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We present a young patient who had to undergo 5 mitral valve replacements (MVR) because of a repetitive immune-mediated noninfectious endocarditis. The patient was treated with multiple anti-inflammatory drugs and high-dose prednisone. After the fifth MVR, the patient remained in stable condition using Anakinra after 22 months of follow-up. (Level of Difficulty: Advanced.), Central Illustration

The heterogeneity of chronic obstructive pulmonary disease (COPD) creates many diagnostic, prognostic, treatment and management challenges, as the pathogenesis of COPD is highly complex and the underlying cellular and molecular mechanisms remain poorly understood. A reliable, easy-to-measure, clinically relevant biomarker would be invaluable for improving outcomes for patients. International and national guidance for COPD suggests using blood eosinophil counts as a biomarker to help estimate likely responsiveness to inhaled corticosteroids (ICS) and, potentially, to aid effective management strategies. However, with the mechanism underlying the association between higher eosinophil levels and ICS effect unknown, use of the blood eosinophil count in COPD continues to be widely debated by the respiratory community.Two international meetings involving respiratory medicine specialists, immunologists and primary and secondary care clinicians were held in November 2018 and March 2019, facilitated and funded by GlaxoSmithKline plc. The aims of these meetings were to explore the role of eosinophils in the disease processes of COPD and as prognostic and diagnostic markers, and to identify areas of deficient knowledge that warrant further research. The consensus views of the attendees on key topics, contextualised with current literature, are summarised in this review article, with the aim of aiding ongoing research into the disease processes of COPD and the development of biomarkers to aid clinical management.Under certain conditions, eosinophils can be recruited to the lung, and increasing evidence supports a role for eosinophilic inflammation in some patients with COPD. Infiltration of eosinophils across the bronchial vascular epithelium into the airways is promoted by the actions of immunoregulatory cells, cytokines and chemokines, where eosinophil-mediated inflammation is driven by the release of proinflammatory mediators.Multiple studies and two meta-analyses suggest peripheral blood eosinophils may correlate positively with an increased likelihood of exacerbation reduction benefits of ICS in COPD. The studies, however, vary in design and duration and by which eosinophil levels are viewed as predictive of an ICS response. Generally, the response was seen when eosinophil levels were 100–300 cells/µL (or higher), levels which are traditionally viewed within the normal range. Some success with interleukin-5-targeted therapy suggests that the eosinophilic phenotype may be a treatable trait.The use of biomarkers could help to stratify treatment for COPD—the goal of which is to improve patient outcomes. Some evidence supports eosinophils as a potential biomarker of a treatable trait in COPD, though it is still lacking and research is ongoing. A unified consensus and a practical, accessible and affordable method of utilising any biomarker for COPD was thought to be of most importance. Challenges around its utilisation may include presenting a clear and pragmatic rationale for biomarker-driven therapy, guidance on ICS withdrawal between primary and secondary care and a lack of financial incentives supporting broad application in clinical practice. Future treatments should, perhaps, be more targeted rather than assuming the primary disease label (COPD or asthma) will define treatment response.

Contains fulltext : 229398.pdf (Publisher’s version ) (Open Access) Acute infection is characterized by eosinopenia. However, the underlying mechanism(s) are poorly understood and it is unclear whether decreased mobilization/production of eosinophils in the bone marrow (BM) and/or increased homing to the tissues play a role. The objective of this study was to investigate the differentiation and activation status of eosinophils in the human BM and blood upon experimental human endotoxemia, a standardized, controlled, and reproducible model of acute systemic inflammation. A BM aspirate and venous blood was obtained from seven healthy volunteers before, 4 h after, and 1 week after intravenous challenge with 2 ng/kg endotoxin. Early progenitors (CD34+/IL-5Rα+), eosinophil promyelocytes, myelocytes, metamyelocytes, and mature eosinophils were identified and quantified in the bone marrow and blood samples using flowcytometry based on specific eosinophil markers (CD193 and IL-5Rα). Activation status was assessed using antibodies against known markers on eosinophils: Alpha-4 (CD49d), CCR3 (CD193), CR1 (CD35), CEACAM-8 (CD66b), CBRM 1/5 (activation epitope of MAC-1), and by plasma cytokine analysis. Four hours after endotoxin administration, numbers of mature eosinophils in the blood and in the BM markedly declined compared with baseline, whereas numbers of all eosinophil progenitors did not change. The remaining eosinophils did not show signs of activation or degranulation despite significantly increased circulating levels of eotaxin-1. Furthermore, the expression of CD49d and CD193 on eosinophils was lower compared to baseline, but normalized after 7 days. Together these data imply that circulatory eosinopenia after an innate immune challenge is mediated by CD49d-mediated homing of eosinophils to the tissues.

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (&lt, 100 cells/μL, p &lt, 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4% (CI95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population (42.21%, CI95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation (7.6%, CI95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation (23.7%, CI95% = 18.5, 27.6, 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue.

At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease.

BACKGROUND Neutrophils and monocytes are key immune effector cells in inflammatory bowel disease (IBD) that is associated with chronic inflammation in the gut. Patients with stable IBD who perform exercise have significantly fewer flare-ups of the disease, but no underlying mechanism has been identified. Therefore, the aim of this study was to compare the responsiveness/refractoriness of these innate immune cells after repeated bouts of prolonged exercise in IBD patients and controls. METHODS Patients with IBD and age- and gender-matched healthy controls were recruited from a cohort of walkers participating in a 4-day walking event. Blood analysis was performed at baseline and after 3 days of walking. Responsiveness to the bacterial/mitochondrial-stimulus N-Formylmethionine-leucyl-phenylalanine (fMLF) was tested in granulocytes and monocytes by measuring the expression of activation markers after adding this stimulus to whole blood. RESULTS In total 38 participants (54 ± 12 years) were included in this study: 19 walkers with and 19 walkers without IBD. After 3 days of prolonged exercise, a significant increase in responsiveness to fMLF was observed in all participants irrespective of disease. However, IBD patients showed significantly less responsiveness in neutrophils and monocytes, compared with non-IBD walkers. CONCLUSIONS Increased responsiveness of neutrophils and monocyte to fMLF was demonstrated after repetitive bouts of prolonged exercise. Interestingly, this exercise was associated with relative refractoriness of both neutrophils and monocytes in IBD patients. These refractory cells might create a lower inflammatory state in the intestine providing a putative mechanism for the decrease in flare-ups in IBD patients after repeated exercise.

