Human Inflammatory Cells Research Articles

C-kit ligand: a unique potentiator of mediator release by human lung mast cells.

Mast cells (MC) play a central role in extrinsic allergic reactions such as asthma and may participate in other inflammatory and fibrotic processes. However, with the exception of immunoglobulin E (IgE) receptor-dependent stimulation, no secretagogues of human lung MC have yet been described. It is also unclear whether mediator release can be regulated by certain cytokines as demonstrated previously in basophils and other human inflammatory effector cells. Here, we show that the c-kit ligand (KL), a recently identified stem cell growth factor, at concentrations 10-100 times lower than that required to promote cell proliferation, enhances the release of histamine and leukotriene C4 in response to IgE receptor crosslinking of human lung MC. KL does not induce mediator release per se, but increases the sensitivity of MC to anti-IgE receptor stimulation and also enhances mediator release to maximally effective concentrations of anti-IgE receptor antibody. By contrast, a large number of cytokines examined, including the mast cell growth factors/agonists in rodents, interleukin 3 (IL-3), IL-4, IL-9, and nerve growth factor, were ineffective in this respect. These findings suggest a unique role of KL in regulating effector functions of human mucosal MC.

Differential synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine and platelet-activating factor by human inflammatory cells.

Abstract The synthesis of platelet-activating factor (PAF) and of the 1-acyl analogue of PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) was examined in seven human cell preparations (lung mast cell, basophil, endothelial cell, neutrophil, eosinophil, lung macrophage, platelet) and in human lung fragments. Cells were activated by either an appropriate receptor-mediated stimulus or by ionophore A23187 in the presence of [3H]acetate. All cell types, with the exception of the platelet, responded to stimulation with at least a twofold increase in the formation of labeled 1-radyl-2-acetyl-GPC as compared with control values. A23187 was the more potent stimulus in all cell types examined except the lung mast cell, in which anti-IgE consistently induced the synthesis of more 1-radyl-2-acetyl-GPC. Human lung fragments stimulated by anti-IgE, Ag Amb a I (after passive sensitization), or A23187 also incorporated [3H]acetate into 1-radyl-2-acetyl-GPC. Subclass analysis of 1-radyl-2-acetyl-GPC produced by each cell indicated that the cell types examined can be divided into two groups according to the predominant type of 1-radyl-2-acetyl-GPC produced. Some cell types (mast cell, basophil, endothelial cell) produced predominantly 1-acyl-2-acetyl-GPC, whereas others (neutrophil, eosinophil, lung macrophage) produced almost exclusively PAF. In some cell types, such as the lung mast cell and the basophil, A23187 stimulation increased the synthesis of PAF relative to 1-acyl-2-acetyl-GPC as compared with anti-IgE stimulation. In the lung fragments, [3H]acetate was predominantly incorporated into 1-acyl-2-acetyl-GPC upon IgE-mediated stimulation (anti-IgE, Amb a I) and into PAF upon A23187 stimulation. The differential production of these two phospholipids was confirmed by determining their sensitivity to lipase A1 and phospholipase A2 hydrolysis and by HPLC. These data demonstrate that 1-acyl-2-acetyl-GPC can be synthesized by a variety of human cells involved in the inflammatory reaction. This finding raises fundamental questions about the biologic role of this molecule and the factors regulating its synthesis within inflammatory cells.

409 Synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in human inflammatory cells

409 Synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in human inflammatory cells

Modulation of mediator release from human basophils and pulmonary mast cells and macrophages by auranofin

