Influx of kininogens into nasal secretions after antigen challenge of allergic individuals.

We have recently demonstrated that kinins are generated in vivo after nasal challenge with antigen of allergic, but not nonallergic, individuals. The present study was undertaken as a first step in determining the mechanism(s) of kinin formation during the allergic reaction and was directed towards establishing the availability and origin of kininogens in nasal secretions. Allergic individuals (n = 6) and nonallergic controls (n = 5) were challenged with antigen; and by using specific radioimmunoassays, nasal washes, obtained before and after challenge, were assayed for high molecular weight kininogen (HMWK), total kininogen (TK), albumin, and kinins. Dramatic increases in HMWK (1,730 +/- 510 ng/ml), TK (3,810 +/- 1035 ng/ml), kinin (9.46 +/- 1.75 ng/ml), and albumin (0.85 +/- 0.2 mg/ml) were observed after challenge of allergic individuals which correlated (P less than 0.001) with increases in histamine and N-alpha-tosyl-L-arginine methyl esterase activity and with the onset of clinical symptoms. For nonallergic individuals, levels of kininogens, albumin, and all mediators after antigen challenge were not different from base line. Linear regression analysis revealed excellent correlations (P less than 0.001 in each case) between increases in HMWK, TK, kinin, and albumin during antigen titration experiments and between the time courses of appearance and disappearance of HMWK, TK, kinin, and albumin after antigen challenge. Gel filtration revealed no evidence of degradation products of kininogens in nasal washes. For each allergic individual the ratio of HMWK/TK in postchallenge nasal washes was similar to the ratio of these two proteins in the same individual's plasma. These data suggest that, during the allergic reaction, there is an increase in vascular permeability and a transudation of kininogens from plasma into nasal secretions, where they can provide substrate for kinin-forming enzymes.

Ramiprilat increases bradykinin outflow from isolated hearts of rat.

To establish that bradykinin is formed in the heart we measured bradykinin in the venous effluent from rat isolated hearts perfused with Krebs-Henseleit buffer. In addition, we examined the effect on bradykinin outflow of the angiotensin converting enzyme (ACE) inhibitor, ramiprilat. From rat isolated normoxic hearts a bradykinin outflow of 0.85 +/- 0.1 ng ml-1 perfusate g-1 wet weight was measured. Perfusion with ramiprilat increased the bradykinin concentration to 2.8 +/- 0.3 ng ml-1 perfusate g-1 wet weight. During ischaemia bradykinin outflow maximally increased 8.2 fold to 7.0 +/- 0.5 ng ml-1 perfusate g-1, and in ramiprilat-perfused hearts 5.8 fold to 16.0 +/- 1.8 ng ml-1 perfusate g-1. In the reperfusion period bradykinin outflow normalized to values measured in the respective pre-ischaemic period. The presents data show that bradykinin is continuously formed in the rat isolated heart. Ischaemia increases bradykinin outflow from the heart. Presumably by inhibiting degradation of kinins, ACE inhibition significantly increased the bradykinin concentration during normoxia, ischaemia and reperfusion.

Substance P enhances antigen-evoked mediator release from human nasal mucosa.

Exogenous substance P (10-80 nmol/ml) induced a dose-dependent increase in nasal symptoms in asymptomatic allergics with rhinitis (n = 15) and controls (n = 8), but did not release any mediators. However, comparing the antigen-evoked release of mediators into nasal secretions with that of a substance P-pretreated antigen challenge, we found a significant enhancement of kinins, TAME esterase activity (p < 0.05-0.01), and histamine (p < 0.001, NS) 10-20 min after antigen challenge. These results suggest 1) that substance P-induced increase in nasal congestion is mediated through direct neurokinin receptor activation independently of mast cell activation, and 2) that during the allergic reaction there is a substance P-mast cell interaction that enhances the mediator response to nasal allergen challenge.

ELISA for the neuropeptide degrading endopeptidase 3.4.24.11 in human serum and leukocytes.

We describe the development of a new ELISA for the detection of neural 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/10(7) 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.

Determination of substance P in human nasal lavage fluid.

The determination of substance P (SP) concentrations in human nasal lavages can be used to monitor physiological and certain pathophysiological processes in human airway mucosa. But, because of the low concentrations, immunoassays of high sensitivity are needed. Two approaches to improve the sensitivity of the radioimmunological determinations of SP are compared: increasing the sample volume and miniaturizing the assay design. The characterization of SP-like immunoreactivity (SP-LIR) in human nasal lavage was performed by investigating the immunological specificity of the antibody used in the radioimmunoassays and by reversed-phase high-performance liquid chromatography separation of the SP-LIR. SP concentrations in nasal lavages can be reliably measured by each of the two introduced RIA methods. Despite the lower detection limit of the miniaturized immunoassay (0.2 in comparison to 1.3 fmol/incubate) it is advisable to increase the sample volume in order to improve the sensitivity because of the higher precision of the determinations. SP-LIR was found in nasal lavage specimens in concentrations between 2 and 10 fmol/ml and consisted of authentic SP and, to a less extent, SP-sulfoxide.