Multicolor Flow Cytometry (MFC)-based gating allows the selection of cellular (pheno)types based on their unique marker expression. Current manual gating practice is highly subjective and may remove relevant information to preclude discovery of cell populations with specific co-expression of multiple markers. Only multivariate approaches can extract such aspects of cell variability from multi-dimensional MFC data. We describe the novel method ECLIPSE (Elimination of Cells Lying in Patterns Similar to Endogeneity) to identify and characterize aberrant cells present in individuals out of homeostasis. ECLIPSE combines dimensionality reduction by Simultaneous Component Analysis with Kernel Density Estimates. A Difference between Densities (DbD) is used to eliminate cells in responder samples that overlap in marker expression with cells of controls. Thereby, subsequent data analyses focus on the immune response-specific cells, leading to more informative and focused models. To prove the power of ECLIPSE, we applied the method to study two distinct datasets: the in vivo neutrophil response induced by systemic endotoxin challenge and in studying the heterogeneous immune-response of asthmatics. ECLIPSE described the well-characterized common response in the LPS challenge insightfully, while identifying slight differences between responders. Also, ECLIPSE enabled characterization of the immune response associated to asthma, where the co-expressions between all markers were used to stratify patients according to disease-specific cell profiles.

Summary Mepolizumab (anti-IL-5) is a successful biological for treatment of T2/eosinophilic asthma by blocking the IL-5-eosinophil axis. The kinetics of human eosinophils in blood and sputum was determined to better understand the underlying mechanism(s). Pulse-chase labeling was performed with 6,6-2H2-glucose in patients with asthma after short term (4 days) and long term (84 days) treatment with mepolizumab (n = 10) or placebo (n = 10). The retention time of eosinophils in sputum was longer than in blood. Treatment with mepolizumab induced a fast and long-lasting eosinopenia with no reduction of eosinophil progenitors. The retention time of eosinophils in blood was delayed only after short-term treatment. This leads to the hypothesis that IL-5 increases the number of IL-5-responsive progenitors and potentiates homing to the tissues, leading to reactive eosinophilia. Long-term treatment is associated with low numbers of IL-5-independent eosinophils in blood and tissues. Therefore, long-term treatment with mepolizumab restores the kinetics of eosinophils as normally found in homeostasis., Graphical abstract, Highlights • Anti-IL-5 (mepolizumab) treatment leads to inhibition of reactive eosinophilia • Reactive blood eosinophils have a high retention time in the absence of IL-5 • Eosinophils are long lived in the sputum of eosinophil asthmatics • Anti-IL-5 reduces proliferating progenitors rather than inhibiting differentiation, Health sciences; Immunology; Respiratory medicine; Clinical medicine; Drugs

Introduction Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated “load & go” flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. Methods Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® “load & go” flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. Results The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p = .028, CD11b: 2.5× (1.7×-3.1×) p = .023, CD10: 2.5× (2.1×-2.7×) p = .009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×–2.2×) p = .058, CD11b: 2.13× (1.6×–2.4×) p = .025) and also a further significant increase in 2 and 3 h was observed. Conclusion This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15–20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. Fund This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Introduction: Cystic fibrosis (CF) is the most common monogenic disease that is eventually lethal. It is characterized by chronic bacterial infection of the lung and affecting additional organs including pancreas and liver. Neutrophils form the largest number of inflammatory cells in the airways of cystic fibrosis patients. Previous studies have shown that the killing function of neutrophils in CF patients is preserved, but maybe influenced by Pseudomonas aeruginosa (PA) colonization during the disease progression. Objective: To evaluate the phagocytosis and bacterial killing of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) by neutrophils in CF patients in comparison with cells of healthy controls. Materials and Methods: Our study was conducted in 10 CF patients (confirmed by clinical examination, sweat test and mutational analysis) in the Masih Daneshvari hospital (Tehran, Iran) and 10 age- and gender-matched healthy control subjects in 2017. Neutrophils were isolated from the peripheral blood of patients with CF patients and control subjects. The cells were incubated with SA and PA -labeled with GFP in a FLUOstar OPTIMA Microplate Fluorescent Reader. The fluorescence was measured over time as a measure of bacterial counts. Results: Neutrophils of CF patients were able to contain SA for long periods of time (72±5 h) that were significantly longer than by cells from healthy control (25±6 h) P≤0.05). Whereas, no differences in releasing of PA was observed. Our data support the hypothesis that neutrophils of CF patients exhibit a primed phenotype that enables them to contain SA longer than unprimed cells isolated from control individuals.

Flow Cytometry is an analytical technology to simultaneously measure multiple markers per single cell. Ten thousands to millions of single cells can be measured per sample and each sample may contain a different number of cells. All samples may be bundled together, leading to a ‘multi-set’ structure. Many multivariate methods have been developed for Flow Cytometry data but none of them considers this structure in their quantitative handling of the data. The standard pre-processing used by existing multivariate methods provides models mainly influenced by the samples with more cells, while such a model should provide a balanced view of the biomedical information within all measurements. We propose an alternative ‘multi-set’ preprocessing that corrects for the difference in number of cells measured, balancing the relative importance of each multi-cell sample in the data while using all data collected from these expensive analyses. Moreover, one case example shows how multi-set pre-processing may benefit removal of undesired measurement-to-measurement variability and another where class-based multi-set pre-processing enhances the studied response upon comparison to the control reference samples. Our results show that adjusting data analysis algorithms to consider this multi-set structure may greatly benefit immunological insight and classification performance of Flow Cytometry data.

Receptors recognizing the Fc-part of immunoglobulins (FcR) are important in the engagement of phagocytes with opsonized micro-organisms, but they also play a major role in the pathogenesis of chronic inflammatory diseases. Different FcRs are specifically recognizing and binding the different classes of immunoglobulins, transmitting different signals into the cell. The function of IgG (FcγR's) and IgA (FcαR) recognizing receptors is controlled by cellular signals evoked by activation of heterologous receptors in a process generally referred to as inside-out control. This concept is clearly described for the regulation of integrin receptors. Inside-out control can be achieved at different levels by modulation of: (i) receptor affinity, (ii) receptor avidity/valency, (iii) interaction with signaling chains, (iv) interaction with other receptors and (v) localization in functionally different membrane domains. The inside-out control of FcRs is an interesting target for novel therapy by therapeutical antibodies as it can potentiate or decrease the functionality of the response to the antibodies depending on the mechanisms of the diseases they are applied for.

Acute inflammation recruits neutrophils with a band-shaped nucleus to the circulation. This neutrophil population was recently shown to have superior antibacterial capacity. Early recruitment of banded neutrophils to an infection site will likely improve the outcome of the immune response, yet it critically depends on efficient migration. However, the current dogma states that the segmentation of the mature neutrophil nucleus has evolved to favor migration through narrow pores as found between endothelial cells and in the interstitium. Therefore, we hypothesized that banded neutrophils migrate less efficiently than neutrophils with segmented nuclei, whereas recently described neutrophils with hypersegmented nuclei would in turn migrate more efficiently. Acute inflammation was evoked in a human model of experimental endotoxemia to recruit neutrophil subsets with different nuclear segmentation to the circulation. To simulate migration toward an infection site, migration of the subsets was studied in in vitro models of transendothelial migration or interstitial chemokinesis and chemotaxis. In both models, nuclear segmentation did not increase migration speed. In dense collagen matrices, the speed of the hypersegmented neutrophils was even reduced compared with the banded neutrophils. Fluorescence microscopy suggested that the hypersegmented neutrophils displayed reduced rear release and deposited more membrane vesicles. Vice versa, migration through narrow pores did not induce nuclear segmentation in the neutrophils. In conclusion, like neutrophils with a segmented nucleus, the banded subset exhibited efficient migration through narrow pores. These findings suggest that the nucleus does not preclude the banded subset from reaching an infection site.

The role of inflammatory cells in bone regeneration remains unclear. We hypothesize that leukocytes contribute to fracture healing by rapidly synthesizing an "emergency extracellular matrix (ECM)" before stromal cells infiltrate the fracture hematoma (FH) and synthesize the eventual collagenous bone tissue. 53 human FHs were isolated at different time points after injury, ranging from day 0 until day 23 after trauma and stained using (immuno)histochemistry. FHs isolated within 48 h after injury contained fibronectin(+) ECM, which increased over time. Neutrophils within the early FHs stained positive for cellular fibronectin and neutrophil derived particles were visible within the fibronectin(+) ECM. Stromal cells appeared at day 5 after injury or later and collagen type I birefringent fibrils could be identified during the second week after injury. Our study suggests that neutrophils contribute to bone regeneration by synthesizing an "emergency ECM" before stromal cells infiltrate the FH and synthesize the eventual bone tissue.

Contains fulltext : 218545.pdf (Publisher’s version ) (Open Access)

Although controlled local inflammation is essential for adequate bone regeneration, several studies have shown that hyper-inflammatory conditions after major trauma are associated with impaired fracture healing. These hyper-inflammatory conditions include the trauma-induced systemic inflammatory response to major injury, open fractures, and significant injury to the surrounding soft tissues. The current literature suggests that increased or prolonged influx of neutrophils into the fracture hematoma may mediate impairment of bone regeneration after hyper-inflammatory conditions. The underlying mechanism remains unclear. We hypothesize that high neutrophil numbers inhibit synthesis of mineralized extracellular matrix (ECM) by bone marrow stromal cells (BMSCs). We therefore studied the effect of increasing concentrations of neutrophils on ECM synthesis by human BMSCs in vitro. Moreover, we determined how high neutrophil concentrations affect BMSC cell counts, as well as BMSC osteogenic activity determined by alkaline phosphatase (ALP) expression and ALP activity. Co-culture of BMSCs with neutrophils induced a 52% decrease in BMSC cell count (p < 0.01), a 64% decrease in the percentage of ALP+ cells (p < 0.001), a 28% decrease in total ALP activity (p < 0.01), and a significant decrease in the amount of mineralized ECM [38% decrease after 4 weeks (p < 0.05)]. Co-cultures with peripheral blood mononuclear cells and neutrophils within transwells did not induce a significant decrease in ALP activity. In conclusion, our data shows that a decreased amount of mineralized ECM became synthesized by BMSCs, when they were co-cultured with high neutrophil concentrations. Moreover, high neutrophil concentrations induced a decrease in BMSC cell counts and decreased ALP activity. Clarifying the underlying mechanism may contribute to development of therapies that augment bone regeneration or prevent impaired fracture healing after hyper-inflammatory conditions.

Neutrophils are one of the most important effector cells of the innate immune response (1). They are traditionally seen as a homogenous population of short‐lived cells mainly involved in the defence against extracellular microorganisms by phagocytosis and intracellular killing (1,2). The cells contain a large armamentarium that aids in this function and ranges from the production of reactive oxygen species by a membrane‐bound NADPH oxidase to cytotoxic proteins and peptides residing in the different granules present in the cytoplasm (3). Recently, the view of neutrophils belonging to a homogenous population of cells has been challenged, and several neutrophil phenotypes have been described that exhibit specialized functions, such as involvement in tissue repair, tumour killing and immune regulation (4). It is not clear whether these cells belong to separate parallel lineages originating from the bone marrow or that neutrophils become instructed in the distant tissues, thus changing their phenotypes. In addition, functional heterogeneity in a phenotypically homogenous population of neutrophils adds to the complexity of neutrophil phenotypes(5). This article will review the current literature describing the heterogeneity within the neutrophil compartment with respect to both phenotype and function in health and disease.

Metaplasia in Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into intestinal-type columnar epithelium. The immune response in BE shares many similarities with the response found in the gut, which is different from the response found in a normal-looking esophagus. Here, we investigated the role of the genes associated with the retinoic acid (RA) pathway in BE, as RA is important not only in shaping the gut's immune response but also in the induction of metaplasia in vitro. mRNA was isolated from esophageal and duodenal biopsies from BE (n = 14), reflux esophagitis patients (n = 9), and controls (n = 12). cDNA was made and qPCR was performed. The expression of RALDH1, CYP26A1, MAdCAM1 were similar for both the BE and duodenum, but different when compared to squamous esophageal epithelium. BE was characterized by a higher expression of RALDH2 and FOXP3, compared to the duodenum. In BE, RALDH2 correlated with expression of the myeloid dendritic cell-specific genes: CD11c and CD1c. Also, RALDH2 expression correlated with RAR-β and FOXP3. Hierarchical clustering on the expression of multiple relevant genes demonstrated that BE, duodenum, and SQ tissues are clustered as three different groups. The differential expression of RA-specific genes and dendritic cell (DC)-subsets indicates that BE resembles duodenal tissue. The higher expression of RALDH2 and FOXP3 in BE points at a mechanism associated with a possible anti-inflammatory microenvironment. This aberrant immune regulation might contribute to the altered tissue and immune responses found in BE.

