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IVIG preparations consisting of pooled IgG are increasingly used for the treatment of autoimmune diseases. IVIG is known to regulate the viability of immune cells, including neutrophils. We report that plasma-derived IgA efficiently triggers death of neutrophils primed by cytokines or TLR agonists. IgA-mediated programmed neutrophil death was PI3K-, p38 MAPK-, and JNK-dependent and evoked anti-inflammatory cytokines in macrophage cocultures. Neutrophils from patients with acute Crohn's disease, rheumatoid arthritis, or sepsis were susceptible to both IgA- and IVIG-mediated death. In contrast to IVIG, IgA did not promote cell death of quiescent neutrophils. Our findings suggest that plasma-derived IgA might provide a therapeutic option for the treatment of neutrophil-associated inflammatory disorders., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Cytokine-primed neutrophils can undergo a nonapoptotic type of cell death using components of the necroptotic pathway, including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase-like (MLKL) and NADPH oxidase. In this report, we provide evidence for a potential role of serine proteases in CD44-mediated necroptotic death of GM-CSF-primed human neutrophils. Specifically, we observed that several inhibitors known to block the enzymatic function of fibroblast activation protein-α (FAP-α) were able to block CD44-mediated reactive oxygen species production and cell death, but not FAS receptor-mediated apoptosis. To understand how FAP-α is involved in this nonapoptotic death pathway, we performed immunoblotting experiments in the presence and absence of inhibitors of RIPK3, MLKL, p38 MAPK, PI3K, and FAP-α. The results of these experiments suggested that FAP-α is active in parallel with RIPK3, MLKL, and p38 MAPK activation but proximal to PI3K and NADPH oxidase activation. Interestingly, neutrophils isolated from the joints of patients suffering from rheumatoid arthritis underwent a GM-CSF-independent necroptosis following CD44 ligation; this effect was also blocked by both FAP-α and MLKL inhibitors. Taken together, our evidence shows that the RIPK3-MLKL pathway activates NADPH oxidase but requires, in addition to p38 MAPK and PI3K, a serine protease activity, whereby FAP-α is the most likely candidate. Thus, FAP-α could be a potential drug target in neutrophilic inflammatory responses to avoid exaggerated nonapoptotic neutrophil death, leading to tissue damage., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Apoptosis is the most common form of neutrophil death under both physiological and inflammatory conditions. However, forms of nonapoptotic neutrophil death have also been observed. In the current study, we report that human neutrophils undergo necroptosis after exposure to GM-CSF followed by the ligation of adhesion receptors such as CD44, CD11b, CD18, or CD15. Using a pharmacological approach, we demonstrate the presence of a receptor-interacting protein kinase-3 (RIPK3)-a mixed lineage kinase-like (MLKL) signaling pathway in neutrophils which, following these treatments, first activates p38 MAPK and PI3K, that finally leads to the production of high levels of reactive oxygen species (ROS). All these steps are required for necroptosis to occur. Moreover, we show that MLKL undergoes phosphorylation in neutrophils in vivo under inflammatory conditions. This newly identified necrosis pathway in neutrophils would imply that targeting adhesion molecules could be beneficial for preventing exacerbation of disease in the neutrophilic inflammatory response., (Copyright © 2016 by The American Association of Immunologists, Inc.)

FcαRI (CD89), the human Fc receptor for IgA, is highly expressed on neutrophil granulocytes. In this study, we show that FcαRI induces different forms of neutrophil death, depending on the inflammatory microenvironment. The susceptibility of inflammatory neutrophils from sepsis or rheumatoid arthritis toward death induced by specific mAb, or soluble IgA at high concentrations, was enhanced. Although unstimulated cells experienced apoptosis following anti-FcαRI mAb stimulation, preactivation with cytokines or TLR agonists in vitro enhanced FcαRI-mediated death by additional recruitment of caspase-independent pathways, but this required PI3K class IA and MAPK signaling. Transmission electron microscopy of FcαRI-stimulated cells revealed cytoplasmic changes with vacuolization and mitochondrial swelling, nuclear condensation, and sustained plasma membrane. Coculture experiments with macrophages revealed anti-inflammatory effects of the partially caspase-independent death of primed cells following FcαRI engagement. Our data suggest that FcαRI has the ability to regulate neutrophil viability and to induce different forms of neutrophils depending on the inflammatory microenvironment and specific characteristics of the ligand-receptor interactions. Furthermore, these findings have potential implications for FcαRI-targeted strategies to treat neutrophil-associated inflammatory diseases., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Basophils are primarily associated with a proinflammatory and immunoregulatory role in allergic diseases and parasitic infections. Recent studies have shown that basophils can also bind various bacteria both in the presence and the absence of opsonizing Abs. In this report, we show that both human and mouse basophils are able to produce mitochondrial reactive oxygen species and to form extracellular DNA traps upon IL-3 priming and subsequent activation of the complement factor 5 a receptor or FcεRI. Such basophil extracellular traps (BETs) contain mitochondrial, but not nuclear DNA, as well as the granule proteins basogranulin and mouse mast cell protease 8. BET formation occurs despite the absence of any functional NADPH oxidase in basophils. BETs can be found in both human and mouse inflamed tissues, suggesting that they also play a role under in vivo inflammatory conditions. Taken together, these findings suggest that basophils exert direct innate immune effector functions in the extracellular space., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Leukotriene B(4) (LTB(4)) is an important proinflammatory lipid mediator generated by neutrophils upon activation. GM-CSF stimulation is known to enhance agonist-mediated LTB(4) production of neutrophils within minutes, a process called "priming". In this study, we demonstrate that GM-CSF also limits the production of LTB(4) by neutrophils via a transcriptional mechanism at later time points. We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient (Rhoh(-/-)) mice demonstrated increased LTB(4) production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB(4) upon activation compared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses.