Auranofln, a new orally absorbable gold compound, inhibits IgE-(anti-IgE) and non-IgE-mediated (f-met-peptide and the Ca 2+ ionophore A23187) histamine release from human basophils. Auranofin inhibits the release of histamine induced by phorbol myristate (TPA) and bryostatin 1 both in the presence and absence of extracellular Ca 2+. Increasing the Ca 2+ concentrations in the extracellular medium does not reduce the inhibitory effect of auranofin on anti-IgE- or A23187-induced secretion. Auranofin inhibits the de novo synthesis of sulfidopeptide leukotriene C 4 (LTC 4) induced by anti-IgE from basophils and mast cells purified from human lung. However, in both systems auranofin has a significantly greater inhibitory effect on LTC 4 release than on histamine secretion. Finally, auranofin induces a concentration-dependent inhibition of A23187-induced leukotrine B 4 (LTB 4) release from purified human lung macrophages. These data suggest that auranofin modulates the release of preformed (histamine) and de novo synthesized (LTC 4 and LTB 4) chemical mediators from human inflammatory cells isolated from peripheral blood and human lung tissues.

Potentiation of the Macrophage 25-Hydroxyvitamin D1-Hydroxylation Reaction by Human Tuberculous Pleural Effusion Fluid*

1,25-Dihydroxyvitamin D [1,25-(OH)2D] is classically viewed as a steroid hormone of renal origin that regulates mammalian and avian mineral ion homeostasis. More recently, 1,25-(OH)2D has been shown to be produced by and act on human inflammatory cells in vitro, suggesting that the hormone may be an important modulator of the host immune response. We have recently detected high concentrations of 1,25-(OH)2D in the pleural fluid (PF) of patients with tuberculous pleuritis. Working on the hypothesis that tuberculous PF contained a soluble cytokine which stimulated 1,25-(OH)2D production by tissue (pleura)-based macrophages, we examined the potential for PF from five patients with tuberculous pleuritis to potentiate 1,25-(OH)2D synthesis by heterologous pulmonary alveolar macrophages (PAM) from patients with sarcoidosis; PAM from patients with active pulmonary sarcoidosis constitutively express a 25-hydroxyvitamin D3-1-hydroxylation reaction in vitro. We demonstrated that tuberculous PF had a concentration-dependent potentiating effect on PAM 1,25-(OH)2D synthesis. The potentiating activity was positively correlated to the interferon-gamma (IFN gamma) concentration of the PF (r = 0.98; P less than 0.01) and was inhibited by 49-89% after coincubation with anti-IFN gamma monoclonal antibody (1:20,000-1:200 dilution). These data suggest that IFN gamma may be an important peripleural regulator of macrophage 1,25-(OH)2D synthesis in patients with tuberculous pleuritis and a high pleural fluid 1,25-(OH)2D concentration.

Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals. Evidence that serum alpha 2-HS glycoprotein is a potent and specific crystal-bound inhibitor.

Tissue deposits of basic calcium phosphate (BCP) crystals are associated with various clinical manifestations of inflammation. We addressed the possibility that native proteins modify the ability of hydroxyapatite (HA) crystals to stimulate human inflammatory cells. Neutrophil superoxide release and chemiluminescence in response to HA crystals (0.3-4.0 mg/ml) were blunted by serum and plasma. Inhibitory activity was progressively removed from serum by sequential adsorption with HA crystals, suggesting that the inhibitors were crystal-bound proteins. Thus, we characterized HA crystal-bound plasma proteins by O'Farrell gels: Fibronectin, transferrin, albumin, alpha 2-HS glycoprotein (AHSG), alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, Gc globulin, haptoglobin, and high density lipoprotein apolipoproteins were major bound species. Of these, AHSG was the most active inhibitor of HA-induced neutrophil superoxide release, and this glycoprotein partially (60%) restored inhibitory activity to HA-adsorbed serum. AHSG also bound in vitro to the related BCP crystal, octacalcium phosphate, but only minimally to calcium pyrophosphate dihydrate crystals and monosodium urate crystals. Suppressive effects on neutrophil stimulation exhibited by AHSG were also specific for BCP crystals. AHSG was present in noninflammatory synovial fluids bound to synthetic HA crystals in vitro, and AHSG could be detected on native synovial fluid HA crystals. We conclude that the binding of AHSG may modulate the inflammatory potential of BCP crystals.

