We have studied human umbilical vein (HUVEC) and bovine aortic endothelial cells (BAEC) for the presence of elements of the kinin-kallikrein system. Kinin generation was measured in homogenates of endothelial cells using a radioimmunoassay with a human bradykinin antibody; it was measured after homogenization and was constant over a time interval of 120 min. Addition of exogenous kallikrein (50 mU) led to a five fold increase in kinin concentrations after 5 min in the homogenates, which declined within 2 h. Pretreatment of BAEC with dextran sulfate (0.1 mg/ml) resulted in a 80% reduction in kinin generation. Staining of endothelial cells using an antiserum against glandular kallikrein showed kallikrein immunoreactivity in all cells. Glandular kallikrein activity was measured in homogenates by a colorimetric method. Activities of 3.5 ± 0.4 mU/106 cells in HUVEC and 7.5 ± 0.8 mU/106 cells in BAEC were detected. These data indicate the presence of all key elements of the kinin-kallikrein system in the vascular endothelium. Thus, our results support the existence of a local kallikrein-kinin system in the vascular wall which may contribute to the regulation of local inflammatory processes and the regulation of vascular tone.

There is accumulating evidence that tachykinins are implicated in inflammation, including asthma. Therefore, we hypothesized that the neutral endopeptidase (NEP), under challenge conditions, could be affected. Serum from 21 asthmatics and six healthy volunteers was sampled before, 30, and 120 min after allergen challenge. NEP-IR was determined using an ELISA and was found in all subjects. Compared to prechallenge, no difference was seen between asthmatics and controls; however, under challenge conditions, NEP-IR in asthmatics was significantly lower (30 min, P = 0.058; 120 min, P = 0.0017, respectively). This finding supports indirectly the hypothesis that tachykinins are released during allergen exposure, and suggests a regulatory role of NEP.

OBJECTIVE:To investigate the kinin generation pathways in acute and chronic airway inflammation.METHODS:BALF from patients with acute, chronic airway inflammation and healthy controls were collected. Kinins, Plasma kallikrein, alpha 2-macroglobulin and toluenesulphonyl-arginine methyl ester esterase activity (TAME-ea) in BALF were studied.RESULTS:Kinins and TAME-ea values were significantly higher in the BALF of patients with acute and chronic airway inflammation than those in the controls, but there was no significant difference between acute and chronic groups; PK and alpha 2-M values were significantly higher in the acute group than the in chronic one. Gel filtration revealed the highest TAME-ea peak at about 800,000 in the acute group, corresponding with the first alpha 2-M peak, whereas at about 40,000 in chronic bronchitis. The inhibition test of the TAME-ea showed that the TAME-ea peak at 800,000 was mainly due to PK and the TAME-ea peak at 40,000 was mainly due to TK.CONCLUSIONS:The results indicated that in acute airway inflammation kinins seem to be mainly generated by PK, whereas in chronic inflammation kininogenases other than PK--such as TK--seem to be more important.

Kinins are potent inflammatory mediators, liberated from kininogens by different kininogenases. The aim of this study was to investigate the kinin generation pathways in acute and chronic inflammation of the lower airways. We studied bronchoalveolar lavage fluid (BALF) of patients with acute pneumonia, patients with chronic bronchitis and healthy controls. Kinins were determined by radioimmunoassay (RIA). Plasma kallikrein (pl-Kal), alpha2-macroglobulin (alpha2-M) and toluenesulphonylarginine methyl ester (TAME) esterase activity (TAME-ea) were studied in BALF before and after gel filtration chromatography. Plasma kallikrein and alpha2-M were measured using two newly developed sandwich enzyme-linked immunosorbent assays (ELISAs). TAME-ea was measured by a radiochemical assay. After gel filtration, inhibition of TAME-ea with benzamidine, soy-bean-trypsin inhibitor (SBTI) and aprotinin was performed. Kinins and TAME-ea did not differ significantly between acute pneumonia and chronic bronchitis, whereas pl-Kal and alpha2-M values were significantly higher in acute pneumonia. Gel filtration revealed the highest TAME-ea peak in acute pneumonia corresponding with the first alpha2-M peak at approximately 800 kDa, whereas in chronic bronchitis the highest peak was found at approximately 40 kDa. The inhibition test showed that the TAME-ea peak at approximately 800 kDa was due to pl-Kal and the TAME-ea peak at approximately 40 kDa was mainly due to tissue kallikrein. High peaks of alpha2-M and pl-Kal were found in pneumonia and only small peaks were seen in chronic bronchitis. We conclude that in acute airway inflammation kinins seem to be mainly generated by plasma kallikrein whereas in chronic inflammation, kininogenases other than plasma kallikrein, such as tissue kallikrein, seem to be more important.

