Chemokine regulation of neutrophil function in tumors

The role of neutrophils in cancer and metastasis is still debated and controversial since they have been shown to be endowed with both pro- and antitumor functions. These contradictory results seem to be now explained by recent discoveries of tumor-associated neutrophils plasticity and multiple neutrophil subsets.Chemokines and chemokine receptors are known to tightly regulate the release of neutrophils from the bone marrow, their passage into circulation and transmigration into the tissues as well as tumor infiltration. It is emerging that chemokine receptors are differentially expressed by neutrophil subsets and they affect not only their recruitment but also their effector functions.Here we are resuming human and murine data suggesting that therapeutic modulation of neutrophil activity through the targeting of specific chemokines or chemokine receptors can improve their anti-tumoral properties.     

Invasion and survival strategies of Anaplasma phagocytophilum

Anaplasma phagocytophilum is an aetiological agent of human granulocytic ehrlichiosis, an emerging tick‐borne zoonosis in the United States and Europe. This obligate intracellular bacterium is unique in that it colonizes polymorphonuclear leucocytes (neutrophils). Neutrophils are key players in innate immunity. These short‐lived phagocytes ingest invading microorganisms and destroy them by various means, which include fusing the bacteria‐containing phagosome with acidic lysosomes as well as directing toxic oxidative and proteolytic compounds into the phagosomal lumen. Its tropism for neutrophils indicates that A. phagocytophilum uses strategies for evading and/or neutralizing these microbicidal activities. This review focuses on some of the mechanisms that A. phagocytophilum uses for neutrophil adhesion, surviving within the hostile intracellular environment of its host neutrophil and for effectively disseminating to naïve host cells.     

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.     

Temporal adaptation of neutrophil oxidative responsiveness to n-formyl-methionyl-leucyl-phenylalanine. Acceleration by granulocyte-macrophage colony stimulating factor

This investigation was undertaken to clarify the mechanism by which purified recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) potentiates neutrophil oxidative responses triggered by the chemotactic peptide, FMLP. Previous studies have shown that GM-CSF priming of neutrophil responses to FMLP is induced relatively slowly, requiring 90 to 120 min of incubation in vitro, is not associated with increased levels of cytoplasmic free Ca2+, but is associated with up-regulation of cell-surface FMLP receptors. We have confirmed these findings and further characterized the process of GM-CSF priming. We found that the effect of GM-CSF on neutrophil oxidative responsiveness was induced in a temperature-dependent manner and was not reversed when the cells were washed extensively to remove the growth factor before stimulation with FMLP. Extracellular Ca2+ was not required for functional enhancement by GM-CSF and GM-CSF alone effected no detectable alteration in the 32P-labeled phospholipid content of neutrophils during incubation in vitro. Our data indicate that GM-CSF exerts its influence on neutrophils by accelerating a process that occurs spontaneously and results in up-regulation of both cell-surface FMLP receptors and oxidative responsiveness to FMLP. Thus, the results demonstrate that, with respect to oxidative activation, circulating endstage polymorphonuclear leukocytes are nonresponsive or hyporesponsive to FMLP; functional responsiveness increases dramatically as surface FMLP receptors are gradually deployed after the cells leave the circulation. Thus, as neutrophils mature, their responsiveness to FMLP changes in a manner which may be crucial for efficient host defense. At 37 degrees C, this process is markedly potentiated by GM-CSF. We conclude that endogenous GM-CSF, released systemically or at sites of infection and inflammation, potentially plays an important role in host defense by accelerating functional maturation of responding polymorphonuclear leukocytes.     

Angiostatin inhibits activation and migration of neutrophils

There is a critical need to identify molecules that modulate the biology of neutrophils because activated neutrophils, though necessary for host defense, cause exuberant tissue damage through production of reactive oxygen species and increased lifespan. Angiostatin, an endogenous anti-angiogenic cleavage product of plasminogen, binds to integrin α_vβ₃, ATP synthase and angiomotin and its expression is increased in inflammatory conditions. We test the hypothesis that angiostatin inhibits neutrophil activation, induces apoptosis and blocks recruitment in vivo and in vitro. The data show immuno-reactivity for plasminogen/angiostatin in resting neutrophils. Angiostatin conjugated to FITC revealed that angiostatin was endocytozed by activated mouse and human neutrophils in a lipid raft-dependent fashion. Co-immunoprecipitation of human neutrophil lysates, confocal microscopy of isolated mouse and human neutrophils and functional blocking experiments showed that angiostatin complexes with flotillin-1 along with integrin α_vβ₃ and ATP synthase. Angiostatin inhibited fMLP-induced neutrophil polarization, as well as caused inhibition of hsp-27 phosphorylation and stabilization of microtubules. Angiostatin treatment, before or after LPS-induced neutrophil activation, inhibited phosphorylation of p38 and p44/42 MAPKs, abolished reactive oxygen species production and released the neutrophils from suppressed apoptosis, as indicated by expression of activated caspase-3 and morphological evidence of apoptosis. Finally, intravital microscopy and myeloperoxidase assay showed inhibition of neutrophil recruitment in post-capillary venules of TNFα-treated cremaster muscle in mouse. These in vitro and in vivo data demonstrate angiostatin as a broad deactivator and silencer of neutrophils and an inhibitor of their migration. These data potentially open new avenues for the development of anti-inflammatory drugs.     

