Characteristics of idiopathic pulmonary fibrosis -associated cough. a case-control study.

BACKGROUND: Most patients with idiopathic pulmonary fibrosis (IPF) complain of cough. IPF-associated cough is widely characterized as dry or non-productive. The aim of this study was to compare chronic cough in early stage IPF patients to cough in subjects with chronic cough from a community-based sample and, especially, to investigate whether cough in IPF is less productive than chronic cough in a community-based sample. METHODS: The IPF cough population consisted of 46 biopsy-confirmed patients who complained of chronic cough. Control population consisted of subjects with chronic cough, gathered by a community-based email survey sent to public service employees and the Finnish Pensioners' Federation. A case-control setting was applied by having four age, gender, and smoking-status matched subjects from the community sample for each IPF cough patient. A cough specific quality of life questionnaire (Leicester Cough Questionnaire (LCQ)) was filled in by all subjects. The LCQ questionnaire contains 19 questions, each question is scored from 1 to 7 and total score from 3 to 21 with a smaller value indicating more severe impairment. RESULTS: The sputum production frequency, as assessed by LCQ question 2, was 5.0 (3.0-6.0) in the IPF chronic cough population and 5.0 (3.0-6.0) in the community-based chronic cough population (median and interquartile range p= 0.72). The LCQ total score was 14.8 (11.5-18.1) in the IPF chronic cough population and 15.4 (13.0-17.5) in the community-based chronic cough population (p=0.76). The domain impact scores were physical, 4.9 (3.9-6.1) vs. 5.1 (4.5-5.6) (p=0.80); psychological, 4.6 (3.7-5.9) vs. 4.7 (3.9-5.7) (p=0.90); and social, 5.5 (3.7-6.5) vs. 5.5 (4.5-6.3) (p=0.84), respectively. Furthermore, cough response to paint or fumes, cough disturbing sleep, and cough frequency per day did not differ between the groups. CONCLUSION: Cough in early stage IPF patients was not distinguishable from chronic cough in the community-based population by LCQ. Especially, there was no difference in the self-reported frequency of cough-associated sputum production.

Virtual communication is commonly used in Finnish interstitial lung disease multidisciplinary meetings.

Multidisciplinary meeting (MDM) is a core element in the diagnosis of interstitial lung diseases (ILD). The aim of the study was to investigate the implementation and key elements related to ILD MDMs in Finnish specialized care, which is characterized by long travel distances and a large number of small centers treating patients suffering from ILDs. An electronic questionnaire was sent to ILD experts working at five academic centers of Finland regarding the implementation of ILD MDMs with the focus on utilization of virtual communication. Responses were received from all academic centers of Finland (n = 5) whose catchment areas cover all of Finland. ILD MDMs were organized in each center approximately every two weeks and the core participants included a radiologist, respiratory physicians, junior staff, pathologist and a rheumatologist. All non-academic centers could refer their patients to be evaluated in ILD MDM of an academic center. Virtual communication was utilized by all academic centers in the implementation of ILD MDMs, being most common among small centers located in Eastern and Northern Finland. Virtual access to ILD MDM of an academic center was available in most parts of Finland, enabling small centers to benefit from the ILD expertise of academic centers.

Reticulation pattern without honeycombing on high-resolution CT is associated with the risk of disease progression in interstitial lung diseases.

BACKGROUND: The disease course of idiopathic pulmonary fibrosis (IPF) is progressive and occasionally, other types of interstitial lung disease (ILD) may progress similarly to IPF. This study aimed to evaluate risk factors for disease progression within 24 months in patients with various ILDs. METHODS: This prospective study obtained 97 patients with a suspected ILD who underwent a transbronchial lung cryobiopsy. The extent of several high-resolution computed tomography (HRCT) patterns was assessed. Due to the inclusion criteria the study population presented a low extent of honeycombing and definite usual interstitial pneumonia (UIP) pattern on HRCT suggesting an early stage of ILD. Disease progression within 24 months despite treatment was defined as a relative decline of ≥ 10% in forced vital capacity (FVC), or a relative decline in FVC of ≥ 5% and one of the three additional criteria: (1) a decline in diffusion capacity to carbon monoxide (DLCO) ≥ 15%; (2) increased fibrosis on HRCT; (3) progressive symptoms, or progressive symptoms and increased fibrosis on HRCT. The same definition was utilized in patients with IPF and other ILDs. Risk factors for disease progression were evaluated in a multivariable logistic regression model. RESULTS: Disease progression was revealed in 52% of the patients with ILD, 51% of the patients with IPF, and 53% of the patients with other types of ILD. A high extent of reticulation on HRCT (Odds ratio [OR] 3.11, 95% Confidence interval [CI] 1.21-7.98, P = 0.019) and never smoking (OR 3.11, CI 1.12-8.63, P = 0.029) were associated with disease progression whereas platelet count (OR 2.06 per 100 units increase, CI 0.96-4.45, P = 0.065) did not quite reach statistical significance. CONCLUSION: Higher extent of reticulation on HRCT and never smoking appeared to associate with the risk of disease progression within 24 months in ILD patients without honeycombing. Approximately half of the patients with ILD revealed disease progression, and similar proportions were observed in patients with IPF and in other types of ILD.

Risk factors of clinically significant complications in transbronchial lung cryobiopsy: A prospective multi-center study.

BACKGROUND: The use of a transbronchial lung cryobiopsy (TBLC) is increasing as a diagnostic method of interstitial lung diseases (ILD). This study aimed to evaluate risk factors associated with clinically significant complications of TBLC in ILD patients. METHODS: Patients referred to Kuopio or Tampere university hospitals, in Finland, for a suspected ILD were included. The TBLC was performed in an outpatient setting for 100 patients. Patients were mechanically ventilated in general anesthesia. Fluoroscopy guidance and prophylactic bronchial balloon were used. Complications, such as bleeding, pneumothorax, infections, and mortality were recorded. Moderate or serious bleeding, pneumothorax, or death ≤90 days were defined as clinically significant complications. A multivariable model was created to assess clinically significant complications. RESULTS: The extent of traction bronchiectasis (Odds ratio [OR] 1.30, Confidence interval [CI] 1.03-1.65, p = 0.027) and young age (OR 7.96, CI 2.32-27.3, p = 0.001) were associated with the risk of clinically significant complications whereas the use of oral corticosteroids ≤30 days before the TBLC (OR 3.65, CI 0.911-14.6, p = 0.068) did not quite reach statistical significance. A history of serious cough was associated with the risk of pneumothorax (OR 4.18, CI 1.10-16.0, p = 0.036). Procedure associated mortality ≤90 days was 1%. CONCLUSION: The extent of traction bronchiectasis on HRCT and young age were associated with the risk of clinically significant complications whereas oral corticosteroid use did not quite reach statistical significance. A history of serious cough was associated with the risk of clinically significant pneumothorax.

Changes in the societal burden caused by sleep apnoea in Finland from 1996 to 2018: A national registry study.

BACKGROUND: In the current century, sleep apnoea has become a significant public health problem due to the obesity epidemic. To increase awareness, improve diagnostics, and improve treatment, Finland implemented a national sleep apnoea programme from 2002 to 2010. Here, we present changes in the societal burden caused by sleep apnoea from 1996 to 2018. METHODS: National register data were collected from the Care Register for Health Care, Statistics Finland, the Social Insurance Institution of Finland, and the Finnish Centre for Pensions. Disease prevalence, use of healthcare and social services, and societal costs were estimated. FINDINGS: The number of sleep apnoea patients increased in secondary care from 8 600 in 1996 to 61 000 in 2018. There was a continuous increase in outpatient visits in secondary care from 9 700 in 1996 to 122 000 in 2018 (1 160%) and in primary care from 10 000 in 2015 to 29 000 in 2018 (190%). Accordingly, the cumulative annual number of days off work for sleep apnoea increased from 1 100 to 46 000. However, disability pensions for sleep apnoea decreased from 820 to 550 (33%) during the observation period. Societal costs per patient decreased over 50% during the observation period (€2 800 to €1 200). INTERPRETATION: The number of sleep apnoea patients in Finland increased remarkably during the observation period. To control this burden, diagnostic methods and treatment were revised and follow up was reorganised. Consequently, there was a significant decrease in societal costs per patient. The decrease in disability pensions suggests earlier diagnosis and improved treatment. The national sleep apnoea programme was one of the initiators for these improved outcomes. FUNDING: The Finnish Institute for Health and Welfare and the Hospital District of Helsinki and Uusimaa (HUH), Helsinki, Finland.

Dexamethasone and RU24858 Induce Survival and Growth Factor Receptor Bound Protein 2, Leukotriene B4 Receptor 1 and Annexin-1 Expression in Primary Human Neutrophils.

Glucocorticoids are widely used anti-inflammatory medication in diseases like asthma and chronic obstructive pulmonary disease. Glucocorticoids can either activate (transactivation) or inhibit (transrepression) transcription. RU24858 was introduced as a "dissociated" glucocorticoid and it has been reported to transrepress but not to transactivate. The aim of this study was to compare the effects of RU24858 and dexamethasone in human neutrophils. RU24858 delayed spontaneous neutrophil apoptosis and further enhanced GM-CSF- induced neutrophil survival to a similar extent as dexamethasone. Like dexamethasone RU24858 also reduced CXCL8 and MIP-1α. Unexpectedly however, RU24858 increased the expression of the glucocorticoid-inducible genes BLT-1, Annexin-1 and Grb-2 in neutrophils to a similar level as seen with dexamethasone. We have shown here that dexamethasone and RU24858 both increase Grb-2, BLT1 and Annexin-1 expression and inhibit CXCL8 and MIP-1α production. This suggests that RU24858 was not able to dissociate between transactivation and transrepression in human neutrophils but enhanced neutrophil survival.

Bacterial DNA delays human eosinophil apoptosis.

Oligodeoxynucleotide (ODN) sequences containing unmethylated cytidine phosphate guanosine (CpG) motifs prevalent in bacterial DNA attenuate allergic lung inflammation in experimental models of asthma but failed to inhibit eosinophilia and improve lung function in patients with asthma. Bacterial respiratory tract infections exacerbate asthma in humans. Increased eosinophil survival is a critical factor leading to persistent eosinophilic airway inflammation. Apoptosis is regarded as a key mechanism in the resolution of eosinophilic inflammation. The aim of this study was to investigate the effects of bacterial DNA and CpG ODNs on human eosinophil apoptosis in vitro and to elucidate the signalling pathway. Eosinophils were isolated from human peripheral blood by CD16- or CD16-, CD19- and CD304-negative selection. Apoptosis was determined by flow cytometric analysis of relative DNA content, Annexin-V staining and/or morphological analysis. Toll-like receptor 9 (TLR9) expression was studied by using western blotting and intracellular flow cytometry. Bacterial DNA and phosphorothioate-modified CpG ODNs, but not vertebrate DNA, were found to delay spontaneous eosinophil apoptosis. The effect of CpG ODNs was dependent on endosomal acidification and reversed by inhibitory ODN, which suggests involvement of TLR9 pathway. Furthermore, we demonstrated TLR9 expression in eosinophils derived from both atopic and healthy donors. Non-CpG ODNs had occasionally parallel but less profound effect on eosinophil apoptosis, which was not dependent on endosomal acidification. The anti-apoptotic effect of CpG ODNs was dependent on phosphatidylinositol 3-kinase (PI3K) and nuclear factor-kappaB (NF-kappaB) but not mitogen-activated protein kinases (MAPKs) as determined by inhibitor studies. Although our results suggest CpG-dependent involvement of TLR9 in the action of phosphorothioate-modified ODNs, we interestingly found that the anti-apoptotic action of native bacterial DNA in eosinophils is not dependent on unmethylated CpG motifs. This suggests that bacterial DNA contains a currently unknown recognition structure lacking from vertebrate DNA. Bacterial DNA-mediated suppression of eosinophil apoptosis is a novel mechanism for exacerbation of eosinophilic lung inflammation associated with bacterial respiratory tract infection.

Phosphodiesterase 4 inhibitors delay human eosinophil and neutrophil apoptosis in the absence and presence of salbutamol.

