K F Austen

Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3.

Human eosinophils were cultured in the presence of recombinant human IL-3 for up to 14 d and their biochemical, functional, and density properties were assessed. After 3 d of culture in 10 pM IL-3, eosinophils had a viability of 70% compared with only 10% in enriched medium alone. Neither IL-1 alpha, IL-2, IL-4, tumor necrosis factor, basic fibroblast growth factor, nor platelet-derived growth factor maintained eosinophil viability. The 7- and 14-d survival of the cultured eosinophils was 55 and 53%, respectively. No other cell type, including neutrophils, was present after culture. After 7 d of culture, the normodense eosinophils were converted to hypodense cells as assessed by density centrifugation. Eosinophils exposed to 1,000 pM IL-3 for 30 min or cultured in 10 pM IL-3 for 7 d generated approximately threefold more leukotriene C4 (LTC4) in response to calcium ionophore than freshly isolated cells. Furthermore, whereas freshly isolated eosinophils killed only 14% of the antibody-coated Schistosoma mansoni larvae, these eosinophils killed 54% of the larvae when exposed to 100 pM IL-3. The enhanced helminth cytotoxicity was maintained for 7 d when eosinophils were cultured in the presence of both 10 pM IL-3 and 3T3 fibroblasts, but not when eosinophils were cultured in the presence of IL-3 alone. IL-3 thus maintains the viability of eosinophils in vitro, augments the calcium ionophore-induced generation of LTC4, enhances cytotoxicity against antibody-sensitized helminths, and induces the eosinophils to become hypodense cells. These phenotypic changes in the eosinophil may be advantageous to host defense against helminthic infections but may be disadvantageous in conditions such as allergic disease.

Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils.

Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.

Effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils.

Exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) have been compared with exogenous arachidonic acid for their capacity to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human neutrophils and for their suitability as parallel substrates in this pathway. The products from specific 14C- or 3H-labeled substrates were isolated by reverse phase high performance liquid chromatography (RP-HPLC) and were identified by elution of radiolabel at the retention times of the appropriate synthetic standards. Each product was also characterized by its ultraviolet (UV) absorption spectrum, and 7-hydroxy-DCHA was defined in addition by analysis of its mass spectrum. The metabolites, 5-hydroxyeicosatetraenoic acid, leukotriene B4 (LTB4), 6-trans-LTB4 diastereoisomers, 5-hydroxyeicosapentaenoic acid, 6-trans-leukotriene B5 diastereoisomers, leukotriene B5 (LTB5), and 7-hydroxy-DCHA were quantitated by integrated UV absorbance during resolution by RP-HPLC. LTB4 and LTB5 were also quantitated by radioimmunoassay of the eluate fractions, and leukotrienes C4 and C5 (LTC4 and LTC5, respectively) were quantitated by radioimmunoassay alone. None of the unlabeled exogenous fatty acids (5-40 micrograms/ml) altered the release of radioactivity from [14C]arachidonic acid-labeled, ionophore-activated neutrophils. The metabolism of 5 and 10 micrograms/ml of exogenous EPA by ionophore-activated, [14C]arachidonic acid-labeled neutrophils not only generated 5-hydroxyeicosapentaenoic acid, 6-trans-LTB5, LTB5, and LTC5, but also stimulated the formation of 5-hydroxyeicosatetraenoic acid, 6-trans-LTB4 diastereoisomers, and LTC4 from membrane-derived arachidonic acid. In contrast, LTB4 production was diminished throughout the EPA dose-response, beginning at 5 micrograms/ml EPA and reaching 50% suppression at 10 micrograms/ml and 84% suppression at 40 micrograms/ml. The selective decrease in extracellular LTB4 concentrations in the presence of EPA was not due to a change in the kinetic appearance of LTB4 or to an increase in conversion to its omega-oxidation metabolites. DCHA was metabolized to 7-hydroxy-DCHA, did not stimulate metabolism of membrane-derived arachidonic acid, did not appreciably inhibit LTB4 formation, and was not a substrate for leukotriene formation. Incremental doses of exogenous arachidonic acid resulted in increased production of 5-hydroxyeicosatetraenoic acid and 6-trans-LTB4 by ionophore-activated, [14C]arachidonic acid-labeled neutrophils without any change in LTB4 production. 5-hydroxyeicosapentaenoic acid and 7-hydroxy DCHA were inactive as chemotactic factors whereas 5-hydroxyeicosatetraenoic acid exhibited 2% of the potency of LBT4. Thus, exogenous DCHA does not appreciably interfere with the metabolism of membrane-derived arachidonic acid by ionophore-activated, [14C]arachidonic acid-labeled neutrophils and is converted only to a monohydroxy derivative. In contrast, exogenous EPA attenuates the generation of LTB4 and is converted to LTB5, which is a weak and partial agonist as compared with LTB4.

