Eileen Uribe-Querol

Neutrophils in Cancer: Two Sides of the Same Coin

Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs) have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Neutrophils: Their Role in Innate and Adaptive Immunity

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes in the blood and constitute the first line of host defense against numerous infectious pathogens, including bacteria, fungi, and protozoa. Neutrophils are the first leukocytes to migrate from the blood to injured or infected sites for killing pathogens and removing cellular debris. Neutrophils migrate to sites of inflammation and infection where they recognize and phagocytose invading microorganisms, in order to kill them via different cytotoxic mechanisms. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of neutrophils migrating toward chemoattractants. Once at sites of infection, neutrophils actively phagocytose microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens. Association of the nicotinamide adenine dinucleotide phosphate (NADPH) reduced oxidase complex at the phagosomal membrane for the production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. In recent years, it has become evident that neutrophils not only have a fundamental role in the acute phase of inflammation when they actively eliminate pathogens, but also are capable of modifying the overall immune response. Neutrophils can do this by exchanging information with macrophages, dendritic cells, and other cells of the adaptive immune system through either soluble mediators or direct cell-cell contact. To illuminate the complex role of neutrophils in infection, inflammation, and immunity, this special issue has gathered original and review articles that will help us expand our knowledge on neutrophil biology. As stated before, any neutrophil response begins with migration of these leukocytes to the site of infection or inflammation. Chemotaxis, the directional movement of the cell guided by extracellular chemoattractant gradients, plays an essential role in the recruitment of neutrophils to sites of inflammation. Chemotaxis is mediated by G protein-coupled receptor (GPCR) signaling pathway. The article by X. Xu and T. Jin describes the novel functions of the PLC/PKC/PKD signaling axis in GPCR-mediated chemotaxis of neutrophils. Similarly, the review by J. Gamara et al. explains how the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) participate in GPCR signaling cascades regulating several neutrophil functional responses. The various cell responses to different chemoattractants are highlighted by the studies of R. Vaivoda et al. and M. A. Hidalgo et al. R. Vaivoda et al. describe how the deficiency of CYP4F18, an enzyme that catalyzes hydroxylation of leukotriene B4 (LTB4), does not alter the chemotactic response to LTB4 but causes a twofold increase response to complement component C5a. M. A. Hidalgo et al. describe that although N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Platelet Activating Factor (PAF) induce similar intracellular signaling profiles, only fMLF induces interleukin-8 (IL-8) release and NADPH oxidase activity in neutrophils. Several microbicidal functions of neutrophils involve the activation of the NADPH oxidase complex for production of reactive oxygen species (ROS) to mediate pathogen killing. The interesting review by R. C. Allen describes the principles of particle physics and quantum mechanics to develop a fundamental explanation of neutrophil microbicidal metabolism based on ROS atomic properties. Myeloperoxidase, the most abundant neutrophil-granule protein, is known to have potent microbicidal properties. But, recently it has become apparent that myeloperoxidase also participates in regulating adaptive immune responses. The review by D. Odobasic et al. presents an overview on how this enzyme has key roles in various functions of neutrophils in innate and adaptive immunity. When neutrophils cannot kill microorganisms by the classical phagocytosis or degranulation mechanisms, they can also form neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins and are induced by several pathogens and also some pharmacological stimuli. Antigen-antibody complexes can also induce NET formation. The paper by O. R. Aleman et al. explores direct stimulation of individual Fcγ receptors to induce NET formation and finds that only FcγRIIIb cross-linking induced NET formation in a NADPH oxidase-, PKC-, and ERK-dependent fashion. An intriguing observation on the microbicidal function of neutrophils of older women is reported by J. Bartholomeu-Neto et al. They find that phagocytic and oxidative activities of neutrophils of healthy older women that exercise regularly are higher than those of sedentary older women. The physical condition of each individual was a significant predictor of phagocytosis potential. Clearly, regular exercise contributes to a better innate immune system. However, no mechanism for this beneficial effect is known. This should be an interesting line of future neutrophil research. Neutrophils are potent regulators of inflammation via the release of proinflammatory factors and several cytokines. The paper by M. R. Tardif et al. describes an alternative secretion pathway in neutrophils for the release of the S100A8/A9 (calprotectin) and S100A12, proinflammatory mediators. The secretion of these cytoplasmic proteins was dependent on the production of ROS and required K+ exchanges through ATP-sensitive K+ channels. Because neutrophils can also cause tissue damage, their downregulation at the inflammatory site is required for proper resolution of inflammation. The paper by M. A. Sugimoto et al. describes how Annexin A1, an endogenous glucocorticoid-regulated protein, inhibits neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. Annexin A1 also induced macrophage reprogramming toward a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules. Similarly, the paper by M. Cohen-Mazor et al. describes how heparin binds to activated neutrophils and induces apoptosis. These results provide an explanation for the long-known anti-inflammatory effects of heparin. Neutrophils seem to have dual roles in promoting and controlling inflammation. The mechanisms that control the final outcome are not completely described, but these opposite functions must be tightly balanced. During sepsis, neutrophils are responsible for both the release of cytokines and the phagocytosis of pathogens. But, in SIRS (systemic inflammatory response syndrome), neutrophils contribute to maintaining of a whole-body inflammatory state. In the review by H. Fang et al., the role of neutrophils in these two clinical conditions is described and the therapeutic effect of G-CSF in sepsis is discussed in relation to its function of regulating neutrophil blood levels. Moreover, because neutrophil-derived products can regulate the action of other immune cells and can contribute to the development and chronicity of inflammatory diseases, I. Naegelen et al. propose in their article an original strategy based on linear fitting, to analyze the link between cytokine release and degranulation time. This method could find correlations between granule proteins and cytokines secreted to the inflammatory site. The idea is to be able in the future to predict the type of inflammatory response that neutrophils could induce under certain conditions. In addition, C. F. M. Morris et al. describe in their review how two apparent opposite models of inflammation may be compatible in the outcome of inflammation. The two-hit model states that a first injury (i.e., hit) can serve as a priming event which sequential insults can build on, culminating in a disproportioned inflammatory response to injury. On the other hand, the ischemic preconditioning (IPC) model states that a mild ischemic event, either remote or local, can be protective and can actually attenuate the inflammatory response to the following insults. This article tries to reconcile both models and presents evidence that each of them brings its own unique perspective onto the biology of inflammation. Finally, the dual role of neutrophil function during inflammation is emphasized again in the review by E. Uribe-Querol et al., where the protumor or antitumor character of the tumor-associated neutrophils (TANs) is revised. Recent findings on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions are presented. These articles together represent the fascinating flexible functions of neutrophils not only in fighting infections, but also in shaping the immune response and the consequences this may have to important health issues such as resolution of inflammation, autoimmunity, and cancer. Carlos Rosales Nicolas Demaurex Clifford A. Lowell Eileen Uribe-Querol