In asthma and chronic obstructive pulmonary disease (COPD), the number of eosinophils and neutrophils in the lung is increased. One described mechanism leading to the impaired clearance of these cells from the lung is the delay in their programmed cell death (apoptosis). Selective inhibitors of phosphodiesterases (PDEs) are under development for the treatment of lung diseases because of their anti-inflammatory and bronchodilator activity. The aim of the present study was to establish whether inhibitors of PDE3, PDE4 and PDE5 modulate human eosinophil or neutrophil apoptosis or beta 2-adrenoceptor agonist- or cytokine-afforded survival. We also evaluated whether a PDE4 inhibitor could modulate the effect of a corticosteroid on eosinophil and neutrophil apoptosis. Apoptosis was measured by using the relative DNA fragmentation assay and Annexin-V binding. Inhibitors of PDE4 (rolipram; 0.1-10 microM) and PDE3 (cilostazol; 0.1-10 microM) delayed spontaneous eosinophil apoptosis maximally by 25% and 15%, respectively. A combination of a PDE4 or PDE3 inhibitor (10 microM) with salbutamol (100 nM) further delayed eosinophil apoptosis maximally by 42-49%. In neutrophils, rolipram (10 microM) also decreased apoptosis with a maximal inhibition of 13%. The combination of rolipram (10 microM) and salbutamol (100 nM) produced a 27% inhibition of neutrophil apoptosis. Inhibitor of cGMP-specific PDE5 (zaprinast; 0.1-10 microM) did not affect eosinophil apoptosis and only slightly increased spontaneous neutrophil apoptosis. The effect of budesonide on apoptosis was not significantly modulated by a PDE4 inhibitor in eosinophils or neutrophils. The present results show that selective inhibitors of cAMP-hydrolyzing PDEs (PDE3 and PDE4) delay eosinophil apoptosis and, thus, increase their survival in vitro. Furthermore, beta 2-adrenoceptor agonists enhance the anti-apoptotic effects of PDE3 and PDE4 inhibitors, suggesting that such drug combinations may prolong eosinophil and neutrophil longevity in the lung.

Histamine reverses IL-5-afforded human eosinophil survival by inducing apoptosis: pharmacological evidence for a novel mechanism of action of histamine.

Eosinophils are essential inflammatory cells in the pathogenesis of asthma and atopic conditions. Histamine, released from mast cells and basophils in response to allergen exposure, is a critical mediator in the allergic response. Histamine exerts its effects via four unequivocally characterized histamine receptors, H(1-4). Several functions of eosinophils have previously been shown to be stimulated by histamine. However, its effects on eosinophil apoptosis are unknown. The aim of the present study was to resolve the effects of histamine on constitutive apoptosis of human eosinophils and on the survival-enhancing action of interleukin (IL)-5. Additional experiments were conducted to elucidate the histamine receptor(s) involved in any response seen and the associated signal transduction cascade. Human isolated peripheral blood eosinophils were cultured in the absence or presence of histamine, IL-5 and receptor antagonists/agonists or mediator inhibitors/analogues. Apoptosis was assessed by measuring the relative DNA content of propidium iodide (PI)-stained cells and the effects were confirmed by morphological analysis with bright field microscopy. Caspase activities were assessed by using commercial Caspase-Glo 3/7, 8 and 9 luminescence assays. Histamine (10-100 microM) partially reversed IL-5-induced human eosinophil survival by enhancing apoptosis as assessed by measuring the relative DNA content of PI-stained cells. This effect was not mediated through any of the known histamine receptors or through non-specific activation of 5-hydroxytryptamine receptors or alpha-adrenoceptors. Moreover, the reversal of IL-5-inhibited eosinophil apoptosis by histamine seemed not to utilize the conventional intracellular second-messenger pathways including cyclic AMP, protein kinase A or phospholipase C. Inhibition of caspase 6 and caspases 1, 10 or 12 reversed the effects of histamine but also inhibited apoptosis in general. In conclusion, the data presented herein indicate that histamine induces human eosinophil apoptosis in the presence of a survival-prolonging cytokine by a mechanism that does not apparently involve the activation of any of the currently known histamine receptor subtypes. The possibility exists that another, as yet unidentified, histamine receptor may exist in human eosinophils that regulates survival, although the participation of histamine receptor-independent mechanisms cannot be excluded.

Levocetirizine and cytokine production and apoptosis of human eosinophils.

Antihistamines are a common therapy for allergic symptoms. Eosinophilic infiltration is considered a hallmark of allergic inflammation. Eosinophils are capable of mediating airway mucosal damage by producing various inflammatory mediators including cytokines, chemokines, basic granule proteins, lipid mediators, and growth factors. Reduced eosinophil apoptosis is thought to be an important feature in the formation of eosinophilia in allergic diseases such as allergic rhinitis, atopic eczema, and asthma. The aim of this study was to investigate the effects of levocetirizine on the production of inflammatory mediators by eosinophils and on eosinophil apoptosis. The production of cytokines and other inflammatory mediators by human eosinophils was measured by a cytokine antibody array. Apoptosis of isolated human eosinophils was assessed by measuring the relative DNA content of propidium iodide-stained cells. Of the 40 cytokines studied, levocetirizine (1 microM) was found to enhance the release of tissue inhibitor of metalloproteinases 1 and 4, matrix metalloproteinase 9, and heparin-binding epidermal growth factor and to attenuate the production of interleukins (IL)-1 beta and IL-7 and stem cell factor in lipopolysaccharide-stimulated human eosinophils. Levocetirizine did not alter constitutive eosinophil apoptosis or eosinophil survival induced by IL-5, granulocyte/macrophage colony-stimulating factor, tumor necrosis factor alpha, or salbutamol. The results of this study suggest that levocetirizine modulates the profile of inflammatory mediators including cytokines, growth factors, proteinases, and antiproteinases produced by eosinophils, which may be of importance in allergic inflammation and airway remodeling. However, eosinophil longevity seems not to be modulated by levocetirizine.

First-generation antihistamines diphenhydramine and chlorpheniramine reverse cytokine-afforded eosinophil survival by enhancing apoptosis.

Antihistamines or histamine H1-receptor antagonists are commonly used to treat a variety of allergic symptoms. Eosinophils are considered to play an essential role in the pathogenesis of allergy. Reduced eosinophil apoptosis is thought to be an important element in the formation of eosinophilia in allergic conditions such as allergic rhinitis, atopic eczema, and asthma. The aim of this study was to investigate the effects of two first-generation antihistamines diphenhydramine and chlorpheniramine on constitutive eosinophil apoptosis and on interleukin (IL)-5-afforded eosinophil survival. The role of c-Jun N-terminal kinase (JNK) in mediating the effects of antihistamines on eosinophil apoptosis was evaluated also. Apoptosis of isolated human eosinophils was assessed by measuring the relative DNA content of propidium iodide-stained cells and confirmed by morphological analysis. The activity of JNK was measured by Western blotting. Antihistamines were found to reverse the survival-prolonging effect of IL-5 in eosinophils by enhancing apoptosis. JNK was found to be activated slowly during diphenhydramine-induced eosinophil apoptosis. An inhibitor peptide specific for JNK, L-JNKI1 (JNK peptide inhibitor 1, L-stereoisomer), inhibited diphenhydramine-mediated eosinophil apoptosis. Our results suggest that first-generation antihistamines diphenhydramine and chlorpheniramine reverse IL-5-afforded eosinophil survival and that the enhanced apoptosis by antihistamines is mediated through activation of JNK. Thus, reversal of IL-5-afforded eosinophil survival may contribute to the antiallergic actions of diphenhydramine and chlorpheniramine.

c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis.

Eosinophils are considered to play an important role in the pathogenesis of asthma. Glucocorticoids are potent anti-inflammatory agents for the treatment of chronic inflammatory diseases and they have been shown to increase the rate of eosinophil apoptosis. c-Jun N-terminal kinase (JNK) has been suggested to participate in the signaling pathways of apoptosis. The aims of the present study were to examine whether JNK is involved in the regulation of constitutive eosinophil apoptosis and whether it mediates dexamethasone-induced apoptosis of human eosinophils. Isolated human eosinophils were cultured with and without dexamethasone and the JNK inhibitor L-JNKI-1. Apoptosis was assessed by measuring the relative DNA content of propidium iodide-stained cells and confirmed by Annexin V-binding and morphological analysis with bright field microscopy. The phosphorylation of both JNK and c-Jun were measured by Western blotting. During a 40h culture, dexamethasone (1muM) enhanced human eosinophil apoptosis by 10-30%. Culture with L-JNKI1 (10muM) inhibited apoptosis in dexamethasone-treated cells by 53%. Furthermore, L-JNKI1 decreased the rate of constitutive eosinophil apoptosis by 64%. However, the enhancement of eosinophil apoptosis by dexamethasone was not reversed by L-JNKI1. Slow activation of JNK in constitutive apoptosis as well as a similar tendency in dexamethasone-induced eosinophil apoptosis could be observed by Western blot analyses. c-Jun was found to be active both in the presence and absence of dexamethasone. However, no further phosphorylation of the serine residue 63 of c-Jun could be seen. Taken together, our present results suggest that JNK is active during apoptosis of human eosinophils both in the presence and absence of glucocorticoids. JNK seems to mediate constitutive human eosinophil apoptosis. However, the activity of JNK is not enhanced by glucocorticoids and the effects of glucocorticoids cannot be reversed by JNK inhibition. JNK therefore seems not to mediate glucocorticoid-induced human eosinophil apoptosis.