Production of granulocyte colony‐stimulating factor (G‐CSF) by human cells: T lymphocyte‐dependent and T lymphocyte‐independent release of G‐CSF by blood monocytes

To evaluate the production of granulocyte colony-stimulating factor (G-CSF) by human immune and inflammatory cells, an assay specifically measuring G-CSF activity in the presence of other cytokines was developed which was based on the proliferation of 32Dcl cells induced by G-CSF. Successful use of the 32Dcl cells to specifically measure G-CSF activity required the selection of cells highly responsive to G-CSF and with reduced responsiveness to interleukin 2 (IL 2) by intermittent culture in medium containing G-CSF. Furthermore, the addition of exogenous IL 2 to standards and experimental samples was necessary to ensure that the concentration of IL 2 was similar in all samples, since IL 2 directly stimulated the proliferation of 32Dcl cells and increased their responsiveness to G-CSF. A variety of stimuli were found to induce G-CSF release by blood monocytes. Lipopolysaccharide and monocyte adherence appeared to directly stimulate G-CSF release, whereas stimulation of G-CSF release from monocytes by mitogenic lectins required the presence of T lymphocytes. In all cases, release of G-CSF was detectable as soon as 4 h after stimulation and was essentially complete after 48 h. These findings indicate that G-CSF release can be initiated by a variety of pathways, and therefore suggest that the production of this mediator may occur in the course of many immune and inflammatory reactions.

Disodium cromoglycate inhibits activation of human inflammatory cells in vitro

Recent clinical studies indicate that disodium cromoglycate (DSCG) may have a direct effect on inflammatory cells because the drug reversed various changes in leukocyte function, such as increased membrane-receptor expression and enhanced cytotoxic capacity observed in peripheral white blood cells from subjects with asthma undergoing allergen-inhalation challenge. In the present study, we have demonstrated that DSCG, at low concentrations (a concentration of drug required to produce 50% inhibition, approximately 10(-8) mol/L) and in a time-dependent fashion, directly inhibited the activation in vitro of human neutrophils, eosinophils, and monocytes. Peripheral blood leukocytes were incubated with the synthetic chemoattractant, formyl-methionyl-leucyl-phenylalanine (at an optimal concentration of 10(-8) mol/L), and activation was assessed by measuring increases in the percentages of complement and IgG (Fc) rosettes as well as the enhanced capacity of these cells to kill target organisms (schistosomula of Schistosoma mansoni). DSCG at a concentration of 10(-7) mol/L totally inhibited both the formyl-methionyl-leucyl-phenylalanine-induced enhancement of complement and IgG rosettes, as well as increased schistosomular killing. These observations indicate that DSCG directly inhibits the secretory properties of inflammatory cells and that in turn might have important implications in modulating mechanisms contributing to the inflammatory component of asthma and allergic disease. It may also help to explain why compounds with considerably greater mast cell stabilizing properties than DSCG have been so disappointing when they are evaluated clinically.

Biomedical applications of thermospray liquid chromatography—mass spectrometry

Thermospray liquid chromatography—mass spectrometry has been applied in the solution of a number of problems of biological and biomedical interest. These include the analysis of phenazines from the Gram negative bacterium Pseudomonas aeruginosa, steroids released by rat adrenals and eicosanoids generated by human inflammatory cells. The application of the technique to leukotrienes in blood is discussed. Isotopic labelling prior to analysis, to facilitate identification and structure elucidation is outlined with reference to the steroids.

