- Email: [email protected]
Release of inflammatory mediators from eosinophils following a hyperosmolar stimulus
ARTICLE IN PRESS Respiratory Medicine (2003) 97, 928–932
Release of inflammatory mediators from eosinophils following a hyperosmolar stimulus E.D. Moloneya,*, S. Griffinb, C.M. Burkea, L.W. Poulterc, S. O’Sullivanc a
Department of Respiratory Medicine, James Connolly Memorial Hospital, Dublin, Ireland Department of Respiratory Research, Beaumont Hospital, Dublin, Ireland c Department of Clinical Immunology, Royal Free University Hospital, London, UK b
KEYWORