Deborah A. Weiler

Establishment of an immature mast cell line from a patient with mast cell leukemia

A cell line showing many characteristics of immature mast cells has been established from the peripheral blood of a patient with mast cell leukemia. Cultured cells contain low levels of histamine, are stained metachromatically by toluidine blue, and contain chloroacetate esterase, aminocaproate esterase and tryptase activities. The cells lack T and B lymphocyte, as well as myeloid cell markers, and do not possess IgE receptors. Solid tumors of metachromatically positive cells have been successfully induced and serially passed in nude mice using 5-azacytidine transformed cells. This cell line may be useful for future studies of mast cells and their constituents.

Mechanism of Endothelial Dysfunction in Apolipoprotein E–Deficient Mice

Abstract—Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis. Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10−9 to 10−5 mol/L) and Ca2+ ionophore (10−9 to 10−6 mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N, N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10−10 to 10−5 mol/L) compared with aortic rings from C57BL/6J mice (P <0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O2−) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn-superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10−5 mol/L), reduced O2− production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (P <0.05). [14C] l-Citrulline assay showed a decrease of Ca2+-dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (P <0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (P <0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O2− and reduced endothelial NO synthase enzyme activity. Thus, chemical inactivation of NO with O2− and reduced biosynthesis of NO are key mechanisms responsible for endothelial dysfunction in aortas of atherosclerotic apoE-deficient mice.

Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes.

Eosinophil‐derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil‐specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112 ± 4 ng/106 cells, and ECP, 163 ± 2 ng/106 cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold‐labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription‐polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross‐reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil‐associated inflammation in human disease. J. Leukoc. Biol. 63: 715–722; 1998.

Ammonium chloride exposure inhibits cytokine-mediated eosinophil survival

To study human eosinophils, their efficient purification from peripheral blood is crucial. Although a number of purification procedures, including discontinuous Percoll and metrizamide density gradient centrifugation, have been used, it has been difficult to isolate eosinophils from normal donors with consistently high yields and purities. Recently, a new isolation technique called magnetic cell separation system (MACS) was reported. To evaluate this procedure, we isolated eosinophils from human peripheral blood using either MACS or the standard discontinuous Percoll density methods, and compared cellular viability, morphology, and response to degranulation stimuli. MACS gave a higher yield of eosinophils than Percoll density centrifugation; for example, 6.6 +/- 1.1 x 10(6) eosinophils were isolated from 20 ml of blood by MACS compared to 6.4 +/- 2.4 x 10(6) from 120 ml by Percoll density gradient. Further, the purity of eosinophils isolated by MACS was 97.1 +/- 0.5% (X +/- SEM) compared to 77.8 +/- 2.9% with Percoll. As part of the MACS protocol, erythrocytes are lysed with either 155 mM ammonium chloride or hypotonic lysis. With 155 mM ammonium chloride treatment, the eosinophils showed a striking reduction in cytokine mediated survival due to interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), marked morphologic abnormalities and a reduced degranulation response. With hypotonic lysis, no differences were observed in survival and morphology between eosinophils purified by MACS and Percoll methods; the degranulation responses to stimuli were essentially the same between the two methods. Taken together, these observations suggest that the exposure of eosinophils to 155 mM ammonium chloride results in cellular damage. Therefore, MACS with hypotonic lysis is a useful technique to isolate eosinophils for biological study.

Purification of tryptase from a human mast cell line.

The neutral protease tryptase has been isolated from a human mast cell line, HMC‐1. The HMC‐1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC‐1‐derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin‐agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS‐PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl‐L‐arginine methyl ester by purified tryptase was inhibited by dansyl‐L‐glutamyl‐glycyl‐L‐arginine chloromethyl ketone 2 HCI, HgCI2, tosyl‐L‐lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl‐L‐phenylalanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary‐derived tryptase.

Immunofluorescent staining for mast cells in idiopathic pulmonary fibrosis: quantification and evidence for extracellular release of mast cell tryptase

In many diseases, retrospective analysis for determining the presence of mast cells has been difficult because of their loss of metachromatic staining properties once tissue has undergone formalin fixation. We quantified mast cells in peribronchiolar tissue of idiopathic pulmonary fibrosis (IPF) and in normal human lung by using rabbit antiserum to human mast cell tryptase. In lung biopsy specimens from 15 patients with IPF, the mean number of mast cells per high-power field in connective tissue directly adjacent to the lumen of small airways (0.5 to 2 mm in diameter) and other fibrotic foci was 29.9 +/- 10.8 in comparison with 13.7 +/- 3.5 in 16 normal controls (P < 0.001). In addition, mast cells in cases of IPF had an altered appearance--irregularity of the plasma membrane and release of extracellular tryptase. We conclude that the number of mast cells is increased in IPF and that the altered appearance of the mast cells suggests that they are activated and undergoing degranulation.

