Neutrophils (polymorphonuclear cells; PMNs) form a first line of defense against pathogens and are therefore an important component of the innate immune response. As a result of poorly controlled activation, however, PMNs can also mediate tissue damage in numerous diseases, often by increasing tissue inflammation and injury. According to current knowledge, PMNs are not only part of the pathogenesis of infectious and autoimmune diseases but also of conditions with disturbed tissue homeostasis such as trauma and shock. Scientific advances in the past two decades have changed the role of neutrophils from that of solely immune defense cells to cells that are responsible for the general integrity of the body, even in the absence of pathogens. To better understand PMN function in the human organism, our review outlines the role of PMNs within the innate immune system. This review provides an overview of the migration of PMNs from the vascular compartment to the target tissue as well as their chemotactic processes and illuminates crucial neutrophil immune properties at the site of the lesion. The review is focused on the formation of chemotactic gradients in interaction with the extracellular matrix (ECM) and the influence of the ECM on PMN function. In addition, our review summarizes current knowledge about the phenomenon of bidirectional and reverse PMN migration, neutrophil microtubules, and the microtubule organizing center in PMN migration. As a conclusive feature, we review and discuss new findings about neutrophil behavior in cancer environment and tumor tissue.

Background: Migration, production of reactive oxygen species (ROS), release of myeloperoxidase (MPO), and NETosis are functional immunological reactions of elementary importance for polymorphonuclear neutrophils (PMN).Unregulated inflammatory response of PMN within tissues plays a key role in the pathophysiology of several diseases. However, little is known about the behavior of PMN after migration through blood vessel walls. Therefore, we investigated the influence of the extracellular matrix (ECM) on PMN function.Materials and Methods: We established an in vitro chemotaxis model of type I and III collagen, fibrin, and herbal agarose tissues using µ‐slide chemotaxis devices and N‐formylmethionine‐leucyl‐phenylalanine (fMLP). PMN within the matrices were assessed with a fluorescent time‐lapse microscope for live‐cell imaging.Results: PMN function was obviously influenced by the ECM. Type III collagen had an inhibitory effect on PMN migration regarding track length, direction, and targeting. Type III collagen also had an accelerating effect on neutrophil ROS production. Agarose had an inhibitory effect on MPO release and fibrin a retarding effect on NETosis.Conclusion: Because of the high abundance of type III collagen in lung and skin matrices, the interaction of PMN with the respective matrix could be an important mechanism in the pathophysiology of acute respiratory distress syndrome and pyoderma gangrenosum.

Acute respiratory distress syndrome (ARDS) is a complex disease pattern in which pathogenesis polymorphonuclear neutrophil granulocytes (PMN) play a key role. In previous experiments, we could show that interaction with collagen III (an important component of pulmonary tissue) is a possible trigger of neutrophil reactive oxygen species (ROS) production. To investigate possible correlations, further elucidate ARDS pathophysiology, and maybe find pharmacological targets, we evaluated PMNs from blood (circulating PMNs: cPMNs) and tracheal secretion (tPMNs) from patients with and without ARDS with regard to function and phenotype. Blood samples and tracheal secretions were obtained from intensive care patients with and without ARDS. Isolation of cPMN was performed by density-gradient gravity sedimentation without centrifugation. For tPMN isolation, endotracheal aspirate was filtered, and tPMNs were separated from the remaining aspirate using a particle filter. Specific surface epitopes (CD66b, CD62L, fMLP-receptor, LOX-1, CD49d, CD29, CD11b) of the isolated PMN cells were labeled with antibody-coupled dyes and analyzed by flow cytometry. Neutrophil ROS production before and after activation with N-formyl-methyl-leucyl-phenylalanine (fMLP) and tumor necrosis factor α (TNFα) was quantified using rhodamine-123. In addition, a qualitative cytological hematoxylin-eosin (HE) staining was performed with a portion of the secretion. tPMNs were observed in both bloody and mucosal tracheal secretions from ARDS patients. The epitope distribution on cPMNs and tPMNs differed significantly in patients with and without ARDS: tPMNs generally showed increased expression of CD66b, LOX-1 and fMLP-receptor compared to cPMNs, and decreased expression of CD62L. The CD49d levels of all cPMNs were at the same level as tPMNs in ARDS, whereas CD49d expression was increased on tPMNs without ARDS. ROS production was significantly stimulated by fMLP/TNFα in cPMNs regardless of the patient group, while it was similarly increased in tPMNs with and without stimulation. Increased expression of CD66b, LOX-1 and fMLP-receptor on tPMNs indicated a higher activity status compared to cPMNs. Increased CD49d expression on tPMNs without ARDS marks different PMN surface changes in lung disease. PMNs appear to be in a more activated state in lung secretions than in blood, as indicated by higher CD66b and lower CD62L expression, higher constitutive ROS production and lower excitability with fMLP and TNFα. In the context of possible CD49d-triggered ROS production, it is noteworthy that CD49d is downregulated in secretion from patients with ARDS compared to patients without. This phenotypic and functional PMN characterization can provide valuable diagnostic and therapeutic information for the intensive care treatment of ARDS patients.