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

Mast cells (MC), supposedly long-lived cells, play a key role in allergy and are important contributors to other inflammatory conditions in which they undergo hyperplasia. In humans, stem cell factor (SCF) is the main regulator of MC growth, differentiation, and survival. Although human MC numbers may also be regulated by apoptotic cell death, there have been no reports concerning the role of the extrinsic apoptotic pathway mediated by death receptors in these cells. We examined expression and function of death receptors for Fas ligand and TRAIL in human MC. Although the MC leukemia cell line HMC-1 and human lung-derived MC expressed both Fas and TRAIL-R, MC lines derived from cord blood (CBMC) expressed only TRAIL-R. Activation of TRAIL-R resulted in caspase 3-dependent apoptosis of CBMC and HMC-1. IgE-dependent activation of CBMC increased their susceptibility to TRAIL-mediated apoptosis. Results suggest that TRAIL-mediated apoptosis may be a mechanism of regulating MC survival in vivo and, potentially, for down-regulating MC hyperplasia in pathologic conditions.

Leptin regulates food intake as well as metabolic, endocrine, and immune functions. It exerts proliferative and antiapoptotic activities in a variety of cell types, including T cells. Leptin also stimulates macrophages and neutrophils, and its production is increased during inflammation. In this study, we demonstrate that human neutrophils express leptin surface receptors under in vitro and in vivo conditions, and that leptin delays apoptosis of mature neutrophils in vitro. The antiapoptotic effects of leptin were concentration dependent and blocked by an anti-leptin receptor mAb. The efficacy of leptin to block neutrophil apoptosis was similar to G-CSF. Using pharmacological inhibitors, we obtained evidence that leptin initiates a signaling cascade involving PI3K- and MAPK-dependent pathways in neutrophils. Moreover, leptin delayed the cleavage of Bid and Bax, the mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase, as well as the activation of both caspase-8 and caspase-3 in these cells. Taken together, leptin is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in inflammatory diseases.

Eosinophils are present in parasitic, allergic, various immunological, and malignant disorders as well as in a variety of idiopathic hypereosinophilic syndromes. However, their exact role in some of these conditions remains elusive. They can be activated both in vivo and in vitro by various agonists, such as Igs, lipid mediators, and cytokines. By phenotyping the surface of the eosinophils, it may be possible to better define their function(s) in different pathophysiological settings. In the present work we screened eosinophils with a panel of Abs recognizing CD2 subfamily receptors usually present on a number of hemopoietic cells. We have demonstrated that human peripheral blood eosinophils, but not basophils or neutrophils, express NTB-A. In addition eosinophils express 2B4, CD84, CD58, and CD48, but not signaling lymphocytic activation molecule or CD2, on their surface (FACS). Cross-linking of 2B4 on eosinophils elicited a significant release of eosinophil peroxidase (30 min), IFN-gamma, and IL-4 (18 h). Moreover, activation of eosinophils via 2B4 induced eosinophil-mediated cytotoxicity toward two malignant cell lines, i.e., mouse mastocytoma P815 and EBV-infected 721.221 B cell lines. Cross-linking of 2B4 on the surface of eosinophils or pervenadate treatment elicited ERK and tyrosine phosphorylation, respectively. Furthermore, we showed that eosinophils express slam-associated protein. The demonstration that human eosinophils express a functional 2B4 receptor indicates a broader role for these cells in health and disease.