Phagocytosis: A Fundamental Process in Immunity

One hundred years have passed since the death of Élie Metchnikoff (1845–1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood.

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen–antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Similar Synapse Density in Layer IV Columns of the Primary Somatosensory Cortex of Transgenic Mice with Different Brain Size: Implications for Mechanisms Underlying the Differential Allocation of Cortical Space

The relative dimension of the areas constituting the cerebral cortex differs greatly in the brains of different mammalian species. The mechanisms by which such an evolutionary remodeling has occurred is not well understood. To begin exploring possible mechanisms, we took advantage of a transgenic mouse model in which the area of the primary somatosensory cortex (S1) shifts, to some extent independent from the area of the cortex as a whole, as a result of differences in the availability of insulin-like growth factor I (IGF-I). Electron microscopy estimations of synapse density in D3 and C3 cortical columns of the S1 layer IV revealed that this parameter was similar among wild type and transgenic mice with higher and lower availability of IGF-I. Because D3 and C3 columns were larger and smaller than normal in mice with higher and lower IGF-I availability, the total number of synapses contained in the average area of D3 and C3 columns increased and decreased, respectively. No differences in the number and overall arrangement of S1 columns were observed among animal groups. These results suggest that: 1) synapse density is a constant factor within the S1 cortical column structure; 2) the mechanisms and factors regulating cell number and synaptogenesis are affected as columns and cortical areas modify their relative dimensions; 3) altered availability of neurotrophic factors might be associated with changes in areal dimensions; and 4) changes in cortical areal dimensions within single lineages might result from the addition of minicolumns to preexisting columns.

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophil Functions in Periodontal Homeostasis

Oral tissues are constantly exposed to damage from the mechanical effort of eating and to microorganisms, mostly bacteria. In healthy gingiva tissue remodeling and a balance between bacteria and innate immune cells are maintained. However, excess of bacteria biofilm (plaque) creates an inflammation state that recruits more immune cells, mainly neutrophils to the gingiva. Neutrophils create a barrier for bacteria to reach inside tissues. When neutrophils are insufficient, bacteria thrive causing more inflammation that has been associated with systemic effects on other conditions such as atherosclerosis, diabetes, and cancer. But paradoxically when neutrophils persist, they can also promote a chronic inflammatory state that leads to periodontitis, a condition that leads to damage of the bone-supporting tissues. In periodontitis, bone loss is a serious complication. How a neutrophil balance is needed for maintaining healthy oral tissues is the focus of this review. We present recent evidence on how alterations in neutrophil number and function can lead to inflammatory bone loss, and how some oral bacteria signal neutrophils to block their antimicrobial functions and promote an inflammatory state. Also, based on this new information, novel therapeutic approaches are discussed.

Control of Phagocytosis by Microbial Pathogens

Phagocytosis is a fundamental process of cells to capture and ingest foreign particles. Small unicellular organisms such as free-living amoeba use this process to acquire food. In pluricellular organisms, phagocytosis is a universal phenomenon that all cells are able to perform (including epithelial, endothelial, fibroblasts, etc.), but some specialized cells (such as neutrophils and macrophages) perform this very efficiently and were therefore named professional phagocytes by Rabinovitch. Cells use phagocytosis to capture and clear all particles larger than 0.5 µm, including pathogenic microorganisms and cellular debris. Phagocytosis involves a series of steps from recognition of the target particle, ingestion of it in a phagosome (phagocytic vacuole), maturation of this phagosome into a phagolysosome, to the final destruction of the ingested particle in the robust antimicrobial environment of the phagolysosome. For the most part, phagocytosis is an efficient process that eliminates invading pathogens and helps maintaining homeostasis. However, several pathogens have also evolved different strategies to prevent phagocytosis from proceeding in a normal way. These pathogens have a clear advantage to perpetuate the infection and continue their replication. Here, we present an overview of the phagocytic process with emphasis on the antimicrobial elements professional phagocytes use. We also summarize the current knowledge on the microbial strategies different pathogens use to prevent phagocytosis either at the level of ingestion, phagosome formation, and maturation, and even complete escape from phagosomes.

Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology.