The mapping of the Lyn kinase binding site of the common beta subunit of IL-3/granulocyte-macrophage colony-stimulating factor/IL-5 receptor.

It has been shown that a membrane-proximal region within common beta (betac) receptor of IL-3/granulocyte-macrophage CSF/IL-5 (amino acids 450-517) is important for Lyn binding. We have shown previously that Lyn kinase is physically associated with the IL-5R betac subunit in unstimulated cells. The result suggests that this association involves binding modules that are not activation or phosphorylation dependent. The objective of this study was to map the exact Lyn binding site on betac. Using overlapping and/or sequential peptides derived from betac 450-517, we narrowed down the Lyn binding site to nine amino acid residues, betac 457-465. The P-->A mutation in this region abrogated the binding to Lyn, indicating a critical role of proline residues. We created a cell-permeable Lyn-binding peptide by N-stearation. This cell-permeable peptide blocked the association of Lyn, but not Jak2 with betac in situ. We also investigated the betac binding site of Lyn kinase. Our results suggest that the N-terminal unique domain of Lyn kinase is important for binding to betac receptor. To our knowledge, this is the first molecular identification of the Lyn binding site of betac receptor. This finding may help develop specific inhibitors of Lyn-coupled signaling pathways.

Lyn, Jak2, and Raf-1 kinases are critical for the antiapoptotic effect of interleukin 5, whereas only Raf-1 kinase is essential for eosinophil activation and degranulation.

Interleukin (IL)-5 has been shown to activate many signaling molecules in eosinophils, but their functional relevance remains unknown. We have examined the functional relevance of Lyn, Jak2, and Raf-1 kinases in eosinophil survival, upregulation of adhesion molecules and degranulation. To this goal we used Lyn and Raf-1 antisense (AS) oligodeoxynucleotides (ODN) to inhibit the expression of these proteins and tyrphostin AG490 to specifically block the activation of Jak2. We have demonstrated that all three kinases are important for IL-5- induced suppression of eosinophil apoptosis. However, Lyn and Jak2 tyrosine kinases are not important for the upregulation of CD11b and the secretion of eosinophil cationic protein. In contrast, Raf-1 kinase is critical for both these functions. This is the first identification of specific signaling molecules responsible for three important functions of eosinophils. We have established a central role for Raf-1 kinase in regulating eosinophil survival, expression of beta2 integrins and degranulation. Further, there appears to be a dissociation between two receptor-associated tyrosine kinases, i.e., Lyn and Jak2, and the activation of Raf-1 kinase. The delineation of the functional relevance of signaling molecules will help design therapeutic approaches targeting specific eosinophil function.

Nasal challenge test in the diagnosis of allergic respiratory diseases in subjects occupationally exposed to a high molecular allergen (flour).

The objective of this study was the evaluation of the usefulness of the nasal challenge test in the diagnosis of allergic respiratory diseases in subjects occupationally exposed to flour. A single-blind, placebo controlled study was conducted in 100 subjects with occupational atopic asthma with rhinitis. The control groups consisted of 20 atopic subjects not sensitized to investigated allergens and 20 healthy subjects. A 'nasal pool' technique was used to evaluate the changes of the cellular response and protein level in nasal washings after topical provocation with allergen or placebo. The concentrations of eosinophil cationic protein and mast cell-derived tryptase in nasal fluid were evaluated in 60 cases. There were significant increases in eosinophil and basophils number, albumin/total protein ratio, eosinophil cationic protein and tryptase levels in occupationally sensitized patients challenged with specific allergens. There were neither severe bronchial reactions or an increase of bronchial hyperreactivity in occupationally sensitized patients after the nasal provocation with flour. The nasal challenge test appears to be a very useful and safe tool for diagnosing occupational allergy.

Respiratory syncytial virus-infected pulmonary epithelial cells induce eosinophil degranulation by a CD18-mediated mechanism.

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the beta2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.

Airway response to formaldehyde inhalation in asthmatic subjects with suspected respiratory formaldehyde sensitization.

The aim of the study was to characterize the mechanism of formaldehyde (FM)-induced nasal and bronchial response in asthmatic subjects with suspected FM allergy. Ten subjects purported to have FM rhinitis and asthma and 10 healthy subjects submitted to an inhalation provocation in an exposure chamber with FM at a dose of 0.5 mg/m3 over 2 hr. Spirometry at rest and following bronchial provocation with histamine (PC20) were recorded before and after FM inhalation. In addition, FM-specific serum IgE antibodies were measured and cellular, biochemical, and mediator changes were assessed in nasal lavage before, and immediately after, provocation and at 4 hr and 24 hr later. Provocation with FM caused only transient symptoms of rhinitis in both groups. None of the subjects supposed to have occupational asthma developed clinical symptoms of bronchial irritation. No specific IgE antibodies to FM were detected in persons with occupational exposure to FM. No differences in the nasal response to FM were found between subjects reporting to have occupational allergic respiratory diseases and healthy subjects (P > 0.05). In summary, inhaled formaldehyde at a level as low as 0.5 mg/m3 did not induce a specific allergic response either in the upper or in the lower part of the respiratory tract. Moreover, there is no difference in nasal response to FM in asthmatic subjects occupationally exposed to FM and healthy subjects.

Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival.

Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.

Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor.

The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.

The activation of the Jak-STAT 1 signaling pathway by IL-5 in eosinophils.

The intracellular signal transduction of IL-5 in eosinophils is unknown. The objective of this study was to investigate the involvement of the newly discovered Jak-STAT pathway in the IL-5 signal transduction mechanism. Eosinophils were purified from peripheral blood by discontinuous Percoll gradients and stimulated with IL-5. The involvement of Jak 2 was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. The activation of Jak 2 was studied by autophosphorylation of the immunoprecipitated kinase. Jak 2 was tyrosine phosphorylated within 1 to 3 min after stimulation of eosinophils with IL-5. Further, the immunoprecipitated Jak 2 obtained from IL-5-stimulated cells underwent autophosphorylation. Jak 2 coprecipitated with the beta-subunit of the IL-5 receptor, suggesting a physical association of the kinase with the receptor. The nuclear factor STAT-1 (p91) was investigated by immunoprecipitation followed by immunoblotting for tyrosine phosphorylation. STAT-1 was tyrosine phosphorylated within 15 min of IL-5 stimulation. The presence of STAT-1 in the nuclear extract was studied by electrophoretic mobility shift assay. IL-5 induced two proteins that bound to the gamma-activating sequence. In the presence of an anti-STAT-1 Ab, the band was supershifted. Thus, we demonstrated that IL-5 activated the Jak 2-STAT 1 signaling pathway in eosinophils. We speculate that the Jak 2-STAT 1 pathway may be involved in the activation of IL-5-inducible genes in eosinophils.

The intracellular signal transduction mechanism of interleukin 5 in eosinophils: the involvement of lyn tyrosine kinase and the Ras-Raf-1-MEK-microtubule-associated protein kinase pathway.

Interleukin 5 (IL-5) regulates the growth and function of eosinophils. The objective of this study was to investigate the intracellular signal transduction mechanism of IL-5 in eosinophils. Purified eosinophils were stimulated with IL-5, and the involvement of various kinases was investigated by immunoblotting, immune complex kinase assay, and in situ denatured/renatured kinase assay. We found that IL-5 induced tyrosine phosphorylation and activation of a number of kinases. Two species of lyn kinases (53 and 56 kD) were present in eosinophils. Both forms were Tyr-phosphorylated and activated rapidly within 1 min. Further, lyn kinase was physically associated with the IL-5 beta receptor in eosinophils. Ras was studied by immunoprecipitation followed by thin-layer chromatography. Ras bound higher quantities of [alpha-32P]guanosine 5'triphosphate upon stimulation with IL-5. Raf-1 kinase showed increased Tyr phosphorylation on immunoblotting and increased activity in the immune complex kinase assay. Two species of MEK (MAP or Erk kinase) (41 and 45 kD) were identified in eosinophils, which underwent autophosphorylation upon stimulation. Microtubule-associated protein (MAP) kinase (p44) was Tyr-phosphorylated on immunoblotting and had increased activity in the immune-complex kinase assay. MAP kinases were also studied after metabolic radiolabeling of the cells with [32P]orthophosphates. IL-5 stimulated phosphorylation of MAP kinases in situ. Thus, we have delineated major components of an important signaling pathway in eosinophils. We believe that one of the signals generated by IL-5 receptor activation is propagated through the lyn-Ras-Raf-1-MEK-MAP kinase pathway.

The interleukin-5/receptor interaction activates Lyn and Jak2 tyrosine kinases and propagates signals via the Ras-Raf-1-MAP kinase and the Jak-STAT pathways in eosinophils.

