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Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule in the cytosol of myeloid cells, required for induction of T-helper cells producing interleukin-17 (Th17 cells) and important in antifungal immunity. In a patient suffering from Candida dubliniensis meningoencephalitis, mutations in the CARD9 gene were found to result in the loss of protein expression. Apart from the reduced numbers of CD4(+) Th17 lymphocytes, we identified a lack of monocyte-derived cytokines in response to Candida strains. Importantly, CARD9-deficient neutrophils showed a selective Candida albicans killing defect with abnormal ultrastructural phagolysosomes and outgrowth of hyphae. The neutrophil killing defect was independent of the generation of reactive oxygen species by the reduced NAD phosphate oxidase system. Taken together, this demonstrates that human CARD9 deficiency results in selective defect in the host defense against invasive fungal infection, caused by an impaired phagocyte killing.

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects

Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.

Accumulation of bioactive lipids during storage of blood products is not cell but plasma derived and temperature dependent

BACKGROUND: Bioactive lipids (lysophosphatidylcholines [lysoPCs]) accumulating during storage of cell‐containing blood products are thought to be causative in onset of transfusion‐related acute lung injury through activation of neutrophils. LysoPCs are thought to be derived from cell membrane degradation products such as phosphatidylcholines (PC) by partial hydrolysis of PC, a process that is catalyzed by phospholipase A2 (PLA2).

Toll-like receptor responses in IRAK-4-deficient neutrophils.

Human neutrophils were found to express all known Toll-like receptors (TLRs) except TLR3 and TLR7. IRAK-4-deficient neutrophils were tested for their responsiveness to various TLR ligands. Essentially all TLR responses in neutrophils, including the induction of reactive oxygen species generation, adhesion, chemotaxis and IL-8 secretion, were found to be dependent on IRAK-4. Surprisingly, the reactivity towards certain established TLR ligands, imiquimod and ODN-CpG, was unaffected by IRAK-4 deficiency, demonstrating their activity is independent of TLR. TLR-4-dependent signaling in neutrophils was totally dependent on IRAK-4 without any major TRIF-mediated contribution. We did not observe any defects in killing capacity of IRAK-4-deficient neutrophils for Staphylococcus aureus, Escherichia coli and Candida albicans, suggesting that microbial killing is primarily TLR independent.

Granulocyte concentrates: Prolonged functional capacity during storage in the presence of phenotypic changes

Granulocyte transfusions may be an effective therapy for neutropenic pediatric patients suffering from life-threatening infections. This study shows that granulocyte concentrates can be stored without loss of in vitro viability and functionality for at least 24 hours. Background Granulocyte transfusion has been proposed as a bridging therapy for patients with prolonged periods of chemotherapy-induced neutropenia, who suffer from severe fungal and bacterial infections. To recover, adequate numbers of granulocytes are required when the patients are refractory to standard treatment. The aim of this study was to assess the functional characteristics and efficacy of granulocyte colony-stimulating factor/dexamethasone-mobilized granulocytes used for transfusions. Design and Methods Granulocytes from the leukapheresis products were tested for the expression of cell-surface antigens, interactions with endothelial cells, motility, killing of microbes and survival. The granulocytes were used in vivo for transfusion in 16 severely ill children, who were – apart from a patient with a granulocyte dysfunction – all suffering from prolonged neutropenia. Results Mobilization of granulocytes with granulocyte colony-stimulating factor and dexamethasone caused phenotypic changes (decreased CD62L expression and increased levels of CD66b and CD177). The ability of the granulocytes to interact with endothelial cells (rolling, adhesion, transmigration) and to kill various types of pathogens was not affected by the mobilization, leukapheresis and irradiation procedures. The granulocytes were functionally indistinguishable from those isolated from untreated donors, even after 24 hours of storage. Granulocyte transfusion seemed to benefit 70% of patients, as 11 out of the 16 children showed clinical recovery within 1–2 weeks after beginning the transfusions. Conclusions Although CD62L expression is downregulated on granulocytes used for granulocyte transfusions, concomitant CD177 upregulation may explain the intact interactions with endothelial cells. All other granulocyte functions tested were intact, including the ability to kill fungi. Granulocyte concentrates can be stored without loss of in vitro viability and functionality for at least 24 hours. As demonstrated in vivo, granulocyte transfusions may be an effective therapy for neutropenic pediatric patients suffering from life-threatening infections.