IL-13 is an immunoregulatory and effector cytokine in allergic diseases such as bronchial asthma. A variety of immune and non-immune cells are known as IL-13 producers. In this study we investigated whether and under what conditions human eosinophils generate IL-13. Freshly isolated highly purified peripheral blood eosinophils from patients with several eosinophilic inflammatory diseases and from normal control individuals were investigated. We observed that blood eosinophils from patients suffering from bronchial asthma, atopic dermatitis, parasitic infections, hypereosinophilic syndrome, and idiopathic eosinophilic esophagitis expressed IL-13, as assessed by ELISA, ELISPOT assay, flow cytometry, and immunocytochemistry. By using nasal polyp tissues and immunohistochemistry, we demonstrated IL-13 expression in eosinophils under in vivo conditions. In contrast, blood eosinophils from control individuals as well as blood neutrophils from both eosinophilic and control patients did not produce detectable IL-13 levels. However, when blood eosinophils from control individuals were stimulated with GM-CSF or IL-5 in vitro, they generated IL-13 mRNA and protein, suggesting that IL-13 expression by eosinophils under inflammatory conditions is a cytokine-driven process. Stimulation of blood eosinophils containing IL-13 by eotaxin resulted in a rapid release of this cytokine. Eosinophil-derived IL-13 was functional, as it increased the surface expression of the low affinity IgE receptor (CD23) on purified B cells. In conclusion, human eosinophils are able to produce and release functional IL-13 in eosinophilic inflammatory responses.

Eosinophils contain in their granules eosinophil cationic protein (ECP) and other basic proteins that have been implicated in immunity to parasites and pathophysiology of chronic allergic responses. In a model of eosinophil degranulation, we show that eosinophils release ECP upon short-term GM-CSF priming and stimulation with either platelet-activating factor (PAF) or the anaphylatoxin C5a, but not eotaxin. Restimulation with the same agonist (PAF or C5a) was unsuccessful as assessed by monitoring intracellular calcium concentration and ECP release. In contrast, upon an intermediate washing step, eosinophils rapidly transduced PAF and C5a signals followed by significant ECP releases. Ligand-binding studies demonstrated that only a proportion of PAF receptors is internalized upon cell stimulation and that washing of the cells removes the agonist from the cell surface. Upon repetitive stimulation, eosinophils with less than 50% of the original ECP content were obtained. Such eosinophils did not increase cellular ECP levels even in the presence of the eosinophil survival factor GM-CSF in overnight cultures. In vivo studies revealed that eosinophils always express detectable amounts of ECP under chronic inflammatory conditions. In conclusion, we have shown that eosinophils maintain their capacity to degranulate upon repetitive stimulation with the same agonist as long as the receptor is not occupied from a previous stimulation. The cellular content of ECP appears to be a no limiting factor in the case of repetitive stimulation, implying that mature eosinophils may not require a significant ECP resynthesis.

In allergic inflammations of the skin, activation of CD4+ T cells was demonstrated to play an important role; however, a minor role for CD8+ T cells is implied. In the present study, we compared cutaneous lymphocyte-associated Ag (CLA)-expressing CD4+ and CD8+ subsets, which were isolated from peripheral blood and lesional skin biopsies in atopic dermatitis (AD) patients. We demonstrated that CD8+CLA+ T cells proliferate in response to superantigen and are as potent as CD4+CLA+ T cells in IgE induction and support of eosinophil survival. In atopic skin inflammation, the existence of high numbers of CD4+ and CD8+ T cells was demonstrated by immunohistochemistry and by culturing T cells from skin biopsies. In peripheral blood, both CD4+ and CD8+ subsets of CLA+CD45RO+ T cells were in an activated state in AD. The in vivo-activated CLA+ T cells of both subsets spontaneously released an IL-5- and IL-13-dominated Th2 type cytokine pattern. This was confirmed by intracytoplasmic cytokine staining immediately after isolation of the cells from peripheral blood. In consequence, both CD4+ and CD8+, CLA+ memory/effector T cells induced IgE production by B cells mainly by IL-13, and enhanced eosinophil survival in vitro by delaying eosinophil apoptosis, mainly by IL-5. These results indicate that in addition to the CD4+ subset, the CD8+CLA+ memory/effector T cells are capable of responding to superantigenic stimulation and play an important role in the pathogenesis of AD.