Dectin-2 Recognition of House Dust Mite Triggers Cysteinyl Leukotriene Generation by Dendritic Cells

House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRγ−/− mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRγ/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.

Acquired C1 inhibitor deficiency in lymphosarcoma

Abstract Two patients with lymphosarcoma and circulating 7S IgM were found to have an unusual complement component profile in which the first, fourth and second components and the inhibitor of the activated first component were diminished in the presence of a normal third component and an elevated ninth component. One of these patients experienced attacks of angioedema which can be distinguished from hereditary angioedema by the absence of any complement system defects in other family members and by the reduced level of C1 in the patients serum. The C 1 inhibitor deficiency in these patients is acquired, most likely as a result of its interaction with activated first component.

Heterogeneity of Mast Cells at Multiple Body Sites: Fluorescent Determination of Avidin Binding and Immunofluorescent Determination of Chymase, Tryptase, and Carboxypeptidase Content

Human mast cells (MCs) from multiple sites were studied to determine heterogeneity of expression of chymase, tryptase, and/or carboxypeptidase and the binding to avidin. Three immunophenotypes were found: MCs positive for tryptase (MCT), no immunodetectable chymase; MCs positive for tryptase and chymase (MCTC); and MCs positive for chymase (MCC), no immunodetectable tryptase. Chymase-positive MCs also bound avidin and contained carboxypeptidase. In breast skin and parenchyma and axillary lymph nodes > 95% were MCTC; a rare MC in skin and lymph nodes was MCT or MCC. In lung alveoli 91% of MCs were MCT, 8% were MCTC and 1% MCC. In bowel mucosa 58% of MCs were MCT, 35% were MCTC and 7% MCC, whereas in bowel submucosa 83% of MCs were MCTC and 17% MCC. Within esophageal epithelium 38% were MCT and 62% MCTC; whereas, in esophageal subepithelium all were MCTC. This study further documents site-dependent diversity among normal human MCs and recognizes a novel third immunophenotype rich in chymase and relatively deficient in tryptase, the MCC cell.

Acquired C1-Inhibitor Deficiency Associated with Antiidiotypic Antibody to Monoclonal Immunoglobulins

The syndrome of acquired angioedema and C1-inhibitor deficiency is associated with B-cell lymphoproliferative disease. It is characterized by accelerated consumption of C1q and C1 inhibitor in vivo and by low levels of serum C2 and C4. Four patients with B-cell malignant diseases (IgA myeloma, macroglobulinemia, chronic lymphocytic leukemia, and B-cell lymphoma, respectively) and acquired C1-inhibitor deficiency were found to have circulating antiidiotypic antibodies to the monoclonal immunoglobulin expressed on the surface of their B cells (three patients) or in the cytoplasm of their bone-marrow cells (one patient). Two of the four patients had circulating M components, and their antiidiotypic antibodies reacted with the M components. In three patients studied the percentage of B cells bearing C1q was 18, 24, and 35 per cent, as compared with 2.3 +/- 1.7 per cent (mean +/- S.D.) in six normal controls. These results suggest that an interaction between the idiotype of monoclonal immunoglobulins and antiidiotypic antibodies causes increased consumption of C1q and C1 inhibitor in patients with acquired angioedema and C1-inhibitor deficiency. We propose that the subsequent activation of the early components of complement leads to increased vascular permeability and to angioedema and that these patients have a disease caused by antiidiotypic antibodies.

