p53; ROS; NCF2/p67phox; NADPH oxidase; NOX2
In the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species–dependent manner. Under inflammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identified a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D.
It has been suggested that Fas ligand–Fas receptor interactions are involved in the regulation of eosinophil apoptosis and that dysfunctions in this system could contribute to the accumulation of these cells in allergic and asthmatic diseases. Here, we demonstrate that nitric oxide (NO) specifically prevents Fas receptor–mediated apoptosis in freshly isolated human eosinophils. In contrast, rapid acceleration of eosinophil apoptosis by activation of the Fas receptor occurs in the presence of eosinophil hematopoietins. Analysis of the intracellular mechanisms revealed that NO disrupts Fas receptor–mediated signaling events at the level of, or proximal to, Jun kinase (JNK), but distal to sphingomyelinase (SMase) activation and ceramide generation. In addition, activation of SMase occurs downstream of an interleukin 1 converting enzyme–like (ICE-like) protease(s) that is not blocked by NO. However, NO prevents activation of a protease that targets lamin B1. These findings suggest a role for an additional NO-sensitive apoptotic signaling pathway that amplifies the proteolytic cascade initialized by activation of the Fas receptor. Therefore, NO concentrations within allergic inflammatory sites may be important in determining whether an eosinophil survives or undergoes apoptosis upon Fas ligand stimulation.
Because of advances in our understanding of the hypereosinophilic syndrome (HES) and the availability of novel therapeutic agents, the original criteria defining these disorders are becoming increasingly problematic. Here, we discuss shortcomings with the current definition of HES and recent developments in the classification of these disorders. Despite significant progress in our understanding of the pathogenesis of some forms of HES, the current state of knowledge is still insufficient to formulate a new comprehensive etiologic definition of HESs. Nevertheless, we suggest a new working definition that overcomes some of the most obvious limitations with the original definition.
Definition; eosinophilia; eosinophilic leukemia; hypereosinophilic syndromes
Asthma is a heterogeneous inflammatory airway disorder which involves eosinophilic and non-eosinophilic phenotypes. Unlike in normal lungs, eosinophils are often present in atopic asthmatic airways although a subpopulation of asthmatic subjects predominantly develops neutrophilic inflammation. Recently, it has been demonstrated that eosinophils and neutrophils generate bactericidal extracellular traps consisting of DNA and cytotoxic granule proteins.
To explore if living eosinophils and neutrophils infiltrating human atopic asthmatic airways actively form extracellular DNA traps in vivo.
Quantitative analysis of eosinophils-releasing DNA was performed in endobronchial biopsies from 20 mild human atopic asthmatics at baseline and after local allergen challenge, and 10 normal subjects. DNA was stained with propidium iodine and major basic protein (MBP) with specific antibody. Differential cell counts and cytokines/chemokines were assessed in bronchoalveolar fluids.
Asthmatic airways were infiltrated with a significantly higher number of eosinophils than normal airways (39.3±4.6 vs. 0.4±0.9, p<0.0001). All asthmatics but only one control subject expressed eosinophils releasing DNA that colocalized with MBP (33.65±20.33 vs. 0.3±0.9 per hpf, p<0.0001). Four asthmatics mostly expressed neutrophilic inflammation and neutrophil DNA traps. Allergen challenge had no significant quantitative effect on eosinophil or neutrophil DNA traps. Airway eosinophils or DNA traps did not correlate with either BAL levels of IL-5, IFN-γ, or eotaxin, or the provoking doses of methacholine or allergen in asthmatics.
Extracellular DNA traps are generated by eosinophils and neutrophils in human atopic asthmatic airways in vivo. The mechanism and role of this new finding will necessitate further investigation.
Eosinophil extracellular traps (EETs) (1) are frequently seen in atopic asthma, (2) consist of DNA and granule proteins, and (3) might be a new useful biomarker reflecting eosinophil activation.
Atopic asthma; BAL fluid; biomarker; eosinophils; extracellular DNA traps; human; neutrophils
Polymorphonuclear neutrophils (PMN) play a key role in host defences against invading microorganisms but can also potentiate detrimental inflammatory reactions in case of excessive or misdirected responses. Intravenous immunoglobulins (IVIg) are used to treat patients with immune deficiencies and, at higher doses, in autoimmune, allergic and systemic inflammatory disorders.
We used flow cytometry to examine the effects of IVIg on PMN functions and survival, using whole-blood conditions in order to avoid artifacts due to isolation procedures. IVIg at low concentrations induced PMN activation, as reflected by decreased L-selectin and increased CD11b expression at the PMN surface, oxidative burst enhancement, and prolonged cell survival. In contrast, IVIg at higher concentrations inhibited LPS-induced CD11b degranulation and oxidative burst priming, and counteracted LPS-induced PMN lifespan prolongation.
IVIg appears to have differential, concentration-dependent effects on PMN, possibly supporting the use of IVIg as either an anti-microbial or an anti-inflammatory agent.
Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.
antisense; cancer; cytokines; differentiation; infection
Clinical and histologic similarities between various eczematous disorders point to a common efferent pathway. We demonstrate here that activated T cells infiltrating the skin in atopic dermatitis (AD) and allergic contact dermatitis (ACD) induce keratinocyte (KC) apoptosis. KCs normally express low levels of Fas receptor (FasR) that can be substantially enhanced by the presence of IFN-γ. KCs are rendered susceptible to apoptosis by IFN-γ when FasR numbers reach a threshold of approximately 40,000 per KC. Subsequently, KCs undergo apoptosis induced by anti-FasR mAb’s, soluble Fas ligand, supernatants from activated T cells, or direct contact between T cells and KCs. Apoptotic KCs show typical DNA fragmentation and membrane phosphatidylserine expression. KC apoptosis was demonstrated in situ in lesional skin affected by AD, ACD, and patch tests. Using numerous cytokines and anti-cytokine neutralizing mAb’s, we found no evidence that cytokines other than IFN-γ participate in this process. In addition, apoptosis-inducing pathways other than FasR triggering were ruled out by blocking T cell–induced KC apoptosis by caspase inhibitors and soluble Fas-Fc protein. Responses of normal human skin and cultured skin equivalents to activated T cells demonstrated that KC apoptosis caused by skin-infiltrating T cells is a key event in the pathogenesis of eczematous dermatitis.
Previous evidence indicates that inhalation of lipopolysaccharide (LPS)-containing with allergens induced mixed Th1 and Th17 cell responses in the airways. Extracellular vesicles (EVs) are nanometer-sized spherical, lipid-bilayered structures and are recently in the public eye as an intercellular communicator in immune responses.
To evaluate the role of EVs secreted by LPS inhalation in the development of airway immune dysfunction in response to allergens.
Extracellular vesicles in bronchoalveolar lavage fluids of BALB/c mice were isolated and characterized 24 h after applications to the airway of 10 μg of LPS for 3 days. To evaluate the role of LPS-induced EVs on the development of airway immune dysfunction, in vivo and in vitro experiments were performed using the isolated LPS-induced EVs.
The inhalation of LPS enhanced EVs release into the BAL fluid, when compared to the application of PBS. Airway sensitization with allergens and LPS-induced EVs resulted in a mixed Th1 and Th17 cell responses, although that with allergens and PBS-induced EVs induced immune tolerance. In addition, LPS-induced EVs enhanced the production of Th1- and Th17-polarizing cytokines (IL-12p70 and IL-6, respectively) by lung dendritic cells. Moreover, the immune responses induced by the LPS-induced EVs were blocked by denaturation of the EV-bearing proteins.
These data suggest that EVs (especially, the protein components) secreted by LPS inhalation are a key intercellular communicator in the development of airway immune dysfunction to inhaled LPS-containing allergens.
asthma; airway immune dysfunction; extracellular vesicles; lipopolysaccharide
Human induced pluripotent stem cells (iPSCs) possess remarkable self-renewal capacity and the potential to differentiate into novel cell types, such as mesenchymal stem cells (MSCs). iPSC-MSCs have been shown to enhance tissue regeneration and attenuate tissue ischaemia; however, their contribution to the immune regulation of Th2-skewed allergic rhinitis (AR) and asthma remains unclear.
This study compared the immunomodulatory effects of iPSC-MSCs and bone marrow-derived MSCs (BM-MSCs) on lymphocyte proliferation, T-cell phenotypes and cytokine production in peripheral blood mononuclear cells (PBMCs) in patients with AR, and investigated the possible molecular mechanisms underlying the immunomodulatory properties of iPSC-MSCs.
In co-cultures of PBMCs with iPSC-MSCs or BM-MSCs, lymphocyte proliferation was evaluated using 3H-thymidine (3H-TdR) uptake, carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) assays; the regulatory T-cell (Treg) phenotype was determined by flow cytometry, and cytokine levels were measured using an enzyme-linked immunosorbent assay. The immunomodulatory properties of both MSCs were further evaluated using NS398 and transwell experiments.
Similar to BM-MSCs, we determined that iPSC-MSCs significantly inhibit lymphocyte proliferation and promote Treg response in PBMCs (P < 0.05). Accordingly, the cytokine milieu (IFN-γ, IL-4, IL-5, IL-10 and IL-13) in the supernatants of PBMCs changed significantly (P < 0.05). The immunomodulatory properties of iPSC-MSCs and BM-MSCs were associated with prostaglandin E2 (PGE2) production and cell–cell contact.
These data demonstrate that iPSC-MSCs are capable of modulating T-cell phenotypes towards Th2 suppression through inducing Treg expansion, suggesting that iPSC-MSCs can be used as an alternative candidate to adult MSCs to treat allergic airway diseases.
allergic rhinitis; immunomodulation; induced pluripotent stem cells; mesenchymal stem cells; T cell
Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.
The protein ATG5 is known to be involved in the formation of autophagosomes. Here, Maskey et al. identify a new role of ATG5 in response to drug-induced DNA damage whereby ATG5 translocates to the nucleus, leading to chromosome misalignment and mitotic catastrophe.
Eosinophils are blood cells that are often found in high numbers in the tissues of allergic conditions and helminthic parasite infections. The pathophysiological roles that eosinophils may serve in other human ‘eosinophil-associated’ diseases remain obscure.
NIH Institutes and the Office of Disease Prevention assembled an international taskforce of clinical and basic scientists with the charge to propose and prioritize unmet research needs in eosinophil-associated diseases.
The taskforce used an organ system approach to dissect out the different and common themes of eosinophil cell involvement in these diseases. In early 2012, a draft document was circulated for review. The document was amended and the prioritizations were set at a NIH-organized workshop in June 2012.
The taskforce identified significant research needs. These needs cross disease entities but some are disease-specific. There are substantial shortcomings to the various preclinical animal models, as well as significant gaps in our epidemiologic, pathophysiologic, diagnostic, prognostic and therapeutic knowledge. The taskforce recognized that recent efforts by patient advocacy groups have played instrumental roles in improving the identification and characterization of these disorders. However, communication amongst the eosinophil interested communities, e.g., governmental funding and regulatory agencies, and industry and clinician scientists need to be more comprehensive.
Significant efforts are required to address our knowledge gaps in order to improve the outcomes of eosinophil-associated diseases. NIH Institutes, other federal agencies, lay organizations and the pharmaceutical industry should consider the taskforce’s recommendations in their future research activities.
eosinophil; apoptosis; therapeutics; research; discovery; fusion oncoproteins; hypereosinophilic syndromes; Churg-Strauss syndrome; coagulopathy
Hypereosinophilic syndromes (HESs) are a diverse group of conditions characterized by clinical manifestations attributable to eosinophilia and eosinophilic infiltration of tissues. HESs are chronic disorders with significant morbidity and mortality. Although the availability of targeted chemotherapeutic agents, including imatinib, has improved quality of life and survival in some patients with HESs, additional agents with increased efficacy and decreased toxicity are sorely needed. The purpose of this review is to provide an overview of eosinophil biology with an emphasis on potential targets of pharmacotherapy and to provide a summary of potential eosinophil-targeting agents, including those in development, in clinical trials, or approved for other disorders.
Hypereosinophilic syndromes; eosinophil-associated gastrointestinal disorders; eosinophilic esophagitis; Churg-Strauss syndrome; IL-5; mepolizumab; reslizumab
Eosinophils and their products play an essential role in the pathogenesis of various reactive and neoplastic disorders. Depending on the underlying disease, molecular defect and involved cytokines, hypereosinophilia may develop and may lead to organ damage. In other patients, persistent eosinophilia is accompanied by typical clinical findings, but the causative role and impact of eosinophilia remain uncertain. For patients with eosinophil-mediated organ pathology, early therapeutic intervention with agents reducing eosinophil counts can be effective in limiting or preventing irreversible organ damage. Therefore, it is important to approach eosinophil disorders and related syndromes early by using established criteria, to perform all appropriate staging investigations, and to search for molecular targets of therapy. In this article, we review current concepts in the pathogenesis and evolution of eosinophilia and eosinophil-related organ damage in neoplastic and non-neoplastic conditions. In addition, we discuss classifications of eosinophil disorders and related syndromes as well as diagnostic algorithms and standard treatment for various eosinophil-related disorders.
classification; eosinophilic leukemia; FIP1L1-PDGFRA; hypereosinophilia; hypereosinophilic syndromes; targeted therapy
eosinophil disorders; hypereosinophilic syndrome (HES); global consensus; classification
Hypereosinophilic syndromes (HES) are a heterogeneous group of rare disorders defined by persistent blood eosinophilia ≥1.5 × 109/L, absence of a secondary cause, and evidence of eosinophil-associated pathology. With the exception of a recent multicenter trial of mepolizumab (anti-IL-5 monoclonal antibody), published therapeutic experience has been restricted to case reports and small case series.
The purpose of the study was to collect and summarize baseline demographic, clinical and laboratory characteristics in a large, diverse cohort of patients with HES and to review responses to treatment with conventional and novel therapies.
Clinical and laboratory data from 188 patients with HES, seen between January 2001 and December 2006 at eleven institutions in the United States and Europe, were collected retrospectively by chart review.
Eighteen of 161 patients (11%) tested were FIP1L1-PDGFRA mutation-positive and 29/168 patients tested (17%) had a demonstrable aberrant or clonal T cell population. Corticosteroid monotherapy induced complete or partial responses at 1 month in 85% (120/141) of patients with most remaining on maintenance doses (median 10 mg prednisone equivalent daily for 2 months-20 years). Hydroxyurea and interferon-alpha (used in 64 and 46 patients, respectively) were also effective, but their use was limited by toxicity. Imatinib (used in 68 patients) was more effective in patients with the FIP1L1-PDGFRA mutation (88%) than in those without (23%; p<0.001).
This study, the largest clinical analysis of patients with HES to date, not only provides useful information for clinicians but should stimulate prospective trials to optimize treatment of HES.
eosinophil; hypereosinophilic syndrome; FIP1L1-PDGFRA
Defects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis.
Human primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule.
The addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80–95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls.
Our findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases.
epidermal differentiation; histamine; keratinocyte; skin barrier function; tight junction