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We report that responses of normal human eosinophils toward the chemokines RANTES and interleukin-8 (IL-8) are modulated and upregulated by priming with IL-5. In a modified Boyden chamber assay, we studied migratory responses toward the members of the chemokine family RANTES, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) (C-C subfamily), and IL-8, platelet factor-4 (PF-4), and neutrophil-activating peptide-2 (NAP-2) (C-x-C subfamily). These chemokines were also studied in terms of actin polymerization and ([Ca2+]i)-mobilizing properties, intracellular signals that are thought to play a role during migratory responses. We found that eosinophils showed significant migratory responses toward RANTES and IL-8 at concentrations of 10(-9) to 10(-7) mol/L only after priming with IL-5 (10 pmol/L). At these concentrations, PF-4, NAP-2, MCP-1, and MIP-1 alpha induced no significant migratory responses after priming. Unprimed eosinophils only showed a significant migratory response toward RANTES (10(-6) mol/L). Changes in [Ca2+]i were found after addition of RANTES, MIP-1 alpha, and NAP-2 (10 nmol/L) to unprimed eosinophils. RANTES (10(-9) to 10(-7) mol/L) significantly induced actin polymerization both in primed and unprimed eosinophils, whereas IL-8 (10(-9) to 10(-8) mol/L) and MIP-1 alpha (10(-8) mol/L) only induced actin polymerization after priming with IL-5. NAP-2, PF-4, and MCP-1 did not affect actin polymerization. These findings are further evidence for the hypothesis that cytokines like IL-5 and locally secreted chemokines like RANTES and IL-8 are both at the basis of specific eosinophil influx into the allergic inflammatory locus.

Transcription regulation of the human intercellular adhesion molecule-1 gene by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor alpha (TNF alpha), and the glucocorticoid dexamethasone was studied using transient transfections in 293 cells with intercellular adhesion molecule-1 promoter-luciferase constructs (together with a glucocorticoid receptor expression vector). TPA and TNF alpha induced promoter activity, which was repressed by dexamethasone. Four TPA-responsive DNA regions were identified, each containing a potential TPA-responsive enhancer sequence: 1) -677/-340 an AP3-like sequence; 2) -290/278 a TPA-response element (TRE); 3) -227/-175 an NF kappa B-like sequence; 4) -105/-38 an AP2-like sequence. TNF alpha enhanced transcription only through region 3. The TRE in region 2 appeared to be functionally coupled to a distal TATA box at -313 and differed from the consensus TRE with respect to binding characteristics for members of the AP1 family. The newly identified NF kappa B enhancer (TGGAAATTCC) is bound by a TNF alpha-induced nuclear protein and appears to be the key element in rapid transcription induction by TNF alpha (and TPA), while transactivation of this element is repressed by the ligand-bound glucocorticoid receptor. We propose a negative cross-talk between the NF kappa B transcription factor and the glucocorticoid receptor.

We report that a transient incubation of human eosinophils with the protein kinase C (PKC) inhibitor CGP39-360 (staurosporine) or the more PKC-specific inhibitors CGP41-251 and CGP44-800 prior to activation of the respiratory burst with opsonized particles results in priming of this response. This priming effect was concentration dependent and occurred in the range in which the phorbol myristate acetate-induced respiratory burst was inhibited. CGP39-360 priming was minimally affected in Ca(2+)-depleted cells, indicating that an increase in [Ca2+]i is not important. Also, the binding of serum-treated zymosan (STZ) particles was strongly enhanced by the inhibitors. On the other hand, the release of platelet-activating factor (PAF) induced by opsonized particles was enhanced only by CGP39-360 and not by CGP41-251 and CGP44-800. Therefore, priming of the respiratory burst is not due to an aspecific enhancing effect of the inhibitors. These data indicate that different signal transduction routes are involved in priming of the STZ-induced respiratory burst and PAF release in human eosinophils.

We report that pretreatment of human eosinophils with GM-CSF, IL-3, or IL-5 enhanced the respiratory burst induced by opsonized particles. In order to gain more insight into the intracellular mechanism(s) involved in cytokine priming, the role of [Ca2+]i and tyrosine kinases was studied. Optimal priming concentrations of GM-CSF, IL-3, and IL-5 did not induce a rise in [Ca2+]i, and Ca(2+)-depleted eosinophils ([Ca2+]i < 20 nM) were still primed after preincubation with these cytokines. GM-CSF, IL-3, and IL-5 induced phosphorylation of two proteins (102 and 122 kd) on tyrosine residues, as deduced from Western blot analysis with an antiphosphotyrosine monoclonal antibody (4G10). This cytokine-stimulated tyrosine phosphorylation was not inhibited under Ca(2+)-depleted conditions. In conclusion, this study demonstrates that GM-CSF, IL-3, and IL-5 priming of the opsonized particle-induced respiratory burst in human eosinophils is completely Ca2+ independent. Moreover the tyrosine phosphorylation of a 102-kd and a 122-kd protein is Ca2+ independent, suggesting that this event might be involved in cytokine priming.

Increased expression of the intercellular adhesion molecule-1 (ICAM-1) on cells present in the airways has been suggested to play a role in the pathogenesis of asthma by enhancing airway inflammation. We used the monocytic U937 cell line, both undifferentiated and differentiated to a macrophage-like phenotype, and the bronchial epithelial cell line NCI-H292 as cellular model systems for human monocytes/macrophages and bronchial epithelial cells, respectively, and studied the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and dexamethasone on ICAM-1 expression. Both cell lines expressed the ICAM-1 protein constitutively. In addition, TPA- or 1,25-dihydroxyvitamin D3-mediated differentiation of the U937 cell line into a macrophage-like phenotype was associated with increased expression of ICAM-1. In both cell lines, two ICAM-1 mRNA transcripts were found, and expression was stimulated to a similar degree within 1 to 2 h after addition of TPA. In both cell lines, the anti-inflammatory corticosteroid dexamethasone repressed both constitutive and TPA-stimulated ICAM-1 expression, within 3 h of its addition. In the presence of cycloheximide, a marked superinduction of ICAM-1 was observed, while the repressive effect of dexamethasone remained, supporting the hypothesis that dexamethasone acts directly at the transcriptional level.

The cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 are important modulators of eosinophilia and eosinophil function. Eosinophil chemotaxis is known to be particularly sensitive for cytokine priming. In the present study, we compared chemotactic responses of eosinophils derived from peripheral blood of allergic asthmatics to responses of eosinophils from peripheral blood of healthy individuals. Eosinophils from allergic asthmatics exhibited a markedly increased sensitivity in their chemotactic response toward platelet-activating factor (PAF) compared with eosinophils from normal donors. In contrast, C5a-induced eosinophil chemotaxis between both groups was similar. This in vivo-primed phenotype could be mimicked in vitro, by preincubating eosinophils from peripheral blood of healthy individuals with picomolar concentrations of either GM-CSF, IL-3, or IL-5. The chemotactic response of eosinophils derived from the circulation of allergic asthmatic patients toward GM-CSF was significantly lower compared with the response of eosinophils of healthy individuals. Our data strongly suggest that release of cytokines may be an important in vivo priming mechanism for eosinophils in the circulation of allergic asthmatic patients. Such an in vivo priming can subsequently result in selective upregulation and downregulation of chemotactic responses toward various chemoattractants release in the lung tissue.

Radioligand binding studies have been developed to determine pharmacologic receptor characteristics in vitro. With this assay, not only the number and dissociation constant (KD) can be studied, but also the interaction of agonists with the receptor. We used this method to study a new beta 2-sympathicomimetic drug, tulobuterol (1-(0-chlorophenyl)-2-butylamino-ethanol hydrochloride). Two sets of experiments were performed. One set of experiments investigated the effects of tulobuterol and terbutaline in chronic administration, while the second set compared the beta-adrenoceptor-stimulating properties of tulobuterol with terbutaline and salbutamol. The effects of 10 days' administration of tulobuterol and terbutaline on beta-adrenergic characteristics in rats were assessed biochemically by means of radioligand binding studies on pulmonary membranes and functionally using isolated tracheal spirals. It was found that: (1) In vivo treatment with both drugs induced a reduction of the number of beta-adrenoceptors bound by 3H-dihydroalprenolol (3H-DHA); however, tulobuterol also induced an increased affinity for beta-adrenoceptor binding. (2) Tulobuterol induced a significant increase in the sensitization of tracheal smooth muscle, facilitating the relaxation of airway smooth muscle. The inhibition of 3H-DHA binding with the three drugs was best fit in a two-binding site model, showing high- and low-affinity binding sites. The high-affinity sites had similar KD values for terbutaline and tulobuterol (1.6 x 10(7) and 1.5 x 10(-7), respectively). The high-affinity sites for salbutamol had a higher KD value (9.4 x 10(-7), suggesting a lower affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of adrenaline on the amount of blood eosinophils (eosinophilia) which had been found in Swiss mice in earlier experiments was lost after a few generations. The hyperglycemic response to adrenaline had not been changed. Sensitivities to noradrenaline and adrenaline were as found earlier, but the sensitivity to isoprenaline was reduced. The reduction of beta-adrenergic responsiveness suggests variations in biochemical parameters of mice or the appearance of mice genetically less responsive to beta-adrenergic drugs in only a few generations.

