Degranulation influences heparin-associated inhibition of RT-PCR in human lung mast cells.

BACKGROUND: The study of gene expression from human lung mast cells (HLMC) has been limited by the ability to reliably detect mRNA transcripts from scant quantities of mast cell RNA contaminated with heparin. OBJECTIVE: As heparin is granule-associated within the mast cell, we examined the role of degranulation in altering the intrinsic ability of this proteoglycan to inhibit reverse transcription polymerase chain reaction (RT-PCR) from HLMC RNA. We also explored alternative means of RNA isolation to eliminate primary heparin contamination. METHODS: Purified HLMC (> 90% pure) were challenged for 2 h with buffer, anti-IgE (3 microg/mL) and/or ionophore A23187 (100 ng/mL) or phorbol 12-myristate 13-acetate (50 ng/mL). Histamine release was measured using a spectroflourometric assay. Following challenge, RNA was isolated by either phenol-chloroform extraction or by nitrocellulose spin column. Parallel samples were either treated with heparinase or placed on ice for 2 h prior to reverse transcription. PCR was performed using primers specific for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: In each of five studies, GAPDH bands were detected at greater intensity in total cellular RNA (tcRNA) derived from degranulated than from non-degranulated mast cells. However, when examining heparinase-treated tcRNA from the same mast cell samples, the intensity of GAPDH bands normalized between degranulated and non-degranulated cells. When comparing parallel samples of column-purified tcRNA, subsequent treatment of samples with heparinase had no effect on the detection of GAPDH, indicating that endogenous heparin was effectively removed by the technique. Moreover, no variability was noted in GAPDH signal detected from resting vs. degranulated mast cells (n = 3). CONCLUSIONS: Degranulation influences the degree of heparin-associated inhibition of RT-PCR in HLMC, and that spin column purification of tcRNA is a time-saving, effective alternative to heparinase pre-treatment.

Effects of purine and pyrimidine nucleotides on intracellular Ca2+ in human eosinophils: activation of purinergic P2Y receptors.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) increases human eosinophil intracellular Ca(2+) concentration; the mechanism of action is not fully known. ATP, a physiologic regulator, acts through 2 purinergic receptor types: cation channels (P2X) and G protein-coupled receptors (P2Y). OBJECTIVE: This study is aimed at identifying the functional purinergic receptors in human eosinophils. METHODS: The relative potency of ATP, uridine (UTP), cytidine (CTP), and inosine (ITP) 5'-triphosphates (P2Y agonists); 2-methylthio-ATP (P2Y(1) agonist); and 2 P2X agonists, alpha,beta-methylene-ATP and beta,gamma-methylene-ATP on intracellular Ca(2+) concentration was examined in Ca(2+)-sensitive Fura-2-labeled human eosinophils. For comparison, ATP effects were similarly studied in human neutrophils. P2X/P2Y mRNA expression in cells was examined by reverse transcription and PCR. RESULTS: The nucleotide potency order was UTP > or = ATP > ITP >>> 2-methylthio-ATP > alpha,beta-methylene-ATP = beta,gamma-methylene-ATP = CTP = 0 in eosinophils. Pertussis toxin (500 ng/mL) pretreatment abolished the effect of lower (10(-6) mol/L) but not higher (10(-5) mol/L) concentrations of ATP in eosinophils, whereas it attenuated the effects of 10(-4) mol/L ATP in neutrophils. The phospholipase C inhibitor U73122 (2 micromol/L) partially inhibited the effect of ATP in eosinophils but totally blocked it in neutrophils. Both cells constitutively express mRNA for P2X(1), P2X(4), P2X(5), P2Y(1), and P2Y(2), but not P2X(7), with much weaker expressions of P2X(4) and P2X(5) in neutrophils. Eosinophils cultured with the T(H)1 cytokine, IFN-gamma, expressed mRNA for P2X(7), a receptor linked to apoptosis. CONCLUSIONS: These results suggest that the P2 purinergic receptor signal transduction pathways in eosinophils and neutrophils are different and are mediated by more than 1 subtype of functional P2Y receptors.

Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.

We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.

ATP modulates anti-IgE-induced release of histamine from human lung mast cells.

Adenosine 5'-triphosphate (ATP) is released from the cytoplasm under physiologic and pathophysiologic conditions and enters the extracellular space, where it acts on a group of recently cloned cell-surface receptors termed P2-purinoceptors (subtypes P2X and P2Y). We examined the effects of extracellular ATP, uridine triphosphate (UTP), the stable ATP analogues alpha,betamethylene-ATP (alpha,betamATP), beta,gammamethylene-ATP (beta,gammamATP), and 2-methylthio-ATP (2mSATP), and adenosine (10(-6)-10(-3) M) on histamine release from human lung mast cells (HLMC) induced by anti-IgE and the calcium ionophore A23187. None of the nucleotides or adenosine directly induced histamine release. Adenosine exhibited a bimodal effect, enhancing histamine release at 10(-6) to 10(-4) M (P > 0.05, NS) and inhibiting it at 10(-3) M (P < 0.05). ATP (10(-4) M) enhanced anti-IgE-induced histamine release (10.9 +/- 2.7% to 19. 2 +/- 2.9%, n = 20, P < 0.01), but not ionophore A23187-induced histamine release (n = 10). The adenine nucleotides consistently enhanced anti-IgE-induced histamine release; the rank order for this action was: ATP > 2mSATP > alpha,betamATP > beta,gammamATP, suggesting mediation by a P2Y-purinoceptor subtype. The selective P2X purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid failed to influence the effect of ATP, further supporting P2Y-purinoceptor mediation of anti-IgE-induced histamine release. UTP, an agonist at P2Y-purinoceptors, also significantly enhanced anti-IgE-induced histamine release. Application of the reverse transcription-polymerase chain reaction indicated that HLMC constitutively express the messenger RNAs encoding the P2Y1- and P2Y2-purinoceptor subtypes, and not that encoding the P2X7-purinoceptor (i.e., P2Z), a subtype implicated in ATP-induced histamine release in rodent peritoneal mast cells. The data produced in the study suggest that ATP plays an important modulatory role in histamine release from HLMC, and that it may therefore be mechanistically involved in human allergic/asthmatic reactions.

A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H2O2 oxidation. Using 50 microM A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respectively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as long as the ratio of A6550:H2O2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. When the concentrations of A6550 and HRP were 10 microM and 0.2 U/ml, respectively, as low as 2 pmol of H2O2 could be reliably measured. The sensitivity of A6550/H2O2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H2O2 release from activated granulocytes can be easily adapted to other cell types which generate H2O2.

Human lung mast cell activation leads to IL-13 mRNA expression and protein release.

Using reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), we studied the generation of the recently described Th2 cytokine interleukin-13 (IL-13) by anti-IgE-activated lung fragments (LF), lung mast cells (LMC), and the mast cell line HMC-1. We found that IL-13 messenger ribonucleic acid (mRNA) was constitutively expressed in LF and rapidly increased after anti-IgE challenge, persisting throughout a 16-h period. Quantitative-competitive PCR (QCPCR) demonstrated an increase from 1.2 fg to 120 fg of IL-13 mRNA/micrograms LF total cellular RNA. Time-course experiments showed that IL-13 protein was not increased in supernatants at 2 h after activation, but was upregulated by 8 h. Anti-IgE-activated LF supernatants contained 592.1 +/- 314.8 pg IL-13/g wet weight of tissue at 24 h (mean +/- SE; n = 11). LMC demonstrated upregulation of IL-13 mRNA expression following treatment with A23187 (n = 4), with maximal upregulation by 3 h; anti-IgE or phorbol myristate acetate (PMA) also led to increased IL-13 mRNA expression. QCPCR analysis of LMC IL-13 mRNA expression at 4 h after activation showed a 7-, 13.8-, and 13.2-fold increase after A23187, anti-IgE, and PMA, respectively. Quantities of IL-13 released from optimally activated LMC and peripheral blood T cells were comparable. HMC-1 also showed enhanced IL-13 mRNA beginning 30 min after A23187 activation, with peak expression from 1 to 10 h, followed by waning over the subsequent 24 h. A23187 stimulation of HMC-1 led to 100-fold upregulation of IL-13 mRNA within 4 h and detectable IL-13 in 24-h supernatants. These results demonstrate that activation of LF and LMC through multiple signal-transduction pathways results in increased IL-13 mRNA and protein expression temporally consistent with a potential role in chronic allergic inflammation.

Human lung mast cell IL-5 gene and protein expression: temporal analysis of upregulation following IgE-mediated activation.

The late-phase of allergic asthma is characterized by infiltration of the airway with eosinophils within 6 h of mast cell activation. Pro-eosinophilic/pro-allergic (TH2) cytokines, originally described as T-lymphocyte products, have recently been ascribed to mast cells as well. To date, however, it is unknown if TH2 cytokine gene expression by the human mast cells is subject to receptor-mediated regulation analogous to that of T-cells, and if messenger RNA (mRNA) expression results in protein secretion occurring in a temporal context consistent with the late-phase response. We examined interleukin-4 (IL-4), IL-5, and IL-6 mRNA expression induced by anti-IgE activation of human lung explants as assessed using reverse transcription/polymerase chain reaction (RT-PCR). Anti-IgE stimulation resulted in rapid and sustained upregulation of IL-5 message, but did not have analogous effects on IL-4 or IL-6. Using quantitative-competitive PCR, we demonstrated that 100 ng of total cellular RNA from human lung contained 1 fg of IL-5 mRNA; this increased to 100 fg 4 h after anti-IgE activation. The source of the anti-IgE-enhanced IL-5 mRNA is likely the mast cell itself, as anti-CD3 activation of lung led to a dissimilar array of cytokine expression. In addition, human lung mast cells purified to near homogeneity expressed IL-5 mRNA after activation, as shown by both RT-PCR and in situ hybridization. In both lung fragments and purified human lung mast cells, the modulation of IL-5 mRNA expression preceded the secretion of IL-5 protein, detected as early as 4 h after activation. Neither isolated purified mast cells nor purified peripheral blood T cells could be induced to secrete detectable amounts of IL-5 protein when activated only with antibodies against IgE or CD3-T cell receptor complex, respectively. However, mast cells (n = 4) and T cells (n = 6) cultured at comparable concentrations (4 x 10(6)/ml) activated through their respective antigen receptors in the presence of phorbol ester yielded comparable IL-5 production (253 +/- 126 pg/ml versus 183 +/- 75 pg/ml, mean +/- SE). We conclude that mast cells are analogous to T cells in the requirement of co-stimuli for the production of IL-5 protein. Moreover, the rapid kinetics of IgE-mediated IL-5 transcription and protein elaboration are consistent with a primary role for mast cell activation directly leading to late-phase airway eosinophilia.

IgE-dependent expression of interleukin-5 mRNA and protein in human lung: modulation by dexamethasone.

mRNA and protein expression of the Th2 cytokines IL-4 and IL-5 from human lung were examined during the first 4 hr following IgE-mediated triggering, a time representative of the evolving late-phase reaction (LPR). Lung explants were incubated for 16 hr at 37 degrees C in culture media alone or with added dexamethasone (10(-6) M), washed, and then challenged with buffer or anti-IgE (3 micrograms/ml). Using RNase protection assays, in 16/16 individual lungs IL-5 mRNA expression was observed at 4 hr following anti-IgE and at no points following buffer challenge. Fragments released 1129 +/- 499 ng of IL-5/g wet wt over a 24-hr period (mean +/- SEM, n = 5). Neither IL-4 transcripts nor protein were detected in any anti-IgE challenges. Both the IgE-mediated IL-5 mRNA and protein responses were below the limits of detection following dexamethasone preincubation, suggesting a mechanism for the potent inhibitory effects of these agents observed in the LPR.

Erythrocytes increase leukotriene C4 release from human eosinophils: characterization and examination of possible mechanisms.

There has been considerable interest in the role of eosinophils in the pathogenesis of asthma and allergic diseases. While examining the conditions necessary for the release of leukotriene C4 (LTC4) from human eosinophils activated by immunoglobulin G-Sepharose (IgG-Seph), we observed that red blood cells (RBCs) potentiated eosinophil LTC4 release. The time course of IgG-Seph-stimulated LTC4 release was prolonged in the presence of RBCs. After 45 min of incubation, eosinophils without RBCs released 0.95 +/- 0.11 ng/10(6) cells, and those with RBCs released 3.69 +/- 0.67 ng/10(6) cells. Control experiments indicated that the effect was not due to platelet contamination of the RBCs and could not be reproduced with RBC supernatants or RBC membrane ghosts. An interesting characteristic of this interaction was that the eosinophils and RBCs had to be in close contact for the enhancement to occur. We also observed that at low calcium concentrations (0.6 mM), the eosinophils had to be primed with fMLP for the RBC effect to occur, but priming was not required at higher calcium concentrations. Several possible mechanisms that would explain the RBC effect on eosinophil LTC4 release were examined: (1) RBCs block the metabolism of LTC4; (2) RBCs protect the eosinophils from oxidative damage; (3) RBCs provide a substrate (or enzyme) that allows increased eosinophil LTC4 production. These mechanisms failed to explain our observation that erythrocytes enhance eosinophil LTC4 release, however, suggesting that alternative mechanisms are responsible for this effect.

Pharmacologic characterization of a novel histamine receptor on human eosinophils.

There is increased recognition that lung mast cell mediators not only produce the symptoms of acute asthma, but also result in the recruitment and activation of additional proinflammatory cells, such as eosinophils. Histamine, one of the major mast cell mediators, is known to have numerous effects on eosinophil function. These effects of histamine are mediated by distinct receptors on the surface of eosinophils, only some of which have been characterized. Prior studies have suggested that eosinophils have non-H1, non-H2 histamine receptors which mediate the chemotactic effects of histamine. We observed previously that the histamine-induced increase in cytosolic calcium in human eosinophils could not be blocked by classic H1 or H2 antagonists, but could be inhibited by the H3 antagonist thioperamide. The purpose of this study was to further characterize the pharmacologic properties of this calcium-linked histamine receptor. Using Fura-2 loaded eosinophils to measure the concentration of cytosolic calcium, we examined the effect of additional histamine receptor antagonists and agonists. We found that the pKb for the H3 antagonists thioperamide, impromidine, and burimamide (8.1, 7.6, and 7.2, respectively), were similar to those reported for H3 receptors in the central nervous system, suggesting that the eosinophil histamine receptor was similar to H3 receptors. However, when the known H3 agonists were tested for activity ([R]-alpha-methylhistamine, N alpha-methylhistamine), the potencies of these compounds were much less than the potency of histamine itself, indicating a significant difference between H3 receptors and this eosinophil histamine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cell mediators prostaglandin-D2 and histamine activate human eosinophils.

Airway damage secondary to eosinophil activation is thought to contribute to the development of asthma. Using the fluorescent dye FURA-2 to measure the concentration of cytosolic calcium, we found that supernatants from anti-IgE-stimulated human lung mast cells increased cytosolic calcium in human eosinophils. We then examined the major mast cell mediators (histamine, PGD2, platelet-activating factor (PAF), eosinophil chemotactic factor of anaphylaxis (ECF-A), leukotriene (LT)C4 and LTB4) for their ability to increase cytosolic calcium in eosinophils. We found that both PAF (5 x 10(-9) to 5 x 10(-6) M) and PGD2 (two of five donors responsive at 1 x 10(-9) M) were potent stimuli for calcium mobilization. LTB4 (10(-8), 10(-7) M) and histamine were also active, although higher concentrations of histamine were required to see a response (3 x 10(-7) to 10(-5) M). LTC4, val-ECF-A, and ala-ECF-A were inactive. The effects of PGD2 and histamine were specific for eosinophils, although LTB4 and PAF increased calcium in both neutrophils and eosinophils. The histamine-induced increase in intracellular calcium was not blocked by the H1 or H2 antagonists pyrilamine or cimetidine (10(-4) M), respectively; however, the response to 10(-6) M histamine was completely blocked by the specific H3 antagonist thioperamide (10(-6) M). To evaluate the relative contribution of these stimulatory mast cell mediators on the calcium mobilizing activity in supernatants from anti-IgE-stimulated human lung mast cell (HLMC), we examined the effect of supernatants from HLMC pretreated with indomethacin and/or the 5-lipoxygenase pathway inhibitor MK886. These supernatants were added to FURA-2-loaded eosinophils that had been preincubated with thioperamide and/or the PAF antagonist WEB-2086. We found that the increase in eosinophil calcium in response to supernatants from anti-IgE-stimulated-HLMC was totally inhibited only when the mast cells were challenged in the presence of indomethacin and MK886, and the eosinophils were preincubated with thioperamide. WEB-2086 had little effect. When we examined the effect of these mediators on eosinophil secretory function, we found that PGD2 (not histamine) primed eosinophils for enhanced release of LTC4 in response to the calcium ionophore A23187. We conclude that the activation of eosinophils by PGD2 and other mast cell products may contribute to airways inflammation that is characteristic of asthma.

Effect of removal of epithelium on antigen-induced smooth muscle contraction and mediator release from guinea pig isolated trachea.

We examined the effect of removal of the epithelium on antigen-induced smooth muscle contraction and the release of mediators of inflammation from superfused, sensitized guinea-pig tracheal spirals in vitro. The epithelium was stripped from one-half of each trachea by mechanical means, and immunologic responses were evaluated by paired analysis. Removing the epithelium potentiated antigen-induced contraction, as reflected by a 5-fold leftward shift in the antigen dose-response curve, but the maximum response to antigen was not altered. This potentiation was not inhibited by pretreating the tissues with indomethacin (5 X 10(-6) M). At maximum concentrations of antigen removing the epithelium had no effect on the magnitude or kinetics of release of immunoreactive sulfidopeptide leukotrienes, prostaglandin (PG) D2, PGF2 alpha or thromboxane B2. Removing the epithelium did, however, significantly decrease the release of PGE and 6-keto-PGF1 alpha, a prostacyclin metabolite. Antigen-induced histamine release was enhanced by removing the epithelium; this effect varied inversely with antigen concentration. Selectively exposing either the luminal or serosal surface of an intact, superfused trachea to antigen resulted in the release of less than 5% of the total tissue histamine. Removing the epithelium from the intact trachea increased histamine release to approximately 25% following luminal but not serosal exposure to antigen. These studies demonstrate that the tracheal epithelium can act to inhibit antigen-induced airway contraction in vitro. This may in part reflect the role of the intact epithelium as a diffusion barrier which can limit the rate of influx of antigen molecules and thereby influence tissue mast cell activation.

Adenylate cyclase and ATPase activities in red cell membranes of patients and genetic carriers of Duchenne muscular dystrophy.

Basal adenylate cyclase activity was increased in red cell ghosts from both patients with Duchenne muscular dystrophy and their mothers when the activities were compared to proper age-matched controls. The activity of ATPase measured in the presence of Na+, K+, and Mg2+ was not found to be different in erythrocyte ghosts from Duchenne dystrophic patients, age-matched controls, or the mothers of Duchenne patients, and ouabain inhibited ATPase in ghosts to the same extent in all membrane preparations.

Adenylate cyclase in muscular dystrophy.

The purpose of this study was to determine whether the previously reported differences in adenylate cyclase activity between the sarcolemma of normal and dystrophic chick muscles are also found in the SR, to search for a possible relationship between the adenylate cyclase changes and the pathophysiology of dystrophy, and to investigate whether the findings can be extended to Duchenne human muscular dystrophy by studying the adenylate cyclase and ATPase activities of erythrocyte ghosts from DMD patients and carriers. Microsomes were separated by standard techniques from the pectoralis muscles of normal and dystrophic ckeckens of various ages. The microsomal yields were significantly larger in dystrophic muscles. Adenylate cyclase activities in dystrophic microsomes were higher than those in matched controls and increased with the progression of the disease. The ratio between the two rose from one at 2 weeks of age to nine at about 9--10 weeks. Kinetic analyses showed that the ks for MgATP2- was about 40 microM (at 3 mM Mg2+ and 0.3 mM Ca2+) both in normal and dystrophic microsomes, that calcium caused umcompetitive inhibition of the enzyme (Ki = 0.2 mM), that the effect of calcium was noncooperative (Hill coefficient, nH = 1), that calcium did not affect the cooperativity for MgATP2-, and that magnesium competitively removed the calcium inhibition and caused additional, cooperative stimulation of the enzymatic activity (ka = 1.5 mM; NH =2). The major difference between normal and dystrophic adenylate cyclase was a higher enzymatic velocity in the latter, suggesting a larger amount of enzyme. We investigated whether altered cAMP levels may effect calcium accumulation. Calcium uptake measured (in the presence of oxalate) at several ages revealed no difference between normal and dystrophic chickens. The extent of calcium binding was also similar, although the kd for Ca2+ was lower in dystrophic microsomes. Binding was enhanced in the presence of exogenous protein kinase, but the responses of normal and dystrophic tissues were similar. We concluded that the elevation of adenylate cyclase in dystrophy was not related to microsomal calcium accumultion. Ivestigation of the localization of microsomal adenylate cyclase supported this view. Separation of calcium-loaded microsomes on a discontinuous sucrose gradient into four fractions demonstrated that adenylate cyclase activity, measured in the presence of Lubrol-PX and EGTA, was inversely related to calcium-accumulating activity. Na+, K+-ATPase comigrated with adenylate cyclase. Highest specific activities were found in the lightest fraction. These observations were confirmed by histochemical studies. The reaction product from adenylate cyclase activity was present predominantly in the terminal cisternae of the SR. In the context of the literature, our findings suggest that the rises in adenylate cyclase and Na+, K+-ATPase in avian dystrophy are compensatory changes, elicited by a defect in ECC at the calcium release step...