Development of IgE-mediated allergic conditions is dependent on the secretion of a Th2 cytokine pattern, including IL-4, IL-5, and IL-13. The induction of anergy would be one mechanism to abrogate cytokine secretion by Th2 cells, which may be pivotal to the allergic response. We demonstrate here that incubation of cloned human CD4+ phospholipase A2 (PLA)-specific Th2 cells with antigenic peptide, in the absence of professional APC, results in a state of nonresponsiveness. The anergic T cells failed to proliferate or secrete IL-4 in response to optimal stimulation with PLA and autologous, professional APC. Secretion of IL-5 and IL-13, however, was only partially inhibited. The anergic state of the Th2 cells was not associated with CD3 or CD28 down-regulation. However, anergy did appear to be closely related to alterations in signaling pathways, mediated through the TCR, of the cells. In contrast to untreated Th2 cells, anergized Th2 cells failed to respond to anti-CD3 mAb with either increased tyrosine kinase activity or increased levels of tyrosine phosphorylation of p56(lck) or ZAP70. A strong and sustained intracellular calcium flux, observed in untreated Th2 cells in response to anti-CD3 mAb, was absent in anergic Th2 cells. Furthermore, the induction of anergy seems to represent an active process, associated with increased levels of basal tyrosine kinase activity, cytokine production, and CD25 up-regulation in anergic Th2 cells. Together, our results indicate that anergy in Th2 cells is associated with defective transmembrane signaling through the TCR.

Nasal polyps, which often occur in association with allergic rhinitis and asthma, are characterized by a marked infiltration of eosinophils. Using a method for detecting eosinophils with DNA strand breaks, we found direct evidence for inhibition of eosinophil apoptosis in this model of tissue eosinophilia. By using Southern blot analysis linked to reverse transcription-PCR, we detected a mRNA signal specific for IL-5 in all nasal polyps. The identification of IL-5 as a major eosinophil survival factor was confirmed by ELISA measurements using tissue homogenates. Moreover, immunohistochemical analysis of the nasal polyp tissues demonstrated that IL-5 was localized in lymphocytes, mast cells, and eosinophils. Treatment of the eosinophil-infiltrated tissue with neutralizing anti-IL-5 mAb induced eosinophil apoptosis and decreased tissue eosinophilia. Therefore, IL-5 may represent an important cytokine responsible for the delay of the death process in eosinophils in nasal polyps. In addition, a previously suggested IL-4-dependent specific recruitment of eosinophils into the inflamed tissue could be excluded by our studies. Taken together, these findings suggest a novel mechanism by which eosinophils specifically accumulate in pathologic human tissues.

Eosinophils possess the capacity to synthesize various cytokines. We demonstrate that IL-8 mRNA and protein are constitutively expressed by freshly isolated resting human eosinophils. Most of the patients with bronchial asthma or atopic dermatitis show evidence for up-regulated IL-8 protein expression in eosinophils but not in neutrophils, suggesting that an eosinophil-specific cytokine may act in these patients. To investigate whether the intracellular IL-8 can be released, eosinophils were stimulated by different cytokines and platelet-activating factor. Priming with granulocyte-macrophage CSF and a subsequent 25-min stimulation with RANTES or platelet-activating factor resulted in release of IL-8 from highly purified human eosinophils in vitro. As the eosinophil is the predominant cell in asthmatic inflammation, we determined IL-8 concentrations in bronchoalveolar lavage fluids from normal individuals and asthmatic patients. Bronchoalveolar lavage fluids from patients with bronchial asthma consistently demonstrated high IL-8 concentration compared with the controls. This suggests that IL-8 is released in vivo by inflammatory bronchial cells in asthma.

Platelet-activating factor (PAF) may play a role in the regulation of immune responsiveness and is a potent mediator in asthmatic inflammation. However, as yet, the mechanisms whereby PAF mediates its pleiomorphic effects on immune cells have not been elucidated. Because PAF is a potent chemotactic factor for eosinophils, the presence of receptors for PAF (PAFR) on lymphocytes may provide a mechanism for the concurrent recruitment of both eosinophils and T lymphocytes into the airways of asthmatic patients. To address this issue, we have examined freshly isolated PBMC and granulocytic cells as well as various T and B lymphocyte lines with regards to PAFR expression and PAF-induced changes in intracellular calcium concentration. Using two-color immunofluorescence techniques and highly purified cell populations, it was not possible to detect surface PAFR protein or functional PAFR on resting and in vivo or in vitro activated T and B cells derived from nonallergic individuals or patients with allergic asthma. In addition, we were unable to detect PAFR mRNA, protein, or functional response to PAF in human or murine T cell lines. In contrast, we found functional PAFR in most B lymphoblastoid cell lines. Within the PBMC population, CD14+ cells respond to PAF. These results suggest that PAF does not interact directly with lymphocytes and thus that previous observations suggestive of such an interaction likely reflect the effects of PAF on monocytes. PAF-induced increases in intracellular calcium concentration were also detected in neutrophils and eosinophils, but were lower in granulocytes relative to the levels detected in monocytes.