A new radioimmunoassay for human mast cell tryptase using monoclonal antibodies.

A solid phase immunoradiometric assay was developed for the quantitation of tryptase released from activated human mast cells. Tryptase exhibits a linear dose-response curve over the standard range of 2-50 micrograms/l in buffer, serum, and plasma. The dose-response curve approached a plateau at a tryptase concentration of 100 micrograms/l and exhibited partial inhibition at concentrations above 10,000 micrograms/l. The sensitivity of the assay was 0.2-0.4 micrograms/l, and the intra-assay and interassay coefficients of variation were below 4% at 2 micrograms/l or higher tryptase concentrations. The recovery of known amounts of purified tryptase added to serum ranged from 91 to 115%. Detection of tryptase was evaluated with several body fluids and was accurate in sera, plasma, bronchoalveolar lavage fluid, nasal lavage fluid, and saliva. The concentration of tryptase was examined in serum samples from 100 healthy controls; in each case the level was less than 2 micrograms/l. The immunoassay also was utilized to examine serum levels of tryptase after the onset of a hypotensive reaction in one patient receiving general anesthesia. A maximally elevated level of tryptase (25 micrograms/l) was detected at the first time point, 0.5 h, and elevated levels persisted to 6 h before a return to normal levels was documented at 24 h. Thus, the involvement of mast cell activation in hypotensive subjects can be ascertained by this new tryptase radioimmunoassay.

Seasonal variation in serum eosinophilic cationic protein (S-ECP) in a general population sample.

The aim of this investigation was to study the seasonal variation in the serum levels of eosinophil cationic protein (S-ECP). The study population comprised a general population sample of 379 individuals (range: 20-45 years) who were investigated with blood sample for the measurement of S-ECP, skin prick test and methacholine challenge. The examination took place between May and October 1991. Of the 379 subjects investigated, 137 (36%) were atopic. A significant seasonal variation in S-ECP was found in the group of birch-pollen-positive subjects (P < 0.05), but not in the non-atopic or birch-negative atopic group. The mean level of S-ECP in birch-positive subjects was about twice as high in June as in birch atopic subjects examined during other months. It is concluded that seasonal variation in birch-pollen-positive subjects must be taken into account when using S-ECP clinically or in epidemiological research.

Eosinophil peroxidase: a new serum marker of atopy and bronchial hyper-responsiveness.

Do markers of eosinophil activation differ in their ability to detect subjects with atopy or bronchial hyper-responsiveness (BHR)? Comparisons of serum levels of eosinophil peroxidase (S-EPO), of eosinophil cationic protein (S-ECP) and the blood eosinophil count (B-Eos) have been made between 154 subjects aged 20-44 years, participating in the European Community Respiratory Health Survey in Uppsala, Sweden. Subjects with atopy had significantly higher levels of S-EPO and S-ECP than those without atopy (P <0 center dot 001). Subjects with BHR had significantly higher levels of S-EPO (P <0 center dot 001) and B-Eos (P <0 center dot 01) than subjects without BHR. Persons reporting asthma-related symptoms had significantly higher levels of S-EPO and B-Eos than subjects without such symptoms (P <0 center dot 001 and P <0 center dot 01, respectively). Asthma symptom score correlated significantly to S-EPO (r = 0 center dot 26, P <0 center dot 01), S-ECP (r = 0 center dot 20, P <0 center dot 05) and B-Eos (r = 0 center dot 18, P <0 center dot 05). Finally, S-EPO was significantly more sensitive than S-ECP for detecting subjects with BHR (P <0 center dot 05) and significantly more sensitive than B-Eos for detecting both subjects with BHR and subjects with a combination of atopy and BHR (P <0 center dot 05). It is concluded that S-EPO is a promising marker with a higher sensitivity for BHR than S-ECP or B-Eos. Further studies are needed to define the value of S-EPO when following disease activity.

Effects of rhinovirus-induced common colds on granulocyte activity in allergic rhinitis.

OBJECTIVE: To explore the activities of mast cells, eosinophils, and neutrophils in patients with allergic rhinitis developing common colds and increased responsiveness to allergen following nasal rhinovirus inoculation. METHODS: We have revisited a nasal lavage material obtained from 17 patients who were successfully inoculated with rhinovirus outside the pollen season and received nasal allergen challenges before and after inoculation. We have examined indices of mast cell activity (tryptase), eosinophil degranulation (eosinophil peroxidase; EPO), and neutrophil activation (myeloperoxidase; MPO). RESULTS: Allergen challenges performed before rhinovirus inoculation increased the nasal output of EPO. Notably, this response was significantly greater after rhinovirus inoculation (cf. before inoculation). The output of MPO was also increased following allergen challenge after, but not before, rhinovirus inoculation. Nasal lavage fluid levels of tryptase were increased following allergen challenge similarly before and after rhinovirus inoculation. Also, the viral infection did not affect the baseline levels of tryptase. CONCLUSIONS: The present data demonstrate that rhinovirus infections activate both eosinophils and neutrophils, but that they may not affect mast cell activity. We suggest that common colds in part through stimulation of granulocyte activity potentiate the airway inflammation in allergic diseases.

Update in allergy testing in childhood asthma: how do you know whether you are successfully controlling the patient's inflammation?

The levels of serum eosinophil cationic protein (ECP) in asthmatic patients have been shown to be increased in acute and undertreated asthma as a result of inflammation. ECP is released during in vitro clotting of peripheral blood. The exposure of the atopic individual to an offending allergen stimulates the activation of the blood eosinophils and their release of ECP into serum. Serum ECP levels reflect avoidance of the allergen, and successful treatment of asthma inflammation with corticosteroids cause a reduction of the inflammation in the lung. When individual patients with asthma are followed, the level of serum ECP can be used (1) as an early indicator of the degree of inflammation, (2) for monitoring the efficacy of corticosteroid therapy, and (3) for possible noncompliance with treatment.

Immune regulation of goblet cell development.

In rats, goblet cells were found in the bronchiole epithelium surrounding enlarged lymphoid noduli. The goblet cell number increased with age. Old rats had goblet cells in the columnar epithelium. Immunization with aerosolized antigen increased goblet cell hyperplasia in bronchioli with a luminal diameter of 0.5-1.0 mm. In mice, goblet cell differentiation in the respiratory epithelium was caused by local delayed-type hypersensitivity reactions. Manipulations of the delayed hypersensitivity reactions with immune suppression and depletion of helper T cells eliminated the increase in goblet cell number.

Mononuclear cells, mast cells and mucous cells as part of the delayed hypersensitivity response to aerosolized antigen in mice.

Mice (BALB/c) exposed to picryl chloride (PiCl) on their shaved abdomen and untreated animals were after 7 days exposed to daily aerosolized trinitrophenylated dog serum albumin (TNP-DSA). Mice exposed to PiCl tended to respond earlier and more strongly with both delayed hypersensitivity (DH) and IgG antibodies in serum and bronchial washings than did mice exposed to aerosol only. Picryl chloride sensitization resulted in spontaneously proliferating axillary lymph node cells, which could not be further stimulated with antigen or mitogen. Histological examination of lung tissue of aerosol-sensitized animals revealed an increase in mononuclear cells and mast cells around bronchioli and mucous cells, particularly in those animals exposed for prolonged periods and sensitized with PiCl prior to aerosol. Sensitization of mice with aerosolized TNP-DSA administrated in two 2- and 1-week periods with a 4-week interval responded with DH and IgG antibody in a dose dependent fashion irrespective of presensitization with PiCl. In bronchial washings IgG antibodies were found particularly after two 2-week periods of exposure. The cells taken from the axillary or brachial lymph nodes showed spontaneous proliferation. Culture of the cells to achieve mast cell maturation resulted in no or very low numbers of mast cells in the lymph nodes.

Regional and systemic immune responses to trinitrophenyl derivatives after intranasal and subcutaneous sensitization of mice.