Equivalence of as-required salbutamol propelled by propellants 11 and 12 or HFA 134a in mild to moderate asthmatics. German Study Group.

This randomized, double-blind, parallel-group study compared the efficacy and tolerability of as-required salbutamol 100 microg administered from either a chlorofluorocarbon (CFC) pressurized metered dose inhaler (pMDI; Ventolin) or from a non-CFC hydrofluoroalkane (HFA) 134a pMDI (Ventolin CFC-free) in patients with mild to moderate asthma. All patients (n = 423) continued with their standard asthma therapy, and recorded their daily use of study medication, morning and evening peak expiratory flow (PEF) and symptom scores, throughout the 4-week treatment period. Clinic lung function was measured at 2-week intervals. The median daily use of inhaled study medication remained constant at four actuations per day throughout the study in both treatment groups and statistical analysis indicated that the two formulations were equivalent. Small improvements in both treatment groups were reported in mean morning and evening PEF, clinic forced expiratory volume in 1 sec and clinic PEF and there were no significant differences between the two groups. Both formulations were well tolerated. This study indicates that as-required salbutamol 100 microg administered via a HFA 134a pMDI is as effective and safe as the currently available CFC-propelled formulation.

Kininogens in nasal secretions after allergen challenge.

Allergic individuals and nonallergic controls were subjected to nasal challenge with allergen; and nasal washes, obtained before and after challenge, were assayed for high molecular weight kininogen (HMWK), total kininogen (TK), albumin and kinins. Following challenge of allergic individuals, HMWK, TK, kinin and albumin all increased dramatically, correlating (p less than 0.001) with the onset of clinical symptoms and with increases in histamine and TAME-esterase activity. No such increases were seen upon challenge of nonallergics. The time course of appearance and disappearance of the kininogens, kinins and albumin were all highly correlated (p less than 0.001 in each case) by linear regression analysis, as were the increases in kinin and each of the proteins during antigen titrations. For each individual, the plasma ratio of HMWK/TK was similar to the ratio of these two proteins in post-challenge nasal washes from the same individual. These findings are consistent with the hypothesis that, during the allergic reaction, vascular permeability increases, allowing a transudation of kininogens from plasma into nasal secretions, where they can provide substrate for kinin-forming enzymes.

[Kinin generation in acute and chronic airway inflammation].

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.

Nasal challenge studies with bradykinin: influence upon mediator generation.

Previous studies have shown that nasal allergen provocation leads to dose-dependent increases of inflammatory mediators, e.g. histamine, kinins, LTC4 and PGD2 in nasal lavages. To investigate further the interaction of these mediators, a titration study with intranasal bradykinin (Bk) application (maximal dose 100 nmol/nostril) and consecutive lavage were performed in eight grass-pollen-allergic patients out of season, and five controls. The nasal lavages were analysed for albumin, N-alpha-tosyl-L-arginine methyl ester (TAME) esterase activity, histamine, 9 alpha,11 beta-PGF2, and LTC4. The clinical reactions were measured with a subjective symptom score. A dose-dependent elevation of albumin was found which was significantly higher in patients with allergic and non-allergic rhinitis compared with normal volunteers. TAME-esterase activity also increased in relation to the dosage of Bk given without significant difference between the various groups. No influence on histamine, LTC4 and 9 alpha,11 beta-PGF2, release (PGD2 metabolite) was seen. Short-lasting clinical symptoms like irritation, sneezing, and obstruction were noticed after the two highest Bk dosages (10 and 100 nmol). We conclude that intranasally applied Bk induces a dose-dependent plasma leakage into the nasal cavity, which is significantly higher in patients with seasonal allergic rhinitis out of season compared to normals. Bk does not seem to affect the mast cell since histamine, LTC4 and 9 alpha,11 beta-PGF2 levels do not alter. The ability to induce relevant symptoms of rhinitis provides strong support for the hypothesis that kinins may be important mediators of inflammatory disorders of the upper airways.

Kinin generation following methacholine nasal airway challenge of non-allergic subjects.

Following nasal challenges with the muscarinic neurotransmitter methacholine in 14 patients with chronic non-allergic rhinitis, kinin generation was induced. This occurrence was inhibited by pretreatment with anticholinergic agents (ipratropium bromide) and by an overdose of neurotransmitter, indicating that a muscarinic receptor is stimulating the liberation of kinin. These observations suggest that patients with chronic non-allergic rhinitis exhibit cholinergic neurotransmitter-induced kinin generation.

Contralateral differences among biomarkers determined by a modified nasal lavage technique after unilateral antigen challenge.

The concentration of biomarkers from vessels and inflammatory cells in nasal lavage fluid reflects the degree of hyperresponsiveness in patients with allergic rhinitis. The lavage has usually been performed of both nasal cavities together after prewashings and administration of decongestants. To improve the technique, we introduced a modification involving lavage of the nasal cavities separately without any prewashings or decongestants. We challenged 20 rhinitic subjects sensitive to timothy unilaterally with timothy extract. In nasal lavages performed before, immediately after, and 6 h after the challenge, we determined the concentrations of albumin, histamine, bradykinin, TAME (N-alpha-tosyl-L-arginine methyl ester)-esterase, and leukotriene C4 (LTC4). In eight subjects, the procedure was repeated 1 and 2 weeks later. After the challenge, albumin, bradykinin, TAME-esterase, and LTC4 in the nasal lavage fluid increased on the ipsilateral side but not on the contralateral side. Histamine did not increase after antigen challenge. After 6 h, the biomarkers were not increased. The concentrations of biomarkers did not differ between sides before the challenge and not between visits. Thus, the modified nasal lavage technique is reliable and improved compared to previous methods because it involves reproducible determinations of different biomarkers, and it is simple and easy to perform.

Bradykinin degrading activity in cultured human endothelial cells.

The role of angiotensin-converting enzyme (ACE), neutral endopeptidase 24.11 (NEP), and other peptidases in the endothelial degradation of bradykinin was investigated in cultured human umbilical vein endothelial cells (HUVEC). The major part of the kininase II activity on intact cells was attributed to ACE activity, the minor part to NEP activity. Amastatin, as aminopeptidase inhibitor, and DL-2-mercaptomethyl-3-guanidinoethyl-thiopropionic acid (MGTA), an inhibitor of kininase I, did not affect endothelial kininase activity. The decline of the bradykinin concentrations in the supernatant of intact endothelial monolayer indicated a total kininase activity of 289 +/- 27 fmol/min/dish. The calculated activity of ACE was 223 fmol/min/dish and the neutral endopeptidase activity was 51 fmol/min/dish. Thus, ACE and neutral endopeptidase are the main kininases in the degradation of bradykinin by intact endothelial cells. In contrast to the intact endothelial monolayers, in homogenates additional kininase activity was found which was not affected by either ACE and NEP inhibitors nor by amastatin and MGTA.

The role of kinins in human allergic disease.

We have provided clear evidence that kinins are generated during local allergic reactions in man and have begun studies on the mechanisms by which kinins are formed during these reactions. Clearly, the extent to which kinins may contribute to the symptomatology of allergic rhinitis remains to be determined, but the levels of kinins detected in the model systems described above are sufficient to cause relatively profound physiologic effects. In addition to its ability to produce vasodilatation and edema, bradykinin has been reported to stimulate fluid production from airway submucosal glands via a reflex action and could function to increase mucus production and cause rhinorrhea. Kinins have also been shown to increase chloride transport in the airway and to stimulate the production of prostaglandin E2 by epithelial cells. Thus we believe kinins must now be considered as potentially important mediators in the pathogenesis of human allergic diseases.

Changes in non-specific nasal reactivity and eosinophil influx and activation after allergen challenge.

It has been suggested that the eosinophilic granulocyte plays a crucial role in the genesis of increased reactivity of the airways. In order to characterize changes in non-specific reactivity in the upper airways following a nasal allergen challenge further 16 subjects with strictly seasonal allergic rhinitis were studied. They were challenged with allergen outside the relevant pollen season and monitored at intervals for a period of 24 hr for nasal symptoms, changes in nasal reactivity, eosinophil influx and activation, and markers of inflammation. The same challenge sequence without an initial allergen challenge was used as a control. A symptom score technique was used to record nasal symptoms and methacholine challenges were used to monitor changes in non-specific reactivity. A nasal lavage was made prior to each methacholine challenge to monitor the influx of cells, specifically eosinophils, and to determine changes in the levels of eosinophil cationic protein (ECP) and TAME-esterase activity. Cells from the mucosal surface were also collected with a Rhinobrush prior to the allergen challenge as well as at the 24-hr follow up. The allergen challenge induced a five-fold increase in non-specific nasal reactivity, as measured by the methacholine challenges, at the 2-hr follow up from 0.051 ml +/- 0.012 (mean +/- s.e.m.) to 0.255 +/- 0.062 (P less than 0.01) and a significant increase was also noted at all observation points, whereas no increases could be observed in the control setting.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversibility and reproducibility of histamine induced plasma leakage in nasal airways.