The αMβ2 integrin and its role in neutrophil function

Neutrophils are the first cell type to arrive at the injury sites and play a critical role in host defense,by virtue of its ability to adhere and transmigrate through endothelium,to phagocytose foreign pathogens,and to produce free oxygen radicals and proteolytic enzymes.Yet,inappropriate neutrophil activation causes tissue damage and various inflammatory diseases.These physiological and pathological functions of neutrophils depend on the engagement of certain surface receptors,especially αMβ2,the major β2 integrin receptor present on neutrophil surface.Understanding of the molecular mechanisms underlying ligand binding by αMβ2,as well as the rolea of αMβ2ligand interactions in neutrophil functions will enable us to regulate more precisely neutrophil activities:that is,to promote their host defense functions,and at the same time to minimize their deleterious effects of normal cells.     

Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.     

The paradox of tumor-associated neutrophils: Fueling tumor growth with cytotoxic substances

Human cancers are comprised of numerous cell types including neutrophils. Although often ignored, neutrophils are frequently present at sites of tumorigenesis including the kidney, breast, colon, and lung. These cells possess substances such as reactive oxygen species and proteinases that are capable of modifying tumor growth and invasiveness. This review addresses recent reports describing both pro-host and pro-tumor roles for neutrophils and neutrophil-derived substances and will examine the alterations in neutrophil behavior that explain this apparent biological discrepancy. Unfortunately, with the exception of investigator driven manipulation of neutrophil function, these cells function overwhelmingly against the host in the setting of cancer. Many cancers are capable of recruiting neutrophils to sites of tumorigenesis where they enhance tumor growth. Identification of the neutrophil-derived substances that promote tumor growth may yield novel therapeutic approaches to inhibit cancer progression. Alternatively, strategies designed to generate highly activated and cytotoxic neutrophils within the tumor microenvironment may provide a means to unleash the tumoricidal potential of the host’s innate immune response.     

The reactivity of the circulating neutrophils in human peripheral blood]

Using cytochemical, biochemical and disc-electrophoretic methods, the degree of extracellular secretion of peroxidase-containing neutrophil granules has been investigated as an index of their functional activity when in contact with antigens of extra- and intra-circulation. It was established that in in vitro contacts of neutrophils with alive and killed microbe culture St. aur., the quantity of the granules in the cells decreased as well as the enzyme activity in them. This is partially due to extracellular secretion of the granules content confirmed by the presence of peroxidase fractions in the solution. Similar results have been obtained for circulating neutrophils and serum of patients with acute pneumonia at the height of the disease. It is hold that antigen-induced extra- and intracellular neutrophil degranulation in the peripheral human blood reflects functional activity of the neutrophils.     

Human Neutrophil Cytoskeletal Dynamics and Contractility Actively Contribute to Trans-Endothelial Migration

Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which “opens the door” in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.     

Human neutrophil defensins increase neutrophil uptake of influenza A virus and bacteria and modify virus-induced respiratory burst responses

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.     

Signal integration in forward and reverse neutrophil migration: Fundamentals and emerging mechanisms

Neutrophils migrate to sites of tissue damage, where they protect the host against pathogens. Often, the cost of these neutrophil defenses is collateral damage to healthy tissues. Thus, the immune system has evolved multiple mechanisms to regulate neutrophil migration. One of these mechanisms is reverse migration — the process whereby neutrophils leave the source of inflammation. In vivo, neutrophils arrive and depart the wound simultaneously — indicating that neutrophils dynamically integrate conflicting signals to engage in forward and reverse migration. This finding is seemingly at odds with the established chemoattractant hierarchy in vitro, which places wound-derived signals at the top. Here we will discuss recent work that has uncovered key players involved in retaining and dispersing neutrophils from wounds. These findings offer the opportunity to integrate established and emerging mechanisms into a holistic model for neutrophil migration in vivo.     

New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and In Vitro Generation

Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.     

Apoptosis and macrophage clearance of neutrophils: regulation by reactive oxygen species

Inflammation is a beneficial host response to foreign challenge involving numerous soluble factors and cell types, including polymorphonuclear granulocytes or neutrophils. Programmed cell death (apoptosis) of neutrophils has been documented in vitro as well as in vivo, and is thought to be important for the resolution of inflammation, as this process allows for engulfment and removal of senescent cells prior to their necrotic disintegration. Studies in recent years have begun to unravel the mechanism of macrophage clearance of apoptotic cells, and evidence has accrued for a critical role of externalization and oxidation of plasma membrane phosphatidylserine, and its subsequent recognition by macrophage receptors, in this process. Activated neutrophils generate vast amounts of reactive oxygen species for the purpose of killing ingested micro-organisms, and these reactive metabolites may also modulate the life-span, as well as the clearance, of the neutrophil itself. This review aims to address the latter topic, as well as to summarize current knowledge on the molecular mechanisms of neutrophil apoptosis and macrophage clearance of these cells at the site of inflammation.     

G-CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity

Uropathogenic Escherichia coli (UPEC), the causative agent of approximately 85% of urinary tract infections (UTI), is a major health concern primarily affecting women. During infection, neutrophils infiltrate the bladder, but the mechanism of recruitment is not well understood. Here, we investigated the role of UPEC-induced cytokine production in neutrophil recruitment and UTI progression. We first examined the kinetics of cytokine expression during UPEC infection of the bladder, and their contribution to neutrophil recruitment. We found that UPEC infection induces expression of several pro-inflammatory cytokines including granulocyte colony-stimulating factor (G-CSF, CSF-3), not previously known to be involved in the host response to UTI. G-CSF induces neutrophil emigration from the bone marrow; these cells are thought to be critical for bacterial clearance during infection. Upon neutralization of G-CSF during UPEC infection, we found fewer circulating neutrophils, decreased neutrophil infiltration into the bladder and, paradoxically, a decreased bacterial burden in the bladder. However, depletion of G-CSF resulted in a corresponding increase in macrophage-activating cytokines, such as monocyte chemotactic protein-1 (MCP-1, CCL-2) and Il-1beta, which may be key in host response to UPEC infection, potentially resolving the paradoxical decreased bacterial burden. Thus, G-CSF acts in a previously unrecognized role to modulate the host inflammatory response during UPEC infection.     

Acute-phase protein α1-antitrypsin inhibits neutrophil calpain I and induces random migration

A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a better understanding of the mechanisms underlying neutrophil influx and activation is of critical importance. We have previously shown that the acute phase protein α1-antitrypsin (AAT) inhibits neutrophil chemotaxis. In this study, we examine mechanisms related to the effect of AAT on neutrophil responses. We report a previously unknown function of AAT to inactivate calpain I (μ-calpain) and to induce a rapid cell polarization and random migration. These effects of AAT coincided with a transient rise in intracellular calcium, increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extra-cellular signal-regulated kinase (ERK1/2). Furthermore, AAT caused a significant inhibition of nonstimulated as well as formyl-met-leu-phe (fMLP)-stimulated neutrophil adhesion to fibronectin, strongly inhibited lipopolysaccharide-induced IL-8 release and slightly delayed neutrophil apoptosis. The results presented here broaden our understanding of the regulation of calpain-related neutrophil functional activities, and provide the impetus for new studies to define the role of AAT and other acute phase proteins in health and disease.     

The role of the neutrophil in inflammatory diseases of the lung

Under certain circumstances, the neutrophil has been implicated in causing disease by damaging normal host tissue. This may occur in the adult respiratory distress syndrome (ARDS). The neutrophil has been implicated since a) substances that activate neutrophils are produced in association with the predisposing risks that lead to ARDS; b) activated neutrophils migrate into the alveolar spaces and their toxic products can be found in lung lavage fluid and in the breath of patients with ARDS; and c) the magnitude of the physiologic alterations correlate with the number of neutrophils in the alveolar space. Additionally, the neutrophils may be primed by substances which are released by activated platelets within the confines of the lung. Both platelet adenine nucleotides and the platelet-derived extracellular matrix protein (ECM), thrombospondin, can prime the neutrophil for subsequent O2- generation following activation of the cells with the chemotactic peptide, F-met-leu-phe (FMLP). Furthermore, neutrophils can be primed or O2- generation by the basement membrane ECM protein, laminin. Since neutrophils express receptors for both laminin and thrombospondin, these constituents may serve to modulate neutrophil behavior for subsequent oxidative metabolism and contribute to exacerbating pulmonary disease.     

Neutrophil granulocytes--Trojan horses for Leishmania major and other intracellular microbes?

Polymorphonuclear neutrophil granulocytes (PMNs) possess numerous effector mechanisms to kill ingested pathogens as the first line of defence. However, several microorganisms evade intracellular killing in neutrophils, survive and retain infectivity. There is increasing evidence that several pathogens even multiply within neutrophils. Taking Leishmania major as a prototypic intracellular pathogen, we suggest an evasion strategy that includes the manipulation of PMNs in such a way that the pathogens are able to use the granulocytes as host cells. The ability to survive and maintain infectivity in PMNs subsequently enables these organisms to establish productive infection. These organisms can use granulocytes as Trojan horses before they enter their definitive host cells, the macrophages.