The glucocorticoid RU24858 does not distinguish between transrepression and transactivation in primary human eosinophils.

BACKGROUND: Glucocorticoids are used to treat chronic inflammatory diseases such as asthma. Induction of eosinophil apoptosis is considered to be one of the main mechanisms behind the anti-asthmatic effect of glucocorticoids. Glucocorticoid binding to its receptor (GR) can have a dual effect on gene transcription. Activated GR can activate transcription (transactivation), or by interacting with other transcription factors such as NF-kappaB suppress transcription (transrepression). RU24858 has been reported to transrepress but to have little or no transactivation capability in other cell types. The dissociated properties of RU24858 have not been previously studied in non-malignant human cells. As the eosinophils have a very short lifetime and many of the modern molecular biological methods cannot be used, a "dissociated steroid" would be a valuable tool to evaluate the mechanism of action of glucocorticoids in human eosinophils. The aim of this study was to elucidate the ability of RU24858 to activate and repress gene expression in human eosinophils in order to see whether it is a dissociated steroid in human eosinophils. METHODS: Human peripheral blood eosinophils were isolated under sterile conditions and cultured in the presence and/or absence RU24858. For comparison, dexamethasone and mometasone were used. We measured chemokine receptor-4 (CXCR4) and Annexin 1 expression by flow cytometry and cytokine production by ELISA. Apoptosis was measured by DNA fragmentation and confirmed by morphological analysis. RESULTS: RU24858 (1 microM) increased CXCR4 and Annexin 1 expression on eosinophils to a similar extent as mometasone (1 microM) and dexamethasone (1 microM). Like dexamethasone and mometasone, RU24858 did suppress IL-8 and MCP-1 production in eosinophils. RU24858 also increased spontaneous eosinophil apoptosis to a similar degree as dexamethasone and mometasone, but unlike dexamethasone and mometasone it did not reverse IL-5- or GM-CSF-induced eosinophil survival. CONCLUSION: Our results suggest that in human eosinophils RU24858 acts as transactivator and transrepressor like classical glucocorticoids. Thus, RU24858 seems not to be a "dissociated steroid" in primary human eosinophils in contrast to that reported in animal cells. In addition, functionally RU24858 seems to be a less potent glucocorticoid as it did not reverse IL-5- and GM-CSF-afforded eosinophil survival similarly to dexamethasone and mometasone.

Ketotifen induces primary necrosis of human eosinophils.

Eosinophils are considered essential in the pathogenesis of allergy. Reduced eosinophil apoptosis is considered to be a key element in the formation of eosinophilia in allergic conditions. Antihistamines are widely used in the treatment of allergic disorders, but their effects on eosinophil apoptosis are poorly understood. The histamine H1-receptor antagonist, ketotifen, is available orally and as eye drops for the treatment of allergic symptoms. The aim of our study was to investigate the possible effect of ketotifen on constitutive eosinophil apoptosis and on interleukin (IL)-5-mediated eosinophil survival. Isolated peripheral blood eosinophils were cultured with or without the survival-prolonging cytokine IL-5 and ketotifen. Apoptosis was assessed by measuring the relative DNA content and by morphological analysis. Ketotifen was found to reverse eosinophil survival induced by interleukin-5. However, the flow cytometry histogram of DNA in propidium iodide-stained cells was not typical to apoptosis. Morphological analysis of the eosinophils by bright-field microscopy suggested that the effect of ketotifen was due to the induction of primary necrosis rather than apoptosis. Histological assessment of eosinophil ultrastructure by transmission electron microscopy confirmed signs of advanced necrosis. In summary, our results suggest that at clinically relevant drug concentrations, ketotifen induces primary necrosis in IL-5-treated human eosinophils.