No abstract

Summary:The presence of neutral endopeptidase 24.11 was demonstrated in human umbilical vein endothelial cells by immunostaining. Enzymatic activity of neutral endopeptidase was determined as 0.167 + 0.02 mU/mg protein in the membrane fraction of human umbilical vein endothelial cells, using the fluorogenic peptide substrate, dansyl-Z>-Ala-Gly-Phe(/?NO 2 )-Gly. No activity was found in the cytosolic fraction of endothelial cells. The role of this peptidase in the degradation of the endogenous vasodilator bradykinin was investigated by incubating human umbilical vein endothelial cell monolayers with bradykinin (10~8 mol/1). The inhibitor of neutral endopeptidase, phosphoramidon (10"" 8 mol/1), decreased the degradation of bradykinin in the supernatant of endothelial cells; the half-life of bradykinin was then increased from 29 + 1 to 46 ± 2 minutes. The angiotensin-converting enzyme inhibitor, lisinopril (10~8 mol/1), increased the half-life of bradykinin to 244 + 20 minutes; the combination of both inhibitors increased the half-life of bradykinin to 381 ± 51 minutes. Inhibitors of aminopeptidase (amastatin) and carboxypeptidase (2-mercaptomethyl-3-guanidinoethylthiopropionic acid) caused no significant effect. The effect of phosphoramidon was small in comparison with that of lisinopril, but was pronounced in combination with lisinopril. Neutral endopeptidase activity is localized in the membranes of human endothelial cells and seems to be involved in the degradation of bradykinin by the vascular endothelium, particularly during angiotensin converting enzyme inhibition.

A new method for the measurement of allergen-specific IgD (as-IgD) was developed by modifying the ImmunoCAP assay (Pharmacia), and amplification of the signal with a goat anti-human/rabbit anti-goat detection system. The assay was sensitive enough to measure as-IgD in serum samples. The specificity of the assay was examined using inhibition tests with excess corresponding and non-corresponding allergens. For the different allergens inhibition rates between 56% (house dust mite) and 88% (cat) could be achieved. Non-corresponding allergens did not inhibit the as-IgD binding. Total IgE and allergen-specific IgE (as-IgE) was measured using the ImmunoCAP system. Total IgD was measured using a sandwich ELISA. As-IgD was measured in serum samples from 51 atopic and 23 non-atopic subjects, and the correlation with as-IgE was examined. As-IgD was detected in both atopics and non-atopics but at higher levels in atopics. As-IgD against birch pollen and timothy pollen allergen was found to be increased in atopics with IgE directed against these allergens compared to atopics without IgE against these allergens (P < 0.02 and P < 0.03). As-IgD against birch pollen allergen was higher in atopics with IgE specific to this allergen than in non-atopics (P < 0.02). In contrast to total IgE and total IgD, significant correlations were observed between as-IgD and as-IgE against timothy pollen (r = 0.34, P < 0.04), birch pollen (r = 0.38, P < 0.05) and cat dander allergen (r = 0.52, P < 0.01). The observed correlations between as-IgD and IgE suggest that IgD and IgE may be similarly regulated, and thus the measurement of as-IgD may give further insight into the regulation of IgE.

Considerable progress has recently been made in the understanding of airway inflammation by cell culture assays and in vivo provocation studies. Inasmuch as ethical considerations limit experimental work in humans, physiologically relevant in vitro models are required to better understand cellular and molecular tissue interactions in human nasal mucosa. Here we describe a human nasal mucosa culture model utilizing a simple gelatin sponge-supported histoculture system at the air-liquid interface. Viable mucosa was preserved for at least 48 h, as shown by morphology and immunohistochemical staining with Ki-67 as marker for proliferation. Pro-inflammatory mediators (kinins, histamine, thromboxane B2, prostaglandin F2 alpha, and substance P) are detectable in serum-containing as well as serum-free culture medium. Incubation with 10(-8) M substance P increases the number of degranulated mast cells after 48 h by 26% (P < 0.01). In this model, biochemical responses can be correlated with histologic alterations of the target tissue. Inflammatory parameters can be examined and compared in various patient groups and different stimulators/inhibitors. This culture method provides a valuable research tool for analyzing all compartments present in nasal mucosa under physiologically relevant conditions, and for studying complex interactions and responses of mucosal cell populations in their natural tissue environment.

Langerhans' cells in the skin have recently been shown to bind IgE molecules via the high-affinity IgE receptor (Fc epsilon RI). Using two highly specific antibodies against the antibody-binding alpha-chain of this receptor, 29C6 and 6F7, we demonstrate by immunohistochemistry and immunoelectron microscopy that Langerhans' cells of diseased nasal mucosa can express the Fc epsilon RI. Tissue sections from hyperplastic nasal conchae and nasal polyps of atopic and nonatopic patients have shown no basic differences in epithelial Fc epsilon RI-bearing cells. Only a few cells expressed the low-affinity IgE receptor (Fc epsilon RII) (Tü1 antibody) in some sections. These findings suggest that Langerhans' cells play an important role in the induction of transepithelial IgE-mediated allergy and in the mediation of inflammation of the nasal mucosa via their Fc epsilon RI.