Control mechanisms of human basophil releasability

Extensive evidence suggests that mediator release from inflammatory cells plays a role in the pathogenesis of allergic disorders in humans.‘.2 Most studies have focused on the identification of new mediators de novo synthesized by human inflammatory cells; two such examples are AGEPC and leukotrienes.‘” In addition, a number of laboratories are striving to understand the immunologic and biochemical mechanisms underlying the release of chemical media-. tors.6-8 However, few studies have been devoted to clarifying the pathophysiologic mechanisms that control the release of mediators from human inflammatory cells (i.e., the concept of “releasability”). The concept and the study of releasability in humans originated in the laboratory of Lichtenstein and Conroy’ almost a decade ago. They foresaw that, in addition to quantitative immunologic parameters such as IgE and IgG antibody levels, the study of allergic disorders should include the evaluation of cellular releasability. Until then, releasability was defined on an almost philosophic plane because it was impossible to characterize and separate immunologic from releasability parameters. Schleimer et al.’ selected the basophil system, a reliable experimental model of human anaphylaxis, to initiate the study of releasability. For example, they showed that histamine release is not correlated to the absolute quantity of IgE antibody present on human basophils challenged with the appropriate antigen. 9 ” Similarly, the release of histamine in response to anti-IgE is not related to the number of IgE molecules on the basophil surface.” In addition, they demonstrated that significant differences in IgEand non-&E-mediated releasability exist in some allergic conditions.“-” These pioneering studies supported the notion that basophil releasability is a real entity that could be of some relevance in the pathogenesis of inflammatory disorders. Stimulated by the lack of information on the factors that affect basophil releasability, we initiated a series of investigations to identify and characterize factors that physiologically control basophil releasability. We also used the ba-

Generation of hydroxyeicosatetraenoic acids by human inflammatory cells: analysis by thermospray liquid chromatography-mass spectrometry

Arachidonic acid was converted to a series of hydroxyeicostatraenoic acids (HETEs) by mixed human inflammatory cells following stimulation with the calcium ionophore A23187. HETEs were purified by a simple one-step extraction procedure followed by HPLC. The HPLC was coupled to a Finnigan quadrupole mass spectrometer using the now commercially available thermospray liquid chromatography-mass spectrometry interface. The HPLC eluant was monitored ‘on line’ by the mass spectrometer. Soft ionisation occurs, generating intense molecular ion species in the negative ion mode ((M − H −: m/ z 319) for each of the isomeric HETEs. The (M + H + − H 2O) ion at m/ z 303 is the major species in the positive ion spectra of HETEs. Mass spectra wer obtained on-line post-HPLC for HETEs formed by the human cells, and the HPLC-MS profile compared with that obtained from standards; species corresponding to the 11-, 9- and 5-HETEs were observed.

Adenosine receptors on human inflammatory cells.

Adenosine is a natural nucleoside that plays a physiological role in the modulation of human inflammatory cells. We have investigated the presence of adenosine A2/Ra and A1/Ri receptors and of the P-site on human inflammatory cells. Human B and T(OKT4+ and OKT8+) lymphocytes, polymorphonuclear leucocytes, monocytes, basophils and platelets possess a membrane adenosine A2/Ra receptor. The activation of adenosine A2/Ra receptor increases the intracellular level of cyclic AMP in these cells. Human lymphocytes and neutrophils possess also an inhibitory adenosine A1/Ri receptor and a P-site whose activation inhibits the effect of many adenylate cyclase agonists including isoproterenol, PGE1, histamine, adenosine, cholera toxin and forskolin.

Immunopharmacology of inflammation: Modulation of the release of chemical mediators from human inflammatory cells by auranofin

Immunopharmacology of inflammation: Modulation of the release of chemical mediators from human inflammatory cells by auranofin

Comparison of live human neutrophil and alveolar macrophage elastolytic activity in vitro. Relative resistance of macrophage elastolytic activity to serum and alveolar proteinase inhibitors.