We have shown that the interaction of interleukin (IL)-5 with the receptor activates Lyn tyrosine kinase within 1 min and Jak2 tyrosine kinase within 1-3 min. IL-5 also stimulates GTP binding to p21ras. The signal is subsequently propagated through the activation of Raf-1, MEK, and MAP kinases as shown by their increased autophosphorylation in vitro and phosphorylation in situ. Jak2 kinase has been shown to phosphorylate STAT nuclear proteins. The activation of STAT nuclear factors was studied by electrophoretic mobility shift assay using a gamma activation site (GAS) probe. We found that IL-5 induces two GAS-binding proteins in eosinophils, one of which is STAT1. We conclude that IL-5 induced signals are propagated through two distinct pathways: (1) Lyn-->Ras-->Raf-1-->MEK-->MAP kinase and (2) Jak2-->STAT1.

Inhibitory effect of levocabastine on allergen-induced increase of nasal reactivity to histamine and cell influx.

For evaluation of the effect of levocabastine pretreatment on allergen-induced rhinitis symptoms, changes in nasal washings, and nasal responsiveness to histamine, 12 asymptomatic patients with documented allergic rhinitis participated in a single-blind, placebo-controlled study. Eight-day treatment with levocabastine (twice in each nostril, four times a day) caused significant reduction in nasal symptoms and inflammatory cell influx after allergen challenge, as compared with placebo administration. Levocabastine inhibited increased nasal reactivity to histamine induced by allergen provocation, as controlled by rhinitis symptoms and albumin level in nasal washings. These data reveal a high effectiveness of levocabastine in the prevention of allergen-induced rhinitis symptoms. Moreover, its inhibitory effect on inflammatory cell influx and hyperresponsiveness to histamine suggest that levocabastine is more than a simple H1-receptor antagonist.

Eosinophil and neutrophil chemiluminescence in patients with atopic asthma and in healthy subjects.

The 10 patients with atopic asthma and 6 healthy subjects were selected for the study. Each person underwent prick testing to common allergens (extracts of house dust, grass, trees, animal danders, feathers and mould). In addition blood was drawn for the specific IgE antibodies to D. pteronyssinus and total serum IgE levels. Neutrophil and eosinophil chemiluminescence induced by PMA and FMLP was measured in both groups. Eosinophils obtained from patients with atopic asthma stimulated with PMA and FMLP generated significantly greater free radicals than obtained from normal subjects. There was no observed significant difference of neutrophil chemiluminescence activated by PMA and FMLP and of the percentage of hypodense eosinophils in the peripheral blood between subjects with asthma and healthy ones.

Effect of ipratropium on nasal reactivity to histamine and eosinophil influx in perennial allergic rhinitis.

In a double-blind, placebo-controlled study nasal saline and histamine provocation tests were performed in patients with perennial allergic rhinitis in order to assess changes in eosinophil influx and non-specific nasal reactivity after 8 days of treatment with ipratropium bromide. A "nasal pool" method was used to trace changes in protein level and eosinophil influx into nasal secretions. Treatment with ipratropium 80 mg q.i.d. caused a significant decrease in the albumin and total protein level in saline washings and induced a five-fold increase in eosinophils as compared to the placebo treatment. The nasal mucosal response to histamine, assessed as the number of sneezes and protein level, was more responsive to ipratropium treatment than the mucosa from placebo-treated subjects. Since eosinophil numbers were correlated with an increase in the vascular and sneezing responses, it appears that ipratropium potentiates inflammatory mechanisms when used in subjects with an allergy in the nasal mucosa.

Changes in nasal lavage fluid due to formaldehyde inhalation.

The aim of the study was to characterize the nature of the formaldehyde-induced nasal response consisting in symptoms of rhinitis and changes in nasal lavage fluid. Eleven healthy subjects and nine patients with specific skin sensitization were provoked in a toxicological chamber with formaldehyde at a dose of 0.5 mg/m3 over 2 h. Nasal lavage was performed prior to and immediately after provocation and 4 and 18 h later. Provocation with formaldehyde caused transient symptoms of rhinitis and prolonged changes in nasal washings. There were increases in the number and proportion of eosinophils and elevated albumin and total protein levels in nasal lavage fluid 4 and 18 h after provocation. No difference in the nasal response to formaldehyde was found between patients with skin sensitization and healthy subjects. These data confirm the irritative effects of formaldehyde and are also suggestive of nonspecific proinflammatory properties when formaldehyde is inhaled at a low (0.5 mg/m3) dose.

[Regulation of immunoglobulin class E synthesis]. / Regulacja syntezy immunoglobuliny klasy e.

IgE production by B cell is T cell dependent. Induction of human IgE synthesis requires IL-4 and T/B cell physical interactions. The pivotal role of cytokines and of Fc epsilon R2 and its soluble fragments in regulation of IgE synthesis are presented.