Plasma exudation is one cardinal factor in airways defence and inflammation. In inflammatory airway diseases such as rhinitis and asthma, however, plasma leakage may also have a pathogenetic role. Experimental data from animals indicate that highly sensitive, active, and reversible processes regulate the vascular and mucosal permeability to macromolecules. With the use of a nasal lavage model for the recovery of liquids on the mucosal surface the effect of histamine on the macromolecular permeability of the airway endothelial-epithelial barriers was studied in normal subjects. The concentrations of albumin, kinins, and N-alpha-beta-tosyl-L-arginine-methyl esterase (TAME) in nasal lavage fluid were measured and nasal symptoms assessed by a scoring technique. The reproducibility of three repeated challenges with 30 minute intervals on the same day was studied in 12 subjects and compared with the same procedure (three challenges) on a different day. Sneezing decreased significantly (p less than 0.05) after the first histamine challenge but was maintained thereafter. Otherwise, the mean values for symptoms and for markers of vascular leakage were very similar both for the three challenges in the same session and for the two challenge sessions on a different day. Sneezing, blockage, and secretions were associated with increased concentrations of TAME esterase (maximum 9000 cpm/ml), kinins (1.4 ng/ml), and albumin (0.3 g/l) in lavage fluid. Both the symptoms and the measures of plasma exudation were reversible and reproducible in the three repeat histamine challenges and at two challenge sessions on different days. These findings support the view that non-injurious, active processes regulate the inflammatory flow of macromolecules across airways endothelial-epithelial barriers. The present experimental approach would be suitable for studies of the modulatory effects of inflammatory stimulus induced plasma leakage and symptoms in human airways.

Concentrations of glandular kallikrein in human nasal secretions increase during experimentally induced allergic rhinitis.

We have previously demonstrated that a mixture of bradykinin and lysylbradykinin is generated in nasal secretions during the immediate allergic response to allergen. The present studies were performed to determine whether glandular kallikrein plays a role in kinin formation during the allergic reaction. Allergic individuals (n = 7) and nonallergic controls (n = 7) were challenged intranasally with appropriate allergen, and nasal lavages obtained before and after challenge were assayed for immunoreactive glandular kallikrein as well as for histamine, kinins, and N-alpha-tosyl-L-arginine methyl esterase (TAME-esterase) activity. The increase in postchallenge immunoreactive glandular kallikrein levels above baseline was significantly greater (p less than 0.01) for the allergic group (16.3 +/- 14 ng/ml; means +/- SD) than for the nonallergic controls (1.0 +/- 1.9 ng/ml). Increased levels of immunoreactive glandular kallikrein correlated with increases in kinins, histamine, and TAME-esterase activity and with the onset of clinical symptoms. Characterization of immunoreactive glandular kallikrein purified from postchallenge lavages by immunoaffinity chromatography confirmed the identity of this material as an authentic glandular kallikrein on the basis of its inhibition by protease inhibitors and by monospecific antibody to tissue kallikrein, its chromatographic behavior on gel filtration, and its ability to generate lysylbradykinin from highly purified human low m.w. kininogen. The specific activity of this purified material, in terms of kinin generation from kininogen, was very similar to that for authentic glandular kallikrein, suggesting that most if not all of the immunoreactive material purified from nasal lavages represented active enzyme. Inhibition studies by using pooled postchallenge lavages suggest that the majority of the kinin generating activity in these samples was due to glandular kallikrein. We conclude, therefore, that glandular kallikrein is secreted during the allergic response and can contribute to the formation of the lysylbradykinin produced during the allergic reaction.

Kinin metabolism in human nasal secretions during experimentally induced allergic rhinitis.