Haas N, Hamann K, Grabbe J, Niehus J, Kunkel G, Kolde G, Czarnetzki M. Demonstration of the high‐affinity IgE receptor (FcɛRI) on Langerhans' cells of diseased nasal mucosa. Langerhans' cells in the skin have recently been shown to bind IgE molecules via the high‐affinity IgE receptor (FcɛRI). Using two highly specific antibodies against the antibody‐binding α‐chain of this receptor, 29C6 and 6F7, we demonstrate by immunohistochemistry and immunoelectron microscopy that Langerhans' cells of diseased nasal mucosa can express the FcɛRI. Tissue sections from hyperplastic nasal conchae and nasal polyps of atopic and nonatopic patients have shown no basic differences in epithelial FcɛRII‐bearing cells. Only a few cells expressed the low‐affinity IgE receptor (FcɛRII) (Tül 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 FcɛRI.

To study the correlation between the reagibility of the bronchial system and the concentration of neutral endopeptidase (NEP) in serum bronchial challenges with acetylcholine (ACH) were performed in 60 patients with history of wheezing. Serum-NEP was measured using a newly established ELISA. NEP was detected in the serum of 38 patients. A highly significant correlation (p < 0,0006, r = 0,53, n = 38) between serum-NEP and FEV1 in the primary lung function was found. Dividing the patients into a group with hyperreactive airways (n = 15) and a group without bronchial hyperreactivity (n = 23) - according to a positive ACH provocation - we found a significant correlation between FEV1 and serum NEP (p < 0,01. r = 0,52, n = 23) in the group without hyperreactivity (FEV1 decrease < 20% and R-aw increase < 100%), whereas this correlation surprisingly was nor seen in the group with bronchial hyperreactivity (FEV1 decrease > 20% and/or R-aw increase > 100%). A correlation between serum-NEP values and FEV1 decrease after ACH provocation was not found. In conclusion, the serum NEP levels do correlate with lung function in patients with respect to FEV1. Bronchial hyperreactivity itself - according to the international definition - does not show this correlation. These results could be interpreted that NEP probably plays a role in the regulation of the bronchomotor tone.

In the lower airways Baraniuk et al. were able to localize neutral endopeptidase 3.4.24.11 (NEP) in epithelium, submucosal glands, smooth muscle cells and endothelium. In this article the distribution of NEP in nasal mucosa is described. Immunohistological stainings of kryostat sections of the nasal mucosa were performed. Hyperplastic nasal mucosa and nasal polyps of 54 patients undergoing surgery for nasal polyposis (n = 27) and hyperplastic nasal mucosa (n = 77) were stained. As primary antibodies the monoclonal antibodies ALB-1, MEK-5 and MEK-10 were used and visualized employing the APAAP method. In none of the cases NEP was detected in the epithelium or in the secretory cells of submucosal glands. There was a distinct staining pattern of a subepithelial cell layer in nasal polyps and hyperplastic mucosa, a strong staining of cells crest to submucosal glands and a clear staining of tile endothelium of postcapillary venules. In cases with shedded epithelium and thickened collagen deposition under the basement membrane no staining of the cell layer close to the epithelium was observed. All monoclonal antibodies used in this study showed the same staining pattern in the tissue.

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.