Adenovirus-Mediated Gene Transfer to Human Cerebral Arteries

Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1000 μm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli β-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant β-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.

Effects of glucocorticoids on eosinophil colony growth

The in vivo and in vitro effects of glucocorticoids on eosinophilopoiesis were examined with soft agar cultures of bone marrow and peripheral blood cells. Prednisone, 10 mg four times daily for three days, administered to normal volunteers, caused a significant drop in circulating eosinophil numbers (p less than 0.005) but did not decrease eosinophil colony numbers in cultures of bone marrow or peripheral blood. A patient with peripheral blood eosinophilia demonstrated a larger percentage decrease in mean eosinophil colony numbers (50%) after prednisone than did any normal volunteer. Incubation of bone marrow cells for 1 hour with either high concentrations of hydrocortisone, up to 3.3 X 10(-4) mol/L, or with postinfusion plasma from volunteers administered intravenous hydrocortisone, plasma levels to 1.6 X 10(-5) mol/L, did not decrease the numbers of eosinophil colonies. At a concentration of 3.3 X 10(-6) mol/L of hydrocortisone, there was a slight but statistically significant stimulation of eosinophil colony numbers. In contrast, incubation with high concentrations of dexamethasone, 3.3 X 10(-4) mol/L or 3.3 X 10(-6) mol/L, significantly reduced eosinophil colony numbers. Prednisone caused a significant reduction in plasma levels of Charcot-Leyden crystal protein but not of eosinophil granule major basic protein. The results indicate that soft agar assay of eosinophil colony growth by blood or bone marrow cells cannot be used to model the in vivo eosinopenic effect of glucocorticoids, levels of dexamethasone in excess of levels commonly administered in clinical practice are required to inhibit eosinophilopoiesis in vitro, and patients with peripheral blood eosinophilia may be more susceptible to the eosinopenic effects of glucocorticoids than normal subjects.

A direct mechanical method for accurate and efficient adenoviral vector delivery to tissues

We describe a mechanical method for delivery of adenoviral vector to the adventitial surface of arteries and to other tissues. Our goal was to characterize, principally in intact carotid artery, the morphological, biochemical, and functional effects of mechanical delivery of a recombinant β-galactosidase-expressing adenoviral vector following its direct application using a small paintbrush. Our ex vivo and in vivo data demonstrate efficient, accurate, and rapid transduction of arteries without compromise of their morphological, biochemical, and functional integrity. We also demonstrate the general applicability of this technique in vivo via transduction of skeletal muscle, fibrotendinous tissue, peritoneum, serosal surface of bowel, and wounded skin. We conclude that direct mechanical delivery of an adenoviral vector to tissues using a suitable paintbrush represents an intuitive, accurate, and effective means of augmenting gene transfer efficiency, and may be a useful adjunct to other delivery methods.

Eosinophil differentiation of human umbilical cord mononuclear cells and prolonged survival of mature eosinophils by murine EL-4 thymoma cell conditioned medium

Umbilical cord mononuclear cells, HL-60 cells, HL-60 clones selected for eosinophil differentiation, and the eosinophil leukemia cell line EoL were tested for their ability to produce eosinophil peroxidase. HL-60 clones selected for eosinophil differentiation produced eosinophil peroxidase, as judged by staining of cells for cyanide-resistant peroxidase activity; however, these cells lost their ability to produce eosinophil peroxidase in long-term culture. In contrast, eosinophil precursors from human umbilical cord blood mononuclear cells stimulated with murine EL-4 conditioned medium (EL-4 CM) were regularly induced to eosinophil protein synthesis, including eosinophil peroxidase, major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin, as assessed by cyanide-resistant peroxidase and immunofluorescence staining. This induction by EL-4 CM is either at the level of gene transcription or mRNA stabilization, as shown by the increase of total mRNA for eosinophil peroxidase, major basic protein, and eosinophil-derived neurotoxin by Northern blot analyses. Purified peripheral blood eosinophils incubated for 4 days with EL-4 CM had increased survival over control eosinophils. Moreover, this enhanced survival was specifically blocked by antiserum to interleukin 5. Our results suggest that the effects of EL-4 CM on human umbilical cord mononuclear cells and mature eosinophils are due to the presence of interleukin 5.