The immune responses and accompanying inflammatory local reactions were analyzed in mice sensitized subcutaneously (SC) or intranasally. Administration of picrylsulfonic acid (PSA) by both routes for two 2- or 1-week periods with a 4-week interval resulted in delayed hypersensitivity responses. Serum antibodies of the IgG and IgE and some of the IgA isotype were found, particularly in SC-sensitized animals. The latter animals also had levels of IgG antibodies in their bronchial fluid. Lymph node cells from mice sensitized for 2-week periods demonstrated high spontaneous proliferation. After culture together with spleen cells, these cells exhibited considerable numbers of mast cells, particularly after stimulation with pokeweed and antigen. The lungs from mice sensitized intranasally revealed increased numbers of mononuclear cells around large vessels and mucous cells in the bronchiolar epithelium, although there were individual differences between the animals. The mast cell numbers in the lungs were only slightly increased compared with numbers in nonimmunized animals subjected to ether anesthesia. Animals injected SC with PSA exhibited increased numbers of mast cells in their lungs compared with control mice. Mice sensitized with trinitrophenylated human serum albumin demonstrated some differences in their immune reactivity compared with animals immunized with PSA. Thus, no obvious delayed hypersensitivity appeared, but the IgG antibody titers were higher, and the IgE antibody titers were lower. The cellular responses assessed histologically demonstrated higher mononuclear cell numbers and lower mast cell numbers compared with those observed in animals sensitized with PSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Contact sensitivity in guinea pigs to different penicillins.

The ability of penicillins of varying lipophilicity to induce and elicit cellular allergic responses were analysed in guinea pigs. Epicutaneous application of the penicillins benzylpenicillin (Bp), cloxacillin (Clox) and Bacampicillin (Bamp) did not cause any unspecific skin irritation. Intracutaneously injected, however, Bamp, Clox and to a lesser extent Bp caused irritation at concentrations of 1.25%. Solutions of 0.12% of penicillins were inactive in this respect. Cellular allergic responses were induced with Bp, Bamp and Clox after repeated epicutaneous application. The magnitude of responses was related to the lipophilic properties of the penicillins, Bamp being superior. In the guinea pig maximization (GPM) test of Magnusson and Kligman employing intradermal injections of the penicillins with Freund's complete adjuvant, similar sensitizing abilities of the three penicillins were observed. The cellular allergic responses were elicited with Bp, Bamp, Clox and in addition ampicillin and the 1'-ethoxycarbonyloxyethyl ester of Bp. An extensive cross-reactivity between the penicillins was seen in Bp- and Bamp-sensitized animals, whereas the Clox-sensitized animals showed a specificity limited to Clox. Bamp was shown to possess a superior activity to elicit reactions, possibly due to its lipophilic properties together with an irritating effect exerted by the NH2 group.

Cyclophosphamide-mediated enhancement of delayed hypersensitivity reactions in the lung.

The appearance of mononuclear cells, mast cells and mucous cells in the airways was studied in Balb/c mice in relation to cell-mediated immunity reflected as delayed hypersensitivity (DH). The animals were pretreated with cyclophosphamide (Cy) to increase cell-mediated immunity and to decrease the influence of antibodies. Mice pretreated with Cy and epicutaneously sensitized with picrylchloride (PiCl) had stronger DH reactions as compared with sensitized mice not pretreated with Cy. The Cy treatment decreased the IgG antibody formation after sensitization. The Cy-pretreated, sensitized and challenged mice had increased numbers of mucus-producing cells and to some extent also mononuclear cells in their lungs compared with sensitized and challenged non-Cy-treated animals. However, the mucous cell numbers were also increased in Cy-treated, but nonsensitized mice in which most of the airway epithelium had differentiated into mucus-containing cells. The present results indicate that local cell-mediated immunity involves the appearance of mononuclear cells and mucus-producing cells in the lung. This inflammatory reaction is enhanced by Cy treatment, although mucous cell differentiation seems to be induced by exposure to Cy alone.

Immunological suppression of delayed hypersensitivity responses in mouse lungs as reflected by numbers of mononuclear cells, mast cells and mucus-producing cells.

The suppression of delayed hypersensitivity (DH) in the lung as reflected by the appearance of mononuclear cells, mast cells and mucus-producing cells was studied in Balb/c mice. Immunosuppression was induced by intravenous and peroral administration of picrylsulfonic acid (PSA) in mice subsequently sensitized with picrylchloride (PiCl). The animals exhibited a decreased DH reactivity as assessed by ear thickness increase compared with mice sensitized but not exposed to PSA pretreatment. In mice exposed to PSA intravenously (suppressed) and sensitized with PiCl there was a decrease in the number of mononuclear cells in the lung after challenge, compared to mice sensitized and challenged only. Similarly, the mast cell and mucus-producing cell numbers were slightly lower in animals immunosuppressed intravenously with PSA compared with sensitized mice. Such a decrease in the numbers of mononuclear cells, mast cells and mucus-producing cells in the lung was not seen in animals treated with PSA perorally, although these animals exhibited suppressed DH reaction in the ears. The present results indicate that induction of mononuclear cells and to some extent mast cells and mucus-producing cells in the lung relates to the DH reaction and its regulation.

Asthma in children less than 5 years of age: eosinophils and serum levels of the eosinophil proteins ECP and EPX in relation to atopy and symptoms.

Children less than 5 years of age with asthma were assessed for total eosinophil counts and serum levels of the eosinophil proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX), to determine whether these measurements would reflect eosinophilic inflammation in the airways. Initially 27 symptomatic patients, 14 atopic and 13 nonatopic were investigated. They had a mean age of 1.8 years and had never been treated with inhaled steroid and had not received Intal for 2 weeks prior to the assessment. The 14 atopic patients proved to have higher mean total eosinophil counts and serum levels of ECP and EPX than the 13 non-atopic patients (eosinophil counts 0.63 x 10(9)/l vs 0.26 x 10(9)/l, P < 0.001; ECP 36.9 micrograms/l vs 10.8 micrograms/l, P < 0.001; EPX 69.0 micrograms/l vs 19.6 micrograms/l, P < 0.01). Thirteen of these patients required treatment with daily doses of inhaled steroid and 11 had a repeat assessment (seven atopic and four non-atopic). The mean serum ECP of the seven atopic patients had fallen significantly (40.6 to 22.9, P < 0.05) while the total eosinophil counts did not. These results suggest a difference in numbers and activity of eosinophils in atopic compared with non-atopic asthma in young children. To determine whether the results were influenced by treatment with inhaled steroids, 31 patients who were being treated with daily inhaled steroid underwent assessment when they were symptomatic (22 samples) or asymptomatic (19 samples). Of the 31 patients, 11 were atopic and 20 non-atopic.(ABSTRACT TRUNCATED AT 250 WORDS)

Solar urticaria: mechanism and treatment.

A patient with solar urticaria induced by wavelengths 290-420 nm is reported. Wheals appeared after a few seconds of exposure to the sun; longer exposure caused general malaise and syncope. Intradermal injection of in vitro irradiated plasma caused a local whealing which was not seen with plasma kept dark. The wheals induced by irradiation could be inhibited by local injection of an antihistamine. Local injection of lidocaine and hydrocortisone was ineffective. Depletion of substance P in the skin by topical application of capsaicin did not change the sensitivity to irradiation with 313 nm and a single PUVA treatment did not change the minimal urticarial dose (MUD). Sunscreens were in practice of limited value with the exception of a protective plastic helmet. Repeated daily irradiation with UVA in increasing doses normalized his response to sunlight.

Familial polymorphous light eruption with aquagenic urticaria: successful treatment with PUVA.

A patient is described with both polymorphous light eruption (PLE) and aquagenic urticaria appearing at 29 yr of age. Her father had the same symptoms after exposure to solar irradiation or water. Four to 6 h after 5-15 min sun-exposure, both had symptoms of general malaise and swelling of various joints. The skin symptoms in our patient were initially urticarial and later mainly papular and vesicular. They were elicited by irradiation with low doses of 300-360 nm and also appeared after 400 and 500 nm. Window glass offered little protection. PUVA treatment improved both conditions remarkably.

Total blood eosinophils, serum eosinophil cationic protein and eosinophil protein X in childhood asthma: relation to disease status and therapy.