To the editor: Monocytes originate from the bone marrow (BM), are distributed in the bloodstream, and can differentiate in the tissue into skin macrophages or intestinal dendritic cells (DCs).[1][1] They play an essential role in the defense against pathogens[2][2] and are implicated in a range of

Introduction – Phagocytosis by neutrophils is a key process in the innate immune response against invading micro-organisms. Despite reported heterogeneity in other neutrophils functions, little is known regarding differences in phagocytosis by individual cells. Therefore, we tested the hypothesis that heterogeneity is present in the neutrophil compartment in its potency to phagocytize bacteria. Methods – Phagocytosis assays were performed in suspension with isolated neutrophils and Staphylococcus aureus expressing different fluorescent proteins at MOI’s between 1 and 10. Repetitive addition of bacteria with different fluorescent proteins and MOI’s was used to compare the phagocytic capacity of S. aureus-GFP positive and negative neutrophils and exclude randomness. Results – The percentage and MFI of S. aureus-GFP positive neutrophils increased with higher MOI’s. The increase in MFI was due to phagocytosis of multiple bacteria per neutrophil as was confirmed by confocal imaging. Sequential phagocytosis of GFP- and mCherry expressing S. aureus showed a non-random process, as S. aureus-GFP positive neutrophils preferentially phagocytized S. aureus-mCherry. Conclusion – All neutrophils were able to phagocytize S. aureus, but some were much more potent than others. Therefore, at physiologically relevant MOI’s these potent phagocytizing neutrophils will outcompete the uptake of bacteria by less competent cells in a process we propose to name ‘competitive phagocytosis’. Keywords – Granulocyte, bacteria, Staphylococcus aureus, innate immunity, PMN

Contains fulltext : 177455.pdf (Publisher’s version ) (Open Access) BACKGROUND: COPD is a heterogeneous chronic inflammatory disease of the airways and it is well accepted that the GOLD classification does not fully represent the complex clinical manifestations of COPD and this classification therefore is not well suited for phenotyping of individual patients with COPD. Besides the chronic inflammation in the lung compartment, there is also a systemic inflammation present in COPD patients. This systemic inflammation is associated with elevated levels of cytokines in the peripheral blood, but the precise composition is unknown. Therefore, differences in phenotype of peripheral blood neutrophils in vivo could be used as a read out for the overall systemic inflammation in COPD. METHOD: Our aim was to utilize an unsupervised method to assess the proteomic profile of peripheral neutrophils of stable COPD patients and healthy age matched controls to find potential differences in these profiles as read-out of inflammatory phenotypes. We performed fluorescence two-dimensional difference gel electrophoresis with the lysates of peripheral neutrophils of controls and stable COPD patients. RESULTS: We identified two groups of COPD patients based on the differentially regulated proteins and hierarchical clustering whereas there was no difference in lung function between these two COPD groups. The neutrophils from one of the COPD groups were less responsive to bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). CONCLUSION: This illustrates that systemic inflammatory signals do not necessarily correlate with the GOLD classification and that inflammatory phenotyping can significantly add in an improved diagnosis of single COPD patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00807469 registered December 11th 2008.

Bloodstream infection is a hallmark of sepsis, a medically emergent condition requiring rapid treatment. However, up-regulation of host defense proteins through Toll-like receptors (TLRs) and nuclear factor B requires hours after endotoxin detection. Using confocal pulmonary intravital microscopy, we identified that the lung provides a TLR4–Myd88 (myeloid differentiation primary response gene 88)–dependent and abl tyrosine kinase–dependent niche for immediate CD11b-dependent neutrophil responses to endotoxin and Gram-negative bloodstream pathogens. In an in vivo model of bacteremia, neutrophils crawled to and rapidly phagocytosed Escherichia coli sequestered to the lung endothelium. Therefore, the lung capillaries provide a vascular defensive niche whereby endothelium and neutrophils cooperate for immediate detection and capture of disseminating pathogens.

Background Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common s-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. Objective We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. Methods Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation, which was then compared to IgG-mediated degranulation of freshly isolated (unstimulated) airway eosinophils activated in vivo by segmental allergen challenge. Results When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMs2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. Conclusions We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMs2-mediated eosinophil degranulation. This article is protected by copyright. All rights reserved.

Background The identification of inflammatory asthma phenotypes, using sputum analysis, has proven its value in diagnosis and disease monitoring. However due to technical limitations of sputum analysis, there is a strong need for fast and noninvasive diagnostics. This study included the activation state of eosinophils and neutrophils in peripheral blood to phenotype and monitor asthma. Objectives To (i) construct a multivariable model using the activation state of blood granulocytes, (ii) compare its diagnostic value with sputum eosinophilia as gold standard and (iii) validate the model in an independent patient cohort. Methods Clinical parameters, activation of blood granulocytes and sputum characteristics were assessed in 115 adult patients with asthma (training cohort/Utrecht) and 34 patients (validation cohort/Oxford). Results The combination of blood eosinophil count, fractional exhaled nitric oxide, Asthma Control Questionnaire, medication use, nasal polyposis, aspirin sensitivity and neutrophil/eosinophil responsiveness upon stimulation with formyl‐methionyl‐leucyl phenylalanine was found to identify sputum eosinophilia with 90.5% sensitivity and 91.5% specificity in the training cohort and with 77% sensitivity and 71% specificity in the validation cohort (relatively high percentage on oral corticosteroids [OCS]). Conclusions The proposed prediction model identifies eosinophilic asthma without the need for sputum induction. The model forms a noninvasive and externally validated test to assess eosinophilic asthma in patients not on OCS.

Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on (1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91–100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.

Neutrophils are recognized as an essential part of the innate immune response, but an active debate still exists regarding the life cycle of these cells. Neutrophils first differentiate in the bone marrow through progenitor intermediaries before entering the blood, in a process that gauges the extramedullary pool size. Once believed to be directly eliminated in the marrow, liver, and spleen, neutrophils, after circulating for less than 1 day, are now known to redistribute into multiple tissues with poorly understood kinetics. In this review, we provide an update on the dynamic distribution of neutrophils across tissues in health and disease, and emphasize differences between humans and model organisms. We further highlight issues to be addressed to exploit the unique features of neutrophils in the clinic. The work in the authors laboratories is funded by (ERC) Medical Research Council, Wellcome Trust, GlaxoSmithKline, MedImmune, the NIHR Cambridge Biomedical Research Centre, British Heart Foundation, National Institute for Health, Cambridge NIHR Biomedical Research Centre, the MCIU (Ministerio de Ciencia, Innovación y Universidades), the Pro-CNIC Foundation, the Dutch Science Agenda (NWA) and the Netherlands Organization for Scientific Research (NWO). The CNIC is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505). Sí

Neutrophils are the most abundant white blood cells and are indispensable for host defense. Recently, they have also been implicated in immune regulation and suppression. The latter functions seem hard to reconcile with the widely held view that neutrophils are very short-lived, with a circulatory half-life of

Integrins are functionally regulated by "inside-out" signaling, in that 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, but the mechanisms that regulate inside out signaling are not completely understood. Previously, we identified gelsolin in a proteomics screen to identify proteins involved in inside-out control of integrins using the lymphocytic leukemia cell line L1210. Furthermore, we showed that gelsolin was involved in affinity regulation of β1 -integrins in the leukemic cell line U937. Here, we examined how gelsolin regulates β1 -integrin affinity in the leukemia cell line L1210. We show that gelsolin is mainly expressed at the cell membrane and is present near β1 -integrins. The role for actin polymerization in integrin affinity regulation was examined using the actin modulating agent jasplakinolide, which decreased β1 -integrin affinity. Similarly, knock-down of gelsolin in L1210 cells also decreased β1 -integrin affinity and cell adhesion to collagen. These data suggest that increased actin polymerization through gelsolin regulates β1 -integrin affinity and cell adhesion.

Neutrophils are essential effector cells in the host defense against invading pathogens. Recently, novel neutrophil functions have emerged in addition to their classical anti-microbial role. One of these functions is the suppression of T cell responses. In this respect, neutrophils share similarities with granulocytic myeloid-derived suppressor cells (G-MDSCs). In this review, we will discuss the similarities and differences between neutrophils and G-MDSCs. Various types of G-MDSCs have been described, ranging from immature to mature cells shaping the immune response by different immune suppressive mechanisms. However, all types of G-MDSCs share distinct features of neutrophils, such as surface markers and morphology. We propose that G-MDSCs are heterogeneous and represent novel phenotypes of neutrophils, capable of suppressing the immune response. In this review, we will attempt to clarify the differences and similarities between neutrophils and G-MDSCs and attempt to facilitate further research.

Inflammation in response to infection or trauma can lead to CARS, which is characterized by leukocyte dysfunction. In this study, we used a human model system for CARS to study the effect of GM-CSF and IFN-γ treatment on this immunoparalyzed state. Healthy human volunteers were treated with GM-CSF (4 μg/kg), IFN-γ (100 μg), or placebo in between two challenges with Escherichia coli LPS/endotoxin (2 ng/kg). Serial leukocyte blood counts were measured. Neutrophil subsets were discriminated using CD16 and CD62L expression. LPS rechallenge resulted in increased mobilization of mature neutrophils, whereas banded neutrophils decreased. GM-CSF and IFN-γ treatment did not restore these changes. GM-CSF treatment did, however, increase the number of CD16bright/CD62Ldim neutrophils that were previously shown be able to suppress T cell proliferation. IFN-γ treatment decreased neutrophilia seen after LPS rechallenge. Our study shows that LPS rechallenge was associated with changes in the distribution of neutrophil subsets, whereas no additional changes in kinetics of other granulocyte populations were observed. GM-CSF and IFN-γ treatment induced a shift in granulocyte composition toward an anti-inflammatory direction by increasing CD16bright/CD62Ldim cells or decreasing neutrophil counts, respectively.

Neutrophils are the predominant inflammatory cells recruited to the respiratory tract as part of the innate immune response to viral infections. Recent reports indicate the existence of distinct functional neutrophil subsets in the circulatory compartment of adults, following severe inflammatory conditions. Here, we evaluated the occurrence of neutrophil subsets in blood and broncho-alveolar lavage fluid during severe viral respiratory infection in infants based on CD16/CD62L expression. We show that during the course of severe respiratory infection infants may develop four heterogeneous neutrophil subsets in blood (mature, immature, progenitor, and suppressive neutrophils), each with distinct activation states. However, while isolated viral respiratory infection was characterized by a relative absence of suppressive neutrophils in both blood and lungs, only patients with bacterial co-infection were shown to produce suppressive neutrophils. These data suggest the occurrence of distinct and unique neutrophil subset responses during severe viral and (secondary) bacterial respiratory infection in infants.

Background Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by chronic airway inflammation and emphysema, and is caused by exposure to noxious particles or gases, e.g. cigarette smoke. Smoking and oxidative stress lead to accelerated formation and accumulation of advanced glycation end products (AGEs), causing local tissue damage either directly or by binding the receptor for AGEs (RAGE). This study assessed the association of AGEs or RAGE in plasma, sputum, bronchial biopsies and skin with COPD and lung function, and their variance between these body compartments. Methods Healthy smoking and never-smoking controls (n = 191) and COPD patients (n = 97, GOLD stage I-IV) were included. Autofluorescence (SAF) was measured in the skin, AGEs (pentosidine, CML and CEL) and sRAGE in blood and sputum by ELISA, and in bronchial biopsies by immunohistochemistry. eQTL analysis was performed in bronchial biopsies. Results COPD patients showed higher SAF values and lower plasma sRAGE levels compared to controls and these values associated with decreased lung function (p

Okan W Bastian,1 Anne Kuijer,1 Leo Koenderman,2 Rebecca K Stellato,3 Wouter W van Solinge,4 Luke PH Leenen,1 Taco J Blokhuis1 1Department of Traumatology, 2Department of Respiratory Medicine, 3Department of Biostatistics and Research Support, Julius Center, 4Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, the Netherlands Abstract: Animal studies have shown that the systemic inflammatory response to major injury impairs bone regeneration. It remains unclear whether the systemic immune response contributes to impairment of fracture healing in multitrauma patients. It is well known that systemic inflammatory changes after major trauma affect leukocyte kinetics. We therefore retrospectively compared the cellular composition of peripheral blood during the first 2 weeks after injury between multitrauma patients with normal (n=48) and impaired (n=32) fracture healing of the tibia. The peripheral blood-count curves of leukocytes, neutrophils, monocytes, and thrombocytes differed significantly between patients with normal and impaired fracture healing during the first 2 weeks after trauma (P-values were 0.0122, 0.0083, 0.0204, and

TNFalpha-induced expression of CD83 in leukocytes is mediated by NF-kappab. The aim of our present study was to investigate the underlying mechanism of a unique functional antagonism between GM-CSF and TNFalpha-induced up-regulation of CD83 in human neutrophils. CD83 was down-regulated by co-stimulation of neutrophils with TNFalpha and GM-CSF compared to TNFalpha alone both at the level of mRNA and protein. In marked contrast, the expression of IL-1RA was up-regulated under the same conditions. The down-regulation of CD83 was not mediated by modulation of the NF-kappab signaling pathway. Neither was it mediated by a decrease in mRNA stability of CD83. NF-kappab was modulated under these conditions as both the expression of the target gene IL-1RA as well as the phosphorylation of IkBalpha were up-regulated. Our results show that co-stimulation with pro-inflammatory cytokines such as TNFalpha and GM-CSF can have differential effects on inflammatory pathways initiated in the same target cell. GM-CSF can both synergize with TNFalpha in the case of expression of IL1-RA and antagonize in the case of CD83. Therefore, expression of CD83 as read out for activation of neutrophils in patients with inflammatory diseases is complicated by the presence of cross-modulating cytokines such as GM-CSF.