Background: The application of normo- and hyperbaric O₂ is a common therapy option in various disease patterns. Thereby, the applied O₂ affects the whole body, including the blood and its components. This study investigates influences of pressure and oxygen fraction on human blood plasma, nutrient media, and the functions of neutrophil granulocytes (PMNs). Methods: Neutrophil migration, reactive oxygen species (ROS) production, and NETosis were examined by live cell imaging. The treatment of various matrices (Roswell Park Memorial Institute 1640 medium, Dulbecco’s Modified Eagle’s Medium, H₂O, human plasma, and isolated PMNs) with hyperbaric oxygen (HBO) was performed. In addition, the expression of different neutrophil surface epitopes (CD11b, CD62L, CD66b) and the oxidative burst were investigated by flow cytometry (FACS). The application of cold atmospheric plasma (CAP) to normoxic and normobaric culture media served as a positive control. Soluble reaction products such as H₂O₂, reactive nitrogen species (RNS: NO₂⁻ and NO₃⁻), and ROS-dependent dihydrorhodamine oxidation were quantified by fluoro- and colorimetric assay kits. Results: Under normobaric normoxia, PMNs migrate slower and shorter in comparison with normobaric hyper- or hypoxic conditions and hyperbaric hyperoxia. The pressure component has less effect on the migration behavior of PMNs than the O₂ concentration. Individual PMN cells produce prolonged ROS at normoxic conditions. PMNs showed increased expression of CD11b in normobaric normoxia, lower expression of CD62L in normobaric normoxia, and lower expression of CD66b after HBO and CAP treatment. Treatment with CAP increased the amount of ROS and RNS in common culture media. Conclusions: Hyperbaric and normobaric O₂ influences neutrophil functionality and surface epitopes in a measurable way, which may have an impact on disorders with neutrophil involvement. In the context of hyperbaric experiments, especially high amounts of H₂O₂ in RPMI after hyperbaric oxygen should be taken into account. Therefore, our data support a critical indication for the use of normobaric and hyperbaric oxygen and conscientious and careful handling of oxygen in everyday clinical practice.

The effectiveness of the immune system decreases with increasing age. This process is known as immunosenescence. Recent studies showed the influence of aging on neutrophil granulocytes (PMNs) and T-cells, with the extent of the influence appearing to depend on various co-factors (such as the primary diseases of a patient). In this study, the PMNs and T-cells of younger and older adult patients were tested for their immunoreactivity before and after an operation in order to examine the consequences of the aging process on the moderately triggered immune system. Whole blood was taken from young patients (aged 18–65 years) and old patients (> 65 years) before and one day after an operation. Previous illnesses and medication intake were taken from the patient’s file. PMNs and T-cells were isolated. Immunoassays, live cell imaging (LCI) and flow cytometric examinations (FACS) were performed in order to assess certain properties of the PMNs (chemotactic migration, ROS production, NET formation, change of surface epitopes), their expression of adhesion molecules as well as their cell viability. In addition, the blood samples were subjected to a laboratory chemical examination. Above all during the initial LCI observation period (< 40 min), the PMNs of old patients covered longer distances than those of young patients. NETosis, ROS production and surface antigen expression were influenced neither by age nor by the surgical procedure. Regardless of age, PMNs´ ROS production started earlier 24 h after the operation compared to the pre OP values. By labeling the translocator protein (TSPO), it was demonstrated that mitochondrial release occurs only during suicidal NETosis. Old patients showed significantly more TSPO-labeled mitrochondria per PMN. The neutrophil-to-lymphocyte ratio (NLR) and the CD4/CD8 ratio were significantly increased in older patients. The share of CD28- and CD8-positive cells was increased in younger patients. All patients showed postoperative leukocytosis caused by an increase in monocytes and PMNs, which was independent of the extent of the trauma. Only young patients showed a postoperative increase in lymphocytes. Old patients had higher IL-6 levels than young patients. The operation did not lead to any increases in the IL-6 and CRP levels. Age influences the function of PMNs and T-cells more strongly than a moderate surgical trauma in combination with anaesthesia. The results advance our understanding of the decreasing effectiveness of the immune system in old age.

Background: There is increasing evidence that nitric oxide (nitrogen monoxide, NO) significantly influences immune cellular responses, including those from polymorphonuclear leukocytes (PMNs). Objective: The aim of this study was to examine a possible effect of NO on PMNs’ function (chemotaxis, production of reactive oxygen species (ROS), and NETosis) using live cell imaging. Moreover, we investigated PMN surface epitope and neutrophil oxidative burst under the influence of NO by flow cytometric analysis. Methods: Whole blood samples were obtained from healthy volunteers, and PMNs were isolated by density centrifugation. Live cell imaging using type I collagen matrix in µSlide IBIDI chemotaxis chambers was conducted in order to observe N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP)-stimulated PMN chemotaxis, ROS production, and NETosis. In the test group, NO was continuously redirected into the climate chamber of the microscope, so the chemotaxis chambers were surrounded by NO. The same experimental setup without NO served as a control. In addition, isolated PMNs were incubated with nitrogen monoxide (NO) or without (the control). Subsequently, flow cytometry was used to analyze neutrophil antigen expression and oxidative burst. Results: Our live cell imaging results demonstrated a migration-promoting effect of NO on PMNs. We observed that in the case of prior stimulation by fMLP, NO has no effect on the time course of neutrophil ROS production and NET release. However, flow cytometric analyses demonstrated an increase in ROS production after pretreatment with NO. No NO-dependent differences for the expression of CD11b, CD62L, or CD66b could be observed. Conclusions: We were able to demonstrate a distinct effect of NO on PMNs’ function. The complex interaction between NO and PMNs remains a major research focus, as the exact mechanisms and additional influencing factors remain elusive. Future studies should explore how varying NO concentrations and the timing of NO exposure relative to PMN activation affect its influence.