved in β₁-integrin affinity regulation and cell adhesion. Here, I summarize the role of neutrophil integrin regulation in the essential steps to reach the site of inflammation and clearance of bacterial pathogens.     

Noncompetitive inhibition of hepatocyte growth factor-dependent Met signaling by a phage-derived peptide

Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like β-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF β-chain (HGF β) and competitively inhibited binding to Met with an IC₅₀ of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC₅₀ of ∼ 20 μM. The 2D ¹H-NMR structure of HB10 revealed a β-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF β mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF β domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.     

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-α, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D₂ (PGD₂) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD₃. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD₂ receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD₂ signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.     

Down-regulation of glutamine synthetase enhances migration of rat astrocytes after in vitro injury

Astrocytes undergo reactive transformation in response to physical injury (reactive gliosis) that may impede neural repair. Glutamine synthetase (GS) is highly expressed by astrocytes, and serves a neuroprotective function by converting cytotoxic glutamate and ammonia into glutamine. Glutamine synthetase was down-regulated in reactive astrocytes at the site of mechanical spinal cord injury (SCI) and in cultured astrocytes at the margins of a scratch wound, suggesting that GS may modulate reactive transformation and glial scar development. We evaluated this potential function of GS using siRNA-mediated GS knock-down. Suppression of astrocytic GS by GS siRNA increased cell migration into the scratch wound zone and decreased substrate adhesion as indicated by the number of focal adhesions expressing the adaptor protein paxillin. Migration was enhanced by glutamine and suppressed by glutamate, in contrast to the result expected if enhanced migration was due solely to changes in glutamine and glutamate concomitant with reduced GS activity. The membrane type 1-matrix metalloproteinase (MT1-MMP) was up-regulated in GS siRNA-treated astrocytes, while a broad-spectrum MMP antagonist inhibited migration in both wild type and GS knock-down astrocytes. In addition, GS siRNA inhibited expression of integrin β1, while antibody-mediated inhibition of integrin β1 impaired direction-specific protrusion and motility. Thus, GS may modulate motility and substrate adhesion through transmembrane integrin β1 signaling to the cytoskeleton and by MMT-mediated proteolysis of the extracellular matrix.     

Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20 days on 3 μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β₂-integrins, but not β1- or β₃-integrins. Migration from the subendothelial compartment was supported by β1- and β₂-integrins for all cultures, but blockade of β₃-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β₃-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.     

β-Arrestin mediates oxytocin receptor signaling, which regulates uterine contractility and cellular migration

Desensitization of the oxytocin receptor (OXTR) in the setting of prolonged oxytocin exposure may lead to dysfunctional labor, which increases the risk for cesarean delivery, and uterine atony, which may result in postpartum hemorrhage. The molecular mechanism for OXTR desensitization is through the agonist-mediated recruitment of the multifunctional protein β-arrestin. In addition to its desensitizing function, β-arrestins have recently been shown to simultaneously activate downstream signaling. We tested whether oxytocin stimulation promotes β-arrestin-mediated OXTR desensitization in vivo and activates β-arrestin-mediated mitogen-activated protein kinase (MAPK) growth signaling. Uterine muscle strips isolated from wild-type mice exhibited diminished uterine contractility following repeated exposure to oxytocin, whereas uterine muscle strips from β-arrestin-1 and β-arrestin-2 knockout mice showed no desensitization. Utilizing siRNA knockdown of β-arrestin-1 and β-arrestin-2 in HEK-293 cells expressing the OXTR, we demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Wild-type and β-arrestin-1 and β-arrestin-2 knockout mice receiving intravenous oxytocin also demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Finally, to test the significance of β-arrestin-mediated signaling from the OXTR, HEK-293 cells expressing the OXTR showed β-arrestin-dependent proliferation in a cell migration assay following oxytocin treatment. In conclusion, β-arrestin is a multifunctional scaffold protein that mediates both desensitization of the OXTR, leading to decreases in uterine contractility, and MAPK growth signaling following stimulation by oxytocin. The development of unique OXTR ligands that prevent receptor desensitization may be a novel approach in the treatment of adverse clinical events secondary to prolonged oxytocin therapy.     

HIV-1 Triggers WAVE2 Phosphorylation in Primary CD4 T Cells and Macrophages,Mediating Arp2/3-dependent Nuclear Migration

The human immunodeficiency virus type 1 (HIV-1) initiates receptor signaling and early actin dynamics during viral entry. This process is required for viral infection of primary targets such as resting CD4 T cells. WAVE2 is a component of a multiprotein complex linking receptor signaling to dynamic remodeling of the actin cytoskeleton. WAVE2 directly activates Arp2/3, leading to actin nucleation and filament branching. Although several bacterial and viral pathogens target Arp2/3 for intracellular mobility, it remains unknown whether HIV-1 actively modulates the Arp2/3 complex through virus-mediated receptor signal transduction. Here we report that HIV-1 triggers WAVE2 phosphorylation at serine 351 through gp120 binding to the chemokine coreceptor CXCR4 or CCR5 during entry. This phosphorylation event involves both Gα_i-dependent and -independent pathways, and is conserved both in X4 and R5 viral infection of resting CD4 T cells and primary macrophages. We further demonstrate that inhibition of WAVE2-mediated Arp2/3 activity through stable shRNA knockdown of Arp3 dramatically diminished HIV-1 infection of CD4 T cells, preventing viral nuclear migration. Inhibition of Arp2/3 through a specific inhibitor, CK548, also drastically inhibited HIV-1 nuclear migration and infection of CD4 T cells. Our results suggest that Arp2/3 and the upstream regulator, WAVE2, are essential co-factors hijacked by HIV for intracellular migration, and may serve as novel targets to prevent HIV transmission.     

Signaling pathways involving adenosine A<Subscript>2A</Subscript> and A<Subscript>2B</Subscript> receptors in wound healing and fibrosis

Collagen and matrix deposition by fibroblasts is an essential part of wound healing but also contributes to pathologic remodeling of organs leading to substantial morbidity and mortality. Adenosine, a small molecule generated extracellularly from adenine nucleotides as a result of direct stimulation, hypoxia, or injury, acts via a family of classical seven-pass G protein-coupled protein receptors, A_2A and A_2B, leading to generation of cAMP and activation of downstream targets such as PKA and Epac. These effectors, in turn, lead to fibroblast activation and collagen synthesis. The regulatory actions of these receptors likely involve multiple interconnected pathways, and one of the more interesting aspects of this regulation is opposing effects at different levels of cAMP generated. Additionally, adenosine signaling contributes to fibrosis in organ-specific ways and may have opposite effects in different organs. The development of drugs that selectively target these receptors and their signaling pathways will disrupt the pathogenesis of fibrosis and slow or arrest the progression of the important diseases they underlie.     

Optimal intensity shock wave promotes the adhesion and migration of rat osteoblasts via integrin β1-mediated expression of phosphorylated focal adhesion kinase

To search for factors promoting bone fracture repair, we investigated the effects of extracorporeal shock wave (ESW) on the adhesion, spreading, and migration of osteoblasts and its specific underlying cellular mechanisms. After a single period of stimulation by 10 kV (500 impulses) of shock wave (SW), the adhesion rate was increased as compared with the vehicle control. The data from both wound healing and transwell tests confirmed an acceleration in the migration of osteoblasts by SW treatment. RT-PCR, flow cytometry, and Western blotting showed that SW rapidly increased the surface expression of α5 and β1 subunit integrins, indicating that integrin β1 acted as an early signal for ESW-induced osteoblast adhesion and migration. It has also been found that a significant elevation occurred in the expression of phosphorylated β-catenin and focal adhesion kinase (FAK) at the site of tyrosine 397 in response to SW stimulation after the increasing expression of the integrin β1 molecule. When siRNAs of integrin α5 and β1 subunit were added, the level of FAK phosphorylation elevated by SW declined. Interestingly, the adhesion and migration of osteoblasts were decreased when these siRNA reagents as well as the ERK1/2 signaling pathway inhibitors, U0126 and PD98059, were present. Further studies demonstrated that U0126 could inhibit the downstream integrin-dependent signaling pathways, such as the FAK signaling pathway, whereas it had no influence on the synthesis of integrin β1 molecule. In conclusion, these data suggest that ESW promotes the adhesion and migration of osteoblasts via integrin β1-mediated expression of phosphorylated FAK at the Tyr-397 site; in addition, ERK1/2 are also important for osteoblast adhesion, spreading, migration, and integrin expression.     

A Highlights from MBoC Selection: Mammalian target of rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton

Chemotaxis allows neutrophils to seek out sites of infection and inflammation. The asymmetric accumulation of filamentous actin (F-actin) at the leading edge provides the driving force for protrusion and is essential for the development and maintenance of neutrophil polarity. The mechanism that governs actin cytoskeleton dynamics and assembly in neutrophils has been extensively explored and is still not fully understood. By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis. Depletion of mTOR and Rictor, but not Raptor, impairs actin polymerization, leading-edge establishment, and directional migration in neutrophils stimulated with chemoattractants. Of interest, depletion of mSin1, an integral component of mTORC2, causes no detectable defects in neutrophil polarity and chemotaxis. In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis. Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils. Together our findings reveal an mTORC2- and mTOR kinase–independent function and mechanism of Rictor in the regulation of neutrophil chemotaxis.     

CHIP-MYTH: A novel interactive proteomics method for the assessment of agonist-dependent interactions of the human β2-adrenergic receptor

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human β₂-adrenergic receptor (β₂AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting β₂AR-agonist. Our results suggest that β₂AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαβγ, into Gα and Gβγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel β₂AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.     

Novel neutrophil inhibitory factor homologue in the buccal gland secretion of Lampetra japonica

Abstract To identify the functional gene fragment, a neutrophil inhibitory factor (NIF) like protein was found in the buccal gland of Lampetra japonica, suggesting that this related lamprey protein represents a novel class of integrin receptor antagonists. The recombinant Lampetra japonica-NIF like (rLj-NIF) was identified by SDS-PAGE and purified by using His·Bind affinity chromatography. Effect of rLj-NIF on neutrophil migration suggested that rLj-NIF can act as a neutrophil inhibitory factor. Besides that, oxidative burst activity of neutriphil was tested by flow cytometry using dihydrorhodamine (DHR123) as a fluorogenic substrate, and the data suggested that the mean fluorescence intensity significantly decreased compared with positive controls (p<0.01). All above results indicated that rLj-NIF could also prevent the binding of β2 integrins to the surface of PMN and its FITC-labeled monoclonal antibodies (p<0.05). These data suggest that Lampetra japonica-NIF like protein is secreted by the stage of the parasite at the site of attachment. rLj-NIF plays an essential role in physiological reaction of neutrophil by a novel class of β2 integrin receptor antagonists. The activity of immunosuppressant of L. japonica-NIF could have potential medicinal value in anti-inflammation and therapy of autoimmune diseases.