Blood eosinophils, and serum levels of the eosinophil proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX) were measured in childhood asthma. Seventeen patients mean age 11.9 years who were symptomatic with asthma, were enrolled in a study examining the eosinophil counts and eosinophil proteins at the onset of study and after treatment in relation to changes in their baseline forced expiratory volume at 1 second (FEV1) and % predicted FEV1. The patients with symptomatic asthma were compared with 17 patients mean age 12.0 years with asymptomatic asthma maintained on daily inhaled steroid and 13 patients, mean age 12.0 years, without asthma but with urticaria who served as non-asthma controls. Patients with symptomatic asthma did not have significantly higher initial eosinophil counts compared with those with asymptomatic asthma (0.43 x 10(9)/l vs 0.26 x 10(9)/l, P = 0.09) but had higher serum ECP levels (28.9 micrograms/l vs 18.5 micrograms/l). Both asthma patient groups had significantly higher serum ECP levels (P < 0.01) than the controls (9.8 micrograms/l). After therapy consisting of increased dose of inhaled steroids and/or oral steroids, patients in the symptomatic asthma group demonstrated a significant rise in FEV1 (1.67 l/sec at Visit 1 vs 2.08 l/sec at Visit 2, P < 0.001). A similar rise was seen for % predicted FEV1.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells and mediators in bronchoalveolar lavage of asthmatic patients: the example of eosinophilic inflammation.

Bronchoalveolar lavage (BAL) greatly improved our understanding of asthma allowing to demonstrate the key role of inflammation in the pathogenesis of the disease. BAL is a safe procedure, even in severe patients when properly performed. BAL samples large and small airways and alveoli. Cells and mediators may be measured in BALF but they only represent an indirect estimation of the bronchial inflammation. Before performing BAL, the clinical status of the patients should be ascertained and drugs taken may have to be withdrawn. BALF markers should follow some requirements: (1) markers should be released by cells that are pertinent to airways inflammation (and reparation) in asthma, and, if possible they should be specific of a single cell type, (2) the enumeration of cells or titration of the marker or of its metabolites should be specific and sensitive, (3) if possible the titration should not be modified by the sampling procedure, (4) pilot studies should have demonstrated that the cell is increased or the secretory product is released during challenge in asthmatic subjects, (5) studies in a large number of patients should have demonstrated that the levels of the marker are increased in chronic asthmatics, that these levels are correlated with the severity of the disease and are decreased during effective anti-inflammatory treatment, and (6) if possible the cell or marker should be specific to asthma (but at present there is no such cell or marker). Eosinophils and granule secretory products follow most of these requirements. BAL represents an important research tool to assess the effects of therapeutic interventions.

Evaluation of mast cell activation (tryptase) in two patients suffering from drug-induced hypotensoid reactions.

Tryptase is predominantly found in mast cells, where it resides in secretory granules, and is released with other mediators during mast cell degranulation. By using a newly developed commercial assay for measurements of tryptase levels we have investigated two cases of suspected drug-induced anaphylaxis. Each patient had a similar clinical presentation, consisting of hypotension and cyanosis after administration of thiopentone and suxamethonium. One of the patients showed a highly elevated serum level of tryptase reaching 26 micrograms/l 30 min after the initial reaction. In addition, slightly elevated levels of specific IgE antibodies to thiopentone were detected. The other patient with similar symptoms showed no increase in the level of tryptase, nor any specific IgE to thiopentone or suxamethonium. These data indicate the patient I suffered from true anaphylaxis, whereas the reaction of patient II occurred by a different mechanism.

Myeloperoxidase and interleukin-8 levels in chronic sinusitis.

We have recently phenotyped inflammation in non-infectious allergic and non-allergic chronic maxillary sinusitis using sinus biopsies and lavage fluids. In this first paper, we have concentrated our work on the eosinophil, T cell, mast cell and macrophage infiltrates. However, many unresolved questions remain and particularly the role of neutrophils needed to be addressed. In the present study, we focused on the neutrophilic inflammation: myeloperoxidase (MPO) and interleukin-8 (IL-8) were measured by immunoassays and neutrophils were enumerated by conventional staining in the sinus lavage fluids of 16 patients with chronic sinusitis and six control subjects. Both MPO and IL-8 levels were significantly higher in patients than in controls (P < 0.01 and 0.005, respectively). There was a significant correlation between MPO levels and neutrophil numbers, and between MPO and IL-8 levels in the sinus lavage fluid (P < 0.0001, Spearman rank correlation). The presence of high levels of IL-8 in the lavage fluids of patients suffering from chronic sinusitis, levels which correlate with those of MPO, suggests that this cytokine may activate neutrophils in this chronic disease.