An intrinsic adrenergic system in mammalian heart.

We have identified a previously undescribed intrinsic cardiac adrenergic (ICA) cell type in rodent and human heart. Northern and Western blot analyses demonstrated that ICA cell isolates contain mRNA and protein of enzymes involved in catecholamine biosynthesis. Radioenzymatic catecholamine assays also revealed that the catecholamine profile of adult rat ICA cell isolates differed from that of sympathetic neurons. Unlike sympathetic neuronal cells, isolated ICA cells have abundant clear vesicles on electron microscopy. Endogenous norepinephrine and epinephrine constitutively released by ICA cells in vitro affect the spontaneous beating rate of neonatal rat cardiac myocytes in culture. Finally, ICA cells could be identified in human fetal hearts at a developmental stage before sympathetic innervation of the heart has been documented to occur. These findings support the concept that these cells constitute an ICA signaling system capable of participating in cardiac regulation that appears to be independent of sympathetic innervation.

Eosinophil hematopoietins antagonize the programmed cell death of eosinophils. Cytokine and glucocorticoid effects on eosinophils maintained by endothelial cell-conditioned medium.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was established as the constitutive and elicited human umbilical vein endothelial cell-derived eosinophil viability-sustaining factor. Stimulation of endothelium cell monolayers with IL-1 alpha (5 U/ml) increased the 48-h elaboration of GM-CSF from a mean of 3.2 to a mean of 8.2 pM (P less than 0.05). Dexamethasone (100 nM) decreased the constitutive GM-CSF elaboration by 49% (P less than 0.001) but did not diminish production by IL-1 alpha-stimulated endothelium. However, eosinophil viability decreased by 21% in dexamethasone-pretreated IL-1 alpha-stimulated endothelial cell-conditioned medium (P less than 0.05), which suggested viability antagonism by glucocorticoids. After 24 h of culture, eosinophil viability for replicate cells in enriched medium alone or with 1 pM GM-CSF decreased from means of 43 and 75% to means of 21 and 54%, respectively, when dexamethasone was included (P less than 0.05). However, 10 pM GM-CSF, IL-3, or IL-5 protected the cells against dexamethasone and against endonuclease-specific DNA fragmentation. In this model system of eosinophil-tissue interactions, dexamethasone prevents the endothelial cells from inducing a pathobiologic phenotypic change in the eosinophil by suppression of GM-CSF elaboration to concentrations that are not cytoprotective. Cytokine priming by GM-CSF, IL-3, or IL-5 may account for the differential responsiveness of select eosinophilic disorders to glucocorticoids.

Renal and systemic hemodynamic responses to intravenous infusion of leukotriene C4 in the rat.

We studied the systemic and renal hemodynamic effects of leukotriene C4 (2 μg/ per min for 5 minutes, iv) in the rat. During the period of its infusion, leukotriene C4 produced a significant elevation of mean arterial pressure and reductions in cardiac output and renal blood flow, as measured by electromagnetic flow probes. These effects were abolished by FPL55712, a putative antagonist of sulfidopeptide leukotrienes, but not by saralasin or indomethacin. Leukotriene C4 also resulted in an average loss of 20% in plasma volume which, during the postinfusion period, perpetuated the low cardiac output state and thus provoked the release of angiotensin II. This vasoactive peptide sustained the elevation in systemic vascular resistance and the reduction in renal blood flow over a 70-minute postinfusion observation period. Consequently, glomerular filtration rate fell by approximately 50%. These angiotensin II-mediated effects were abolished by saralasin. Indomethacin prevented the leukotriene C4-induced loss of plasma volume and, thus, allowed for the significant recovery of cardiac output and renal blood flow during the postinfusion period, thereby preserving glomerular filtration rate. We conclude that leukotriene C4 exerts direct systemic and renal vasoconstrictor, as well as cardiodepressant effects, during the period of its infusion. By virtue of its vasopermeability enhancing effect, leukotriene C4 also results in an immediate loss of plasma volume, an effect which requires the presence of secondarily generated cyclooxygenase products and which perpetuates the hemodynamic abnormalities observed beyond the period of leukotriene C4 infusion.