Changes in gene expression of granulocytes during in vivo granulocyte colony-stimulating factor/dexamethasone mobilization for transfusion purposes

The treatment of healthy donors with granulocyte colony-stimulating factor (G-CSF) and dexamethasone results in sufficient numbers of circulating granulocytes to prepare granulocyte concentrates for clinical purposes. Granulocytes obtained in this way demonstrate relatively normal functional behavior combined with a prolonged life span. To study the influence of mobilizing agents on granulocytes, we used oligonucleotide microarrays to identify genes that are differentially expressed in mobilized granulocytes compared with control granulocytes. More than 1000 genes displayed a differential expression pattern, with at least a 3-fold difference. Among these, a large number of genes was induced that encode proteins involved in inflammation and the immune response, such as C-type lectins and leukocyte immunoglobulin-like receptors. Because mobilized granulocytes have a prolonged life span, we focused on genes involved in the regulation of apoptosis. One of the most prominent among these was CAST, the gene encoding calpastatin. Calpastatins are the endogenous inhibitors of calpains, a family of calcium-dependent cysteine proteases recently shown to be involved in neutrophil apoptosis. Transcriptional activity of the CAST gene was induced by G-CSF/dexamethasone treatment both in vivo and in vitro, whereas the protein expression of CAST was stabilized during culture. These studies provide new insight in the genotypic changes as well as in the regulation of the immunologic functions and viability of mobilized granulocytes used for clinical transfusion purposes.

Regulation and kinetics of platelet-activating factor and leukotriene C4 synthesis by activated human basophils

Background Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet‐activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils.

Platelet-activating factor (PAF) acts as an intercellular messenger in the changes of cytosolic free Ca2+ in human neutrophils induced by opsonized particles

Additon of opsonized particles to human neutrophils in suspension leads to a biphasic elevation in the cytosolic free Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i, during the second phase (> 3 min) is pronounced (about 400 nM), in contrast to the rise during the first phase, which is relatively small (< 100 nM). The second and large rise in [Ca2+]i is brought about by messenger(s) released from the cell after addition of opsonized particles. This second rise in [Ca2+]i, is not observed in the presence of the platelet‐activating factor (PAF) antagonist WEB 2086, indicating that PAF can act as an intercellular messenger affecting Ca2+ homeostasis in human neutrophils.

Defects in neutrophil granule mobilization and bactericidal activity in familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) syndrome caused by STXBP2/Munc18-2 mutations

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by genetic defects in cytotoxic granule components or their fusion machinery, leading to impaired natural killer cell and/or T lymphocyte degranulation and/or cytotoxicity. This may accumulate into a life-threatening condition known as macrophage activation syndrome. STXBP2, also known as MUNC18-2, has recently been identified as the disease-causing gene in FHL type 5 (FHL-5). A role for STXBP2 in neutrophils, and for neutrophils in FHL in general, has not been documented thus far. Here, we report that FHL-5 neutrophils have a profound defect in granule mobilization, resulting in inadequate bacterial killing, in particular, of gram-negative Escherichia coli, but not of Staphylococcus aureus, which rather depends on intact reduced NAD phosphate oxidase activity. This impairment of bacterial killing may contribute to the apparent susceptibility to gastrointestinal tract inflammation in patients with FHL-5.

Cytokine-induced protein tyrosine phosphorylation is essential for cytokine priming of human eosinophils

BACKGROUND Human eosinophils are strongly modulated by the eosinophilotrophic cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). A clear intracellular effect of these cytokines is the induction of tyrosine phosphorylation of multiple cellular substrates. However, the relevance of tyrosine phosphorylation for eosinophil functioning has not been established. OBJECTIVE In this study we have investigated dose-response and time curves of IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation in eosinophils. Moreover, we have evaluated the importance of IL-5-induced tyrosine phosphorylation for priming of human eosinophils. METHODS Cytokine-induced tyrosine phosphorylation was monitored on western blot with an antiphosphotyrosine antibody (4G10). To probe the relevance of tyrosine phosphorylation for priming, eosinophils were primed with IL-5 in the presence of the tyrosine kinase inhibitor herbimycin A. Platelet activating factor (PAF) was used as a control priming agent. Subsequently, the eosinophils were incubated with serum-treated zymosan (STZ) to activate the respiratory burst. Binding of STZ was determined by FACS analysis. RESULTS IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation was found at concentrations that primed eosinophil effector mechanism (median effective dose values: approximately 5.10(-11) mol/L, approximately 5.10(-10) mol/L, and approximately 5.10(-12) mol/L for IL-5, IL-3, and GM-CSF, respectively). Cytokine-induced tyrosine phosphorylation was transient with an optimum value at 15 minutes. IL-5 priming of STZ-induced activation of the respiratory burst was blocked by herbimycin A, whereas PAF still primed this response. In fact, herbimycin A inhibited IL-5 priming of STZ binding to human eosinophils. On the other hand, PAF priming of STZ binding was not affected by herbimycin A. Both IL-5-induced and PAF-induced tyrosine phosphorylation were inhibited by herbimycin A. CONCLUSION These data demonstrate for the first time that IL-5 priming of opsonized particle-induced responses is mediated by tyrosine kinase activity in human eosinophils.