We describe the development of a new ELISA for the detection of neutral endopeptidase 3.4.24.11 (NEP). Neutral endopeptidase 3.4.24.11 was determined in preparations of human granulocytes, mononuclear cells (MNC), and in serum. Human recombinant NEP was used as reference. Specificity of the mAbs was tested using APAAP, FACS analysis, and Western blot analysis. Lysis of the blood cells was performed by incubating the cells with 0.4% Tween-20 and repeated freezing cycles. The minimal detectable dose for recombinant NEP was 15 pg/ml. The recovery was 94 ± 9%. The NEP was detectable in 15 out of 20 serum samples of 20 volunteers (mean ± SEM, 245 ± 88 pg/ml, n = 20)) and in all granulocyte preparations (1176 ± 138 pg/107 cells, n = 20)). The results were reproducible among replicates (CV = 3 ± 1%, n = 40), dilutions (CV = 8 ± 2%, n = 5), and assays (CV = 12 ± 4%, n = 5). With this new ELISA, a simple and reproducible method for the measurement of NEP 3.4.24.11 is described.

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 noncorresponding 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 nonatopic 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.

A new hypothesis integrates neuropeptides such as vasointestinal peptide, neurokinin A, calcitonin gene-related peptide, and substance P (SP) into the pathophysiological considerations of the development of bronchial-inflammatory diseases. Since bronchial hyperreactivity is one of the main diagnostic features of bronchial asthma, it is important to evaluate its underlying causes. This chapter discusses allergic bronchial asthma and describes substance P in bronchoalveolar lavage (BAL) fluid in grass pollen-allergic patients before and after allergen provocation to obtain more information about the role of substance P and ß-endorphin. Comparing the baseline concentrations for substance P, there was a significant difference between patients and volunteers. The baseline concentration of SP was 6.8 times higher in allergic persons. The same finding was observed for ß-endorphin, where the baseline was 14.3 times higher in patients than in volunteers. The results provide evidence that baseline concentrations of substance P and ß-endorphin in BAL fluid of allergic asthmatics was significantly elevated.

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-D-Ala-Gly-Phe(pNO2)-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-guanidinoethyl-thiopropionic 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.

In recent years it has become evident that neuropeptides make an important contribution to inflammation in respiratory disease, especially rhinitis and asthma. In the neural-control of the airways cholinergic, adrenergic, and non-adrenergic non-cholinergic mechanisms are involved. It is known today that type C sensory nerves, parasympathetic and sympathetic nerves release—depending on their stimulation—neuropeptides in specific combinations (CGRP, SP, NKA, GRP for sensory nerves, VIP and PHM for parasympathetic and NPY for sympathetic nerves). Upon stimulation, neuropeptides are released locally and act via antidromic stimulation in the form of local axon reflexes augmenting the tissue response, resembling the symptoms of rhinitis and asthma like hypersecretion, itching, sneezes, nasal blockage, coughs, sputum production, shortness of breath. The neuropeptides best examined so far are SP and VIP. There is a clear indication that these peptides are involved in nasal and bronchial airway response to allergens. Their close relationship to mediators of inflammation like histamine, bradykinin is described. The role of GRP, CGRP, NKA and NPY in allergic rhinitis and asthma is still not yet clear.

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-D-Ala-Gly-Phe(pNO2)-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/l). The inhibitor of neutral endopeptidase, phosphoramidon (10(-8) mol/l), 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/l), 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-guanidinoethyl-thiopropionic 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 novel procedure for determining the growth fraction of cell suspensions by flow cytometry is described. This method identifies proliferating cells by binding the monoclonal antibody Ki-67 to a nuclear antigen present in all cells that are in the G1, S, G2, and M phase of the cell cycle, but not in the G0 phase. In a kinetic study of Na cell line U937 using concanavalin A for stimulation of peripheral blood mononuclear cells, a steady increase of Ki-67 positive cells evaluated by flow cytometry was observed. Simultaneously, the [3H]thymidine uptake of the ConA blasts was measured and compared to the expression of Ki-67. A linear correlation between the percentage of Ki-67 positive cells and the log transformed counts per minute was demonstrated, and staining with Ki-67 detected cell proliferation with the same sensitivity as 3H-TdR uptake. In addition, it was possible to stain Ki-67-labelled cells with a second cell marker if a second fluorescent dye coupled to an antibody was used. This provided the opportunity to define precisely the phenotype of proliferating cells. Conversely, the number of proliferating cells expressing certain preselected surface markers could be easily determined.