Background Allergic asthma is associated with chronic airway and systemic immune responses. Systemic responses include priming of peripheral blood eosinophils, which is enhanced after allergen challenge. In a subpopulation of asthmatic subjects, neutrophils are associated with bronchial inflammation. Objective We sought to monitor systemic granulocyte priming in allergic asthmatic subjects as a consequence of chronic and acute inflammatory signals initiated by allergen challenge. Methods Blood was taken at baseline and 6 to 24 hours after allergen challenge in asthmatic subjects with and without late asthmatic responses. Systemic granulocyte priming was studied by using expression of cellular markers, such as α-chain of Mac-1 (αm)/CD11b, L-selectin/CD62L, and an activation epitope present on FcγRII/CD32 recognized by monoclonal phage antibody A17. Results Eosinophils of asthmatic subjects have a primed phenotype identified by cell-surface markers. Neutrophils of these patients were subtly primed, which was only identified after activation with N-formyl-methionyl-leucyl-phenylalanine. After allergen challenge, an acute increase in eosinophil priming characterized by enhanced expression of activated FcγRII was found in patients experiencing a late asthmatic response and not in patients with a single early asthmatic response. In contrast, expression of αm/CD11b and L-selectin on granulocytes was not different between control and asthmatic subjects and was not affected by allergen challenge. Interestingly, expression of both adhesion molecules was positively correlated, and αm expression on eosinophils and neutrophils correlated positively with bronchial hyperresponsiveness. Conclusion Different phases, phenotypes, or both of allergic asthma are associated with distinct priming profiles of inflammatory cells in peripheral blood. Clinical implications Insight in differences of systemic innate responses will lead to better definition of asthma subtypes and to better designs of new therapeutic options.

Summary Inflammatory phenotypes of asthma are associated with differences in disease characteristics. It is unknown whether these inflammatory phenotypes are reflected by the activation status of neutrophils in blood and sputum. We obtained peripheral blood and induced sputum from 21 asthma patients and stratified our samples based on sputum eosinophilia resulting in two groups (>3% eosinophils: n = 13

Introduction Inflammation after trauma is thought to be aggravated by intramedullary nailing (IMN) and predisposes to acute respiratory distress syndrome. Polymorphonuclear granulocytes (PMNs) are the main effector cells in this process. However, in patients with a femur fracture, the injury severity was the decisive factor for the PMN phenotype. A tibia fracture is often caused by a more moderate injury and might allow for a window to assess the innate immune response caused by IMN. Methods A consecutive series of patients with a tibia fracture were included. The innate immune response was measured before and after IMN by plasma interleukin 6, PMN Mac1, and active FcγRII (FcγRII*) expression both before and after fMLF (N-formylmethionyl-leucyl-phenylalanine) stimulation. Furthermore, HLA-DR on monocytes was analyzed. Results Twenty-five consecutive patients were included. Polymorphonuclear granulocyte fMLF-induced Mac1 and FcγRII* were decreased. In concordance, HLA-DR expression on monocytes was decreased in patients compared with control subjects. Intramedullary nailing was associated with a further decrease of HLA-DR-positive monocytes, whereas no changes in PMN phenotype or plasma interleukin 6 levels were observed. Conclusion Intramedullary nailing of a tibial fracture did not affect the PMN phenotype. The impact from injury determined the PMN phenotype. In contrast, the monocyte phenotype changed after the additional insult by IMN in patients with an isolated tibial fracture.

Summary Chronic inflammation of the airways is a hallmark of chronic obstructive pulmonary disease (COPD). We investigated the kinetics of priming of inflammatory cells in peripheral blood during exacerbations of COPD and during the resolution phase. Modulation of the leukocyte compartment as a consequence of systemic activation by cytokines/chemokines was determined by measuring the expression of priming-associated epitopes by novel antibodies designated A17 and A27. Furthermore, H 2 O 2 was determined in breath condensate as a read out for local inflammation. Leukocytes were obtained from COPD patients (GOLD II–IV) during and after an exacerbation of their disease. During an exacerbation the expression of priming epitopes on leukocytes was increased. This priming phenotype disappeared upon treatment with intravenous corticosteroids. Similarly, H 2 O 2 levels in breath condensate were also increased during an exacerbation and decreased upon treatment. We conclude that the activation status of neutrophils in the systemic compartment can be used as a read-out for systemic innate immune signals involved in the pathogenesis of COPD. The correlation between H 2 O 2 in exhaled air with A27 priming on neutrophils showed that local inflammation has systemic effects on cells of the innate immune system.

Interleukin (IL)-5 is a hematopoietic cytokine able to regulate differentiation, survival, and effector functions of eosinophils. It binds specifically to its receptor, which is composed of a cytokine-specific α-chain and a β-chain shared with the receptors for IL-3 and the granulocyte macrophage-colony stimulating factor. The molecular mechanisms by which IL-5 modulates eosinophil survival remain unclear. In this study, we demonstrate that IL-5 withdrawal induces eosinophil apoptosis through a mitochondria-dependent pathway, independently of Fas receptor activation. The lipid kinase phosphatidylinositol-3 kinase plays a crucial role in the maintenance of eosinophil survival, as inhibition of its activity results in apoptosis. IL-5 induces phosphorylation and thus, inhibition of the Forkhead transcription factor FOXO3a and glycogen synthase kinase 3 (GSK-3). We analyzed expression of FOXO3a-dependent transcriptional targets: Fas ligand or Bim (a proapoptotic Bcl-2 family member), but neither was detected in apoptotic eosinophils. We further show that GSK-3 is activated after IL-5 withdrawal, and inhibition of its activity rescues eosinophils from apoptosis. β-catenin, a direct GSK-3 substrate, is present in the nucleus of IL-5-stimulated eosinophils, but it is translocated to the plasma membrane in the absence of cytokine in a GSK-3-dependent manner. This is the first report describing a potential role for GSK-3 and β-catenin in regulating eosinophil survival and suggests a novel mechanism by which IL-5 inhibits the constitutive apoptotic program in these cells.

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in children. It is associated with increased neutrophil numbers in the airway. In this study, we assessed whether this ssRNA virus can directly influence granulocyte longevity. By culturing RSV with granulocytes, it was observed that virus delays both constitutive neutrophil and eosinophil apoptosis. Using pharmacological inhibitors, the RSV-induced delay in neutrophil apoptosis was found to be dependent on both PI3K and NF-κB, but not p38 MAPK or MEK1/MEK2 activation. Using blocking Abs and a reporter cell line, we were able to exclude TLR4 as the receptor responsible for mediating RSV-induced delay in neutrophil apoptosis. The antiapoptotic effect was abrogated by preincubation with the lysosomotropic agent chloroquine, indicating the requirement for endolysosomal internalization. Furthermore, addition of ssRNA, a ligand for the intracellular TLR7/TLR8, also inhibited neutrophil apoptosis, suggesting that intracellular TLRs could be involved in induction of the antiapoptotic effect. Using the BioPlex cytokine detection assay (Bio-Rad), we found that IL-6 was present in supernatants from RSV-exposed neutrophils. IL-6 was found to inhibit neutrophil apoptosis, suggesting that there is an autocrine or paracrine antiapoptotic role for IL-6. Finally, RSV treatment of neutrophils resulted in increased expression of the antiapoptotic Bcl-2 protein Mcl-1. Taken together, our findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intracellular TLRs in mediating these effects.

SummaryRespiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease (LRTD) in infants. Eosinophils have been suggested to play a role in the disease pathogenesis of LRTD. Inflammation can induce functional and morphological alterations of peripheral blood granulocytes. In patients with RSV LRTD, we aimed to investigate the eosinophil activation status by analysing surface markers. In vitro stimulation of eosinophils with cytokines leads to up-regulation of CD11b and priming markers recognized by the recently developed priming markers A17 and A27, whereas interleukin (IL)-5Rα is being down-regulated. In 51 patients and 10 controls we examined the expression of these surface markers on eosinophils in moderate to severe RSV-induced LRTD patients at the time of admission and 6 weeks later during the convalescence phase. RSV-patients were characterized by a higher eosinophil CD11b expression compared to controls. Although basal A17 and A27 expression was not increased, we observed a significantly higher expression of these priming epitopes on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated cells of RSV patients compared with cells of controls, indicative of prior in vivo priming. Furthermore, IL-5Rα expression was down-regulated on peripheral blood eosinophils of these patients. Follow-up blood samples showed normalization of all markers but CD11b, which was persistently increased. Utilizing cellular markers, we observed that peripheral blood eosinophils from infants with RSV LRTD are in a more activated state compared to eosinophils of controls, which normalizes only partially during convalescence.

Background Eosinophils isolated from the blood of patients with allergic asthma exhibit enhanced responsiveness to multiple stimuli compared with cells from normal controls, a phenomenon generally referred to as priming . This priming response is essential for optimal activation with augmented responses including chemotaxis, cytotoxicity, respiratory burst, and the release of proinflammatory lipid mediators. Objective To monitor the kinetics of priming of eosinophils in the peripheral blood and in the bronchoalveolar lavage fluid of patients with allergic asthma before and after allergen challenge. Methods Priming of blood eosinophils obtained from patients with allergy and donors without allergy was measured by labeling with monoclonal phage antibodies A17 and A27 recognizing priming-associated epitopes on phagocytes. In addition, blood and bronchoalveolar lavage fluid eosinophils from subjects with allergy after segmental and whole lung allergen challenge were similarly analyzed. Results A dose-dependent cytokine-induced upregulation of priming-associated epitopes on blood eosinophils was found. Patients with allergic asthma exhibited an in vivo partially primed eosinophil phenotype, which is further primed in vitro after cytokine or chemokine incubation. Priming was increased in peripheral blood 6 hours after whole lung challenge as well as after segmental allergen challenge. Interestingly, eosinophils obtained from the bronchoalveolar lavage fluid 48 hours after segmental allergen challenge exhibited a higher primed phenotype. Conclusion These data are consistent with a model in which local allergic inflammatory reactions induce partial systemic eosinophil priming in the peripheral blood. Eosinophils found in the airway are highly primed, consistent with the markedly upregulated inflammatory capacity observed in these cells.

During the late-phase asthmatic response, eosinophils migrate to the bronchial tissue and cause severe damage. In this study we compared in vivo primed eosinophils from patients with allergic asthma with eosinophils from healthy control subjects in their adhesion behavior to tumor necrosis factor-alpha-activated endothelium under flow conditions (0.8 dyn/cm2). More eosinophils from patients with asthma adhered to activated endothelium, compared with cells from healthy control subjects (1,237 +/- 126 versus 887 +/- 94 cells/mm2, respectively). In the presence of blocking antibodies directed against very late antigen-4 and E-selectin, the residual binding of the cells of individuals with allergic asthma was significantly higher than that of the healthy control subjects (353 +/- 64 versus 123 +/- 31 cells/mm2, respectively, P < 0.02). In addition, secondary tethering or formation of clusters of the eosinophils of patients with allergic asthma was significantly increased compared with the healthy control subjects (cluster indices 1.8 +/- 0.3 versus 0.8 +/- 0.2, respectively, P < 0.05). Because patient cells showed an enhanced interaction with platelets during the perfusions, the role of P-selectin on platelets was investigated. Blocking antibodies directed against P-selectin reduced the enhanced binding and clustering of eosinophils of patients with allergic asthma. We conclude that P-selectin-bearing platelets contribute to secondary tethering processes of eosinophils to activated endothelium. Therefore, platelets might play an important role in the chronic inflammatory processes of these patients.

Signal transducers and activators of transcription (STATs) have been reported to play a critical role in the differentiation of several myeloid cell lines, although the importance of STATs in the differentiation of primary human hematopoietic cells remains to be established. Terminal eosinophil differentiation is induced by interleukin-5 (IL-5), which has also been demonstrated to activate STAT5. We have investigated whether STAT5 plays a critical role during eosinophil differentiation using umbilical cord blood–derived CD34+ cells. In this ex vivo system, STAT5 expression and activation are high early during differentiation, and STAT5 protein expression is down-regulated during the final stages of eosinophil differentiation. Retroviral transductions were performed to ectopically express wild-type and dominant-negative STAT5a (STAT5aΔ750) in CD34+ cells. Transduction of cells with STAT5a resulted in enhanced proliferation compared with cells transduced with empty vector alone. Interestingly, ectopic expression of STAT5a also resulted in accelerated differentiation. In contrast, ectopic expression of STAT5aΔ750 resulted in a block in differentiation, whereas proliferation was also severely inhibited. Similar results were obtained with dominant-negative STAT5b. Forced expression of STAT5a enhanced expression of the STAT5 target genes Bcl-2 andp21WAF/Cip1, suggesting they may be important in STAT5a-mediated eosinophil differentiation. These results demonstrate that STAT5 plays a critical role in eosinophil differentiation of primary human hematopoietic cells.