We have previously shown that both bradykinin and lysylbradykinin are generated in nasal secretions upon nasal challenge of allergic individuals with appropriate allergen and have suggested that these potent pro-inflammatory peptides may contribute to the pathogenesis of the allergic response. In this study we used a variety of synthetic substrates together with both thin layer and high performance liquid chromatography systems to examine the metabolism of these peptides in nasal secretions obtained by lavage. We now demonstrate that in addition to low levels of angiotensin-converting enzyme, nasal lavages contain an aminopeptidase activity that converts lysylbradykinin to bradykinin, and a carboxypeptidase that removes the C-terminal arginine from bradykinin and lysylbradykinin. The levels of all these activities are significantly increased after allergen challenge of allergic, but not nonallergic, individuals. The aminopeptidase and carboxypeptidase activities present in post-challenge lavages from allergic individuals convert lysylbradykinin to intermediate products (bradykinin and des (Arg10) lysylbradykinin) and eventually to des (Arg9) bradykinin. The nasal carboxypeptidase was activated 475% by 0.1 mM CoCl2 and was inhibited by the carboxypeptidase N inhibitor, MERGETPA (D-L-mercaptomethyl-3-guanidino-ethylthiopropanoic acid) (IC50 = 10 microM). The aminopeptidase activity was not affected by MERGETPA but was potently inhibited by amastatin and bestatin (IC50 = 0.05 microM and 3.0 microM, respectively). The activity of the aminopeptidase against its synthetic substrate was also inhibited by lysylbradykinin (IC50 = 50 microM). Both the carboxypeptidase and aminopeptidase activities had neutral pH optima and were inhibited by o-phenanthroline, but were unaffected by inhibitors of neutral endopeptidases (phosphoramidon) or angiotensin-converting enzyme (Captopril). The Km of bradykinin for the nasal carboxypeptidase was 139 +/- 14 microM (n = 3). We conclude that during the allergic response, nasal secretions contain aminopeptidase and carboxypeptidase activities that convert lysylbradykinin and bradykinin (B2 agonists) to des (Arg9) bradykinin (a B1 agonist). Because the nature of the kinin receptors in the nasal mucosa are currently unknown, it remains to be determined whether this metabolism results in the termination of biologic activity or the production of a biologically active moiety.

Topical vasoconstrictor (oxymetazoline) does not affect histamine-induced mucosal exudation of plasma in human nasal airways.

Mucosal exudation of almost unfiltered plasma proteins, plasma-derived mediators and fluid has recently been advanced as a major respiratory defence mechanism. Oxymetazoline chloride is a commonly used decongestant agent. By reducing blood flow it may reduce mucosal exudation and thus compromise the mucosal defence capacity. This study examines the effect of topically applied oxymetazoline on histamine-induced plasma exudation into human nasal airways. Twelve normal volunteers participated in a double-blind, randomized, cross-over and placebo-controlled study with pretreatment with a single dose oxymetazoline chloride (5 micrograms or 50 micrograms; a dose previously known to reduce nasal mucosal blood flow by almost 50%) prior to the histamine challenge sequence. Nasal lavages were performed every 10 min for 140 min, and three histamine challenges were performed at 30-min intervals during this period. The concentrations of two exudative indices, N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity and albumin, were measured in the nasal lavage fluids. Nasal symptoms (sneezing, nasal secretion and blockage) were assessed by a scoring technique. Histamine induced all three symptoms with correlatively raised levels of the biochemical markers for plasma exudation. Oxymetazoline chloride caused a significant decrease in nasal stuffiness, but did not influence the other nasal symptoms or the histamine-induced plasma exudation. It is concluded that histamine-induced plasma exudation is not influenced by topical oxymetazoline. Thus, an important airway defence reaction such as plasma exudation may be little affected by topical alpha-adrenoreceptor-mediated vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

Tryptase in nasal lavage fluid after local allergen challenge. Relationship to histamine levels and TAME-esterase activity.

The activation of mast cells is generally considered to be an important trigger mechanism in the immediate allergic response. This study focused on the determination of three markers of mast cell activation after an allergen challenge. Nasal allergen challenges were performed in 25 subjects with seasonal allergic rhinitis using three allergen doses increasing in 10-fold steps in a standardised nasal lavage model for the subsequent recovery of the markers of mast cell activation. The levels of histamine and tryptase in the nasal lavage fluid were determined using radioimmunoassays, while the TAME-esterase activity was determined using a radiochemical technique. The nasal symptoms obtained on challenge were assessed using a scoring technique. The allergen challenge resulted in significant increases in the levels of all three markers, tryptase, histamine and TAME-esterase. In the individual measurements after the challenges there was a highly significant correlation between the TAME-esterase levels and the tryptase levels (r = 0.71; P less than 0.001), while the generation of histamine and tryptase was not significantly correlated. When comparing the cumulative generation of the three markers, significant correlations were found between all three. Allergen challenges in six non-allergic controls using the same technique did not result in any increase in tryptase levels. The findings suggest that the determination of tryptase in nasal lavage fluid may be a valuable indicator of mast cell activation in the upper airways.