Adrenoceptor (sub)populations (alpha 1, beta and beta 1/beta 2 ratio) in human lung tissue were characterized by means of radioligand binding assays. Lung tissue specimens for these assays were obtained at thoracotomy from normal individuals and COLD patients. The patients were not receiving therapy known to influence adrenoceptor characteristics. In COLD patients, the number of alpha 1-adrenoceptors appeared to be markedly enhanced while the relative amount of beta 2-adrenoceptors was decreased. Furthermore, some adrenoceptors seemed to be correlated to pulmonary function both in the normal individuals and COLD patients. In-vitro modulation with disodium cromoglycate and ketotifen revealed therapeutic possibilities for these substances based on other pharmacological mechanisms than hitherto assumed.

Histidine decarboxylase activity and histamine levels of peritoneal mast cells were enhanced 4 days after intraperitoneal Haemophilus influenzae vaccination of rats. Incubation of the cells with propranolol (3.4 x 10(-4) M) resulted in histamine release and an increased histidine decarboxylase activity. Histidine decarboxylase activity and histamine release were more increased in the presence of propranolol in mast cells obtained from H. influenzae-vaccinated rats. An increased mediator release is also suggested by the increase of the number of peritoneal eosinophils. These data might explain the earlier observed enhanced plasma and lung histamine levels in H. influenzae-vaccinated rats.

An isolation procedure to obtain inflammatory cells from normal human lung tissue is described, consisting of four steps: an enzymatic digestion, centrifugal elutriation, gradient centrifugation and additional affinity chromatography (to obtain mast cells). The dispersed cell population consisted mainly of macrophages, pneumocytes type II, neutrophils and lymphocytes. Further separation by elutriation yielded pure macrophages and pure lymphocytes. The heterogeneous cell mixtures obtained at elutriation were separated by gradient centrifugation. This method yielded pure macrophages, lymphocytes and pneumocytes, and fractions containing eosinophils, mast cells or neutrophils were enriched considerably. Additional affinity chromatography with anti-human-IgE yielded a mast-cell fraction of 75%.

1. Rats and mice were vaccinated with Haemophilus influenzae in different vaccination schedules whereafter blood eosinophils were counted. In rats a single vaccination resulted in a dose-dependent effect on the blood eosinophil count in a pattern comparable with that after Bordetella pertussis vaccination. In a long-term vaccination schedule (five times a week for 5 weeks) rats developed a constant eosinophilia. In mice a single vaccination resulted in an eosinopenia of a consistent pattern which differed from the response after Bordetella pertussis vaccination; in a long-term vaccination schedule, eosinophilia was evoked for a period of about 13 days. 2. Thirty minutes after an adrenaline injection in vaccinated rats and mice with Haemophilus influenzae, hyperglycaemic and eosinophilic responses were measured. The eosinophilic response after adrenaline was inhibited in both species; the hyperglycaemic response in rats was unaltered, in mice the response was slightly but significantly (P less than 0.05) decreased. 3. The sensitivity to several drugs was tested in mice, 5 days after vaccination with Haemophilus influenzae or Bordetella pertussis. Haemophilus influenzae vaccination reduced the isoprenaline sensitivity and increased the noradrenaline sensitivity. Bordetella pertussis vaccination reduced the isoprenaline sensitivity while the sensitivity to histamine and adrenaline was raised. 4. The Haemophilus influenzae vaccinated experimental animal provides a model that is possibly more related to human atopy than the Bordetella pertussis vaccinated animal.

Rats were treated with terbutaline in order to specify the in vivo effects of beta 2-sympathicomimetic treatment in relation with desensitization phenomena on the level of pulmonary adrenoceptors. Maximal binding of 3H-dihydroalprenolol (3H-DHA) to lung membrane preparations, binding affinity and the relative amount of beta 2-adrenoceptors were used as parameters. These were established 1, 3, and 6 days after a 10 days terbutaline treatment and compared with control values. Ten days treatment did not cause significant changes in the parameters measured. However, maximal binding was enhanced 3 and 6 days after the last treatment, binding affinity was enhanced 3 days after the last treatment, while the relative amount of beta 2-adrenoceptors was diminished 1 and 6 days after termination of the treatment. When the amount of beta 2-adrenoceptors was calculated in terms of 3H-DHA binding, an increase could be shown 3 days after the last terbutaline treatment. Therefore the direct influence of the treatment on the beta-adrenoceptor population appeared to be small, dependent on the parameter used and probably not eliciting a functional desensitization.

A test to determine quantitatively the lectin binding sites in brush-border membranes has been developed. Highly purified bovine small intestinal brush-border membranes were prepared, and subsequently coated directly to the bottom of a microtiter plate. Soybean agglutinin conjugated with peroxidase was coupled to its binding sites in the brush-border membranes and the peroxidase activity was determined in a spectrophotometer. The number of soybean agglutinin binding sites in the brush-border membranes has been established by means of iterized computer fit analysis of the data, indicating values for maximal binding of 7.10(-7) M soybean agglutinin per mg of brush-border membrane protein and a dissociation constant of 1.5.10(-5) M.