Three human monocyte subsets are recognized with different functions in the immune system: CD14++/CD16- classical monocytes (CM), CD14++/CD16+ intermediate monocytes (IM) and CD14+/CD16++ non-classical monocytes (NCM). Increased IM and NCM percentages have been reported under inflammatory conditions, yet little is known about monocyte subsets at the onset of inflammation. The human endotoxemia model is uniquely capable of studying the first phases of acute inflammation induced by intravenous injection of 2 ng/kg bodyweight lipopolysaccharide (LPS) into healthy volunteers. After that, monocyte subset counts, activation/differentiation status and chemokine levels were studied over 24 h. The numbers of all subsets were decreased by >95% after LPS injection. CM numbers recovered first (3- 6 h), followed by IM (6-8 h) and NCM numbers (8-24 h). Similarly, increased monocyte counts were observed first in CM (8 h), followed by IM and NCM (24 h). Monocytes did not display a clear activated phenotype (minor increase in CD11b and CD38 expression). Plasma levels of CCL2, CCL4 and CX3CL1 closely resembled the cell numbers of CM, IM and NCM, respectively. Our study provides critical insights into the earliest stages of acute inflammation and emphasizes the necessity to stain for different monocyte subsets when studying the role of monocytes in disease, as neither function nor kinetics of the subsets overlap.

Chronic obstructive pulmonary disease (COPD) is a smokinginduced chronic inflammatory disease that affects mainly the lungs. In susceptible subjects airways inflammation becomes autonomous in part through pathogen-associated molecular patterns and tissue-derived damage-associated molecular patterns activating mainly Toll-like receptors expressed by multiple inflammatory cells. The current consensus is that the chronic inflammatory process in patients with COPD requires treatment to stop the ongoing cycle of infection, inflammation, and host tissue damage. The inflammatory response in patients with COPD is dominated by neutrophils, which circulate in greater numbers in patients with COPD. Moreover, there are now compelling data to indicate that these cells play a key pathogenic role in this condition and represent an important therapeutic target. Critically, the neutrophils in patients with COPD are different compared to cells isolated from healthy subjects. In particular, they display (1) aberrant chemotactic responses, (2) enhanced protease content, (3) intrinsic ‘‘priming’’ leading to greater agonist-induced reactive oxygen species generation, (4) enhanced IL-8 and leukotriene B4–driven migration to the airways and lung parenchyma, 3 and (5) a seemingly acquired resistance to the normal antiinflammatory effects of corticosteroids. As a consequence, it is generally agreed that inhibition or ‘‘normalization’’ of neutrophil function in patients with COPD might represent an important therapeutic strategy. Compared with other chronic inflammatory diseases, COPD is characterized by a further and possibly unique feature: oxidative stress, which originates from the inhalation of smoke and cellular sources, such as the phagocyte nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2). This oxidative environment is thought to have a negative effect on myeloid cell function (including the induction of corticosteroid resistance). However, it

Inflammation in allergic asthma is characterized by an influx of eosinophils and the presence of eosinophil products in the bronchial tissue. Orchestration of this inflammatory response is in part mediated by cytokines and chemoattractants, but final activation can require additional stimuli. IgA, the most abundant immunoglobulin at mucosal surfaces, is potentially a potent trigger for eosinophil activation. Previously, we have shown that binding IgA-coated targets is dependent on in vitro stimulation of cells with cytokines. Here, we demonstrate that eosinophils isolated from the blood of allergic asthmatic patients bind IgA beads independently of prior in vitro stimulation. Furthermore, we found that the proinflammatory cytokine, TNF-α, is a potent enhancer of IgA binding to eosinophils from allergic asthmatics, and it does not activate FcαR on eosinophils isolated from normal donors. The difference in IgA binding by FcαRs on normal and patient eosinophils might be explained by the activation of different signal transduction pathways. Studying intracellular signaling, we found an enhanced basal activity of phosphatidylinositol 3-kinase (PI3K) in eosinophils derived from allergic asthmatics. Moreover, inhibition of PI3K in these cells blocked the background and the TNF-α-induced IgA binding completely. In summary, these data demonstrate that the responsiveness of human eosinophils to TNF-α might be an important contribution for fine-tuning the allergic inflammatory reaction. Furthermore, the preactivation of PI3K results in a broader sensitivity to subsequent challenge with inflammatory cytokines.

Increased numbers of eosinophils have been found in bronchoalveolar lavage (BAL) fluid obtained from patients with idiopathic pulmonary fibrosis (IPF). This suggests the presence of one or more cytokines in the lung tissue of patients with IPF, which are involved in the induction of migration of eosinophils towards the pulmonary compartment. To evaluate this hypothesis, we studied migratory responses of blood eosinophils towards BAL fluid. Migratory responses were tested by means of a modified Boyden chamber assay in 21 patients with IPF and 14 healthy controls. Experiments were performed with unprimed eosinophils and in vitro primed eosinophils (preincubated with 10(-11) M granulocyte macrophage-colony-stimulating factor). Changes in intracellular free Ca2+ ([Ca2+]i) in eosinophils in response to BAL fluid were also investigated, to characterize putative chemotaxins further. Chemotactic responses of eosinophils were observed towards BAL fluid from both patients with IPF and controls, provided that the eosinophils were primed. No changes in [Ca2+]i in eosinophils were detected in response to BAL fluid. Furthermore, neither a blocking antibody against interleukin-8 nor one against regulated on activation, normal T-cell, expressed and secreted (RANTES) influenced the response. Since a chemotactic response of in vitro primed eosinophils was also observed towards bronchoalveolar lavage fluid from normals, it was concluded, that in idiopathic pulmonary fibrosis, apart from the presence of a chemotactic factor in the lung tissue, other mechanisms such as priming of eosinophils in the peripheral blood are responsible for the extravasation of eosinophils into the pulmonary compartment. As no changes in [Ca2+]i were observed in the eosinophils after incubation with bronchoalveolar lavage fluid, the chemotaxin responsible for the migratory responses is probably not one of the known eosinophil-activating chemokines.

Although it is known that many stimuli can activate mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinases (PI3K) in human neutrophils, little is known concerning either the mechanisms or function of this activation. We have utilized a selective inhibitor of MAPK kinase (MEK), PD098059, and two inhibitors of PI3K, wortmannin and LY294002, to investigate the roles of these kinases in the regulation of neutrophil effector functions. Granulocyte/macrophage colony-stimulating factor, platelet-activating factor (PAF) and N-formylmethionyl-leucyl-phenylalanine are capable of activating both p44ERK1 and p42ERK2 MAPKs and phosphotyrosine-associated PI3K in human neutrophils. The activation of extracellular signal-related protein kinases (ERKs) is correlated with the activation of p21ras by both tyrosine kinase and G-protein-coupled receptors as measured by a novel assay for GTP loading. Wortmannin and LY294002 inhibit, to various degrees, superoxide generation, neutrophil migration and PAF release. Incubation with PD098059, however, inhibits only the PAF release stimulated by serum-treated zymosan. This demonstrates that, while neither MEK nor ERK kinases are involved in the activation of respiratory burst or neutrophil migration, inhibition of PAF release suggests a potential role in the activation of cytosolic phospholipase A2. PI3K isoforms, however, seem to have a much wider role in regulating neutrophil functioning.