Elastin is an extracellular matrix protein critical to the normal structure and function of human lung. Recently reported data indicate that live human alveolar macrophages can degrade purified elastin in vitro. In this study, we directly compared the elastolytic activity of alveolar macrophages with that of human neutrophils. In the absence of proteinase inhibitors, human neutrophils degrade much more elastin than do human alveolar macrophages. However, macrophages cultured in 10% human serum and in contact with purified 3H-elastin degraded 4.7 micrograms elastin/10(6) cells per 24 h, as compared to less than 1 microgram/10(6) cells/24 h for neutrophils. We observed a similar pattern when the two cells were cultured in human alveolar fluid. We determined that the relative resistance of macrophage elastolytic activity to serum or alveolar proteinase inhibitors was not simply due to phagocytosis of substrate by the larger macrophages. Live macrophages as well as neutrophils degrade 125I-elastin coupled to noningestible sepharose beads. Again in serum-free media, neutrophils degraded eight-fold more elastin than macrophages but only macrophages degraded sepharose-coupled elastin in the presence of 10% serum. Because of these findings, we compared the enzymatic mechanisms of elastin breakdown by macrophages with that of neutrophils. Macrophage elastolytic activity is largely (65-80%) due to a cysteine proteinase(s), at least part of which is Cathepsin B. Approximately half of the cysteine proteinase activity appeared to be expressed at or near the cell surface. These experiments defined two enzymatically distinct pathways of elastin breakdown by human inflammatory cells: the classic, neutrophil derived soluble elastase(s) that is sensitive to serum and alveolar proteinase inhibitors, and a macrophage-mediated pathway that is largely cell associated and relatively resistant to inhibitors. The function of the two pathways depends on the relative excess or deficiency of soluble inhibitors. At inflammatory sites rich in proteinase inhibitors, tissue macrophages may degrade more extracellular matrix elastin than neutrophils. In smokers without antiproteinase deficiency, pulmonary macrophages, which are known to be increased in number, may be the more important cause of elastin breakdown and emphysema.

Neutral Proteinases from Human Inflammatory Cells: A Critical Review of Their Role in Extracellular Matrix Degradation

The cell biology and in vitro activity of neutral proteinases derived from inflammatory cells and the mechanisms by which these enzymes might injure connective tissue are outlined. Current work directed toward understanding local control of tissue degradation may lead to effective strategies for assessing and reducing connective tissue injury in human disease.

Inhibition of histamine release from human basophils in vitro by calmodulin antagonists

Calmodulin is a ubiquitous and versatile Ca 2+-binding protein that plays a pivoting role in cellular metabolism. We have investigated the possibility that calmodulin plays a role in immediate hypersensitivity reactions by evaluating the effects of two agents, trifluoperazine dihydrochloride (TFP) and the sulfonamide derivative N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) which selectively bind to calmodulin. TFP and W-7 cause a dose-dependent inhibition of histamine secretion from human basophils in vitro induced by several immunological (i.e., antigen and anti-IgE) and nonimmunological (i.e., formyl-methionine-containing peptide and the Ca 2+ ionophore A23187) stimuli. These results indicating that two specific calmodulin antagonists are potent inhibitors of the secretory response of human basophils support the hypothesis that calmodulin may play a role in the control of the release of preformed mediators from human inflammatory cells.

734 THE EFFECTS OF CROMOLYN SODITIM ON NEUTROPHIL CHEMOTAXIS

Cromolyn Sodium (CS) is an effective therapeutic agent in the prophylaxis of bronchial asthma. Although its specific mechanism of action has not been elucidated, CS has been shown to inhibit mediator release in the immediate hypersensitivity reaction. The effects of CS upon the chemotactic response of human inflammatory cells have not been extensively investigated. We have demonstrated that CS is not chemotactic for either human neutrophils (HN) or eosinophils. Incubation of HN with CS for 30′ (37°C) prior to chemotaxis did not affect the response of HN toward standard chemotactic factors: zymosan activated serum (ZAS), C5a or normal human serum (NHS) which was allowed to clot at 37°C for 1 hr. and then heat inactivated at 56° for 30′. Further chemotactic studies were performed utilizing untreated HN and incremental concentrations of CS added to each of the above standard chemotactants. Incremental cone. of CS (10−2M to 10−6M) added to ZAS or C5a did not affect the chemotactic response of HN, while after identical molar cone. of CS were added to HNS the chemotactic stimulus was diminished. The role of the neutrophil in immediate hypersensitivity has become prominent with the identification of a neutrophil chemotactic factor (NCF). The data suggest that CS may inhibit serumderived chemotactic activity, perhaps complement-independent factors, with resultant modification of the immediate hypersensitivity response.