The contribution of mast cell subtypes and their different mediators to the pathogenesis of chronic obstructive lung diseases (COLD) has not yet been established. In the present study, enzymatic digestion, centrifugal elutriation and Percoll gradient centrifugation were used to obtain two populations of mast cell subtypes from human lung tissue. Mast cell subtypes were challenged with anti-human IgE, propranolol, compound 48/80, or opsonized zymosan. Both subtypes were able to release histamine, but differed in the amount of the amine release. Only the formalin-sensitive and alcian blue-positive type (FS-AB) released histamine on challenge with opsonized zymosan. The same subtype was able to release leukotriene C4 (LTC4) after challenge with anti-human IgE. The other subtype, the formalin-insensitive and alcian blue-positive type (FI-AB), did not respond to opsonized zymosan and did not release LTC4 after challenge with anti-human IgE. Stimulation with propranolol or compound 48/80 did not release histamine from the FS-AB mast cells while the FI-AB mast cells released only about 10% of their histamine content upon challenge with these secretagogues.

To determine whether Haemophilus influenzae could be a factor in human atopy its effects were studied on the (para-)Sympathic Cyclic nucleotide-histamine axis in rats. Haemophilus influenzae vaccination induced changes in the cholinergic system compatible with higher cyclic GMP levels and enhanced histamine release. The authors suggest an involvement of the cholinergic system in Haemophilus influenzae vaccination effects.

The B. pertussis model of atopy as proposed by Szentivanyi in 1968 has been a starting point for much research involving the pathogenesis of COLD. Moreover, it supplied more insight into the pharmaco-therapeutic approach toward this group of diseases. In this review, it is shown that products of bacteria considered to be a constituent of the normal flora of the human upper respiratory tract, such as H. influenzae, elicit changes in adrenoceptor responsiveness which are compatible with an enhanced tendency toward bronchoconstriction. One of the features of human atopy is enhanced mediator release after appropriate stimuli resulting in bronchoconstriction. This phenomenon can be mimicked in an animal model, the H. influenzae-vaccinated rat or guinea pig; enhanced histamine synthesis and release are found in vivo as well as in vitro. The effects point in the direction of a beta-adrenergic defect which is not only demonstrable in biochemical but also in physiologically oriented parameters. Pulmonary smooth muscle tissue appears to be less responsive to beta-adrenergic agonists and has an enhanced tendency to contract. The view that these changes are indeed the reflection of changes in adrenoceptor systems has been investigated in guinea pigs and rats. In both species impairment of beta-adrenergic systems together with a reduction in the number of beta 2-adrenoceptors was found after vaccination. Also the involvement of other factors, e.g., catecholamines, has been demonstrated. Comparable changes occur within the pulmonary adrenoceptor populations of COLD patients, suggesting disturbed homeostasis in the autonomic nervous system, possibly leading to bronchoconstriction. The question whether a bacterial factor is important in these changes and might induce, sustain or enhance the effects of other factors or even have a role in the pathogenesis of COLD is discussed in this review.

In this study the, in vitro, influence of arachidonic acid metabolites on human beta-adrenoceptors was investigated. Incubation of normal human pulmonary membranes with PAF, LTB4 and LTC4 affected pulmonary beta-adrenoceptor properties, as was shown in radioligand binding studies. The same mediators were able to induce a decreased lymphocyte cAMP synthesis. It is concluded that beta-adrenoceptor deficiencies, that can be demonstrated in peripheral lung tissue of COLD patients, may result from pathological processes such as inflammation.

The influence of Haemophilus influenzae on anaphylactic mediator release from ovalbumin-sensitized isolated guinea pig lungs was investigated. Lungs from H. influenzae-vaccinated animals released prostaglandins and thromboxanes following a smaller dose of ovalbumin than was effective in non-vaccinated animals. Histamine release was significantly increased in 4 day-vaccinated animals but not 1 or 10 days after vaccination, while broncho-constriction was potentiated in 1 and in 4 day-vaccinated animals. This increased histamine release was achieved following 2 micrograms ovalbumin. In contrast, doses of 10 micrograms and 1 mg ovalbumin respectively did not affect and decreased histamine release in the vaccinated group. The inhibition of anaphylactic mediator release by an infusion of 6 x 10(-9) M isoprenaline was significantly attenuated by H. influenzae vaccination. These results indicate an increased sensitivity to antigenic challenge and suggest that the functioning of beta-adrenoceptors was decreased as a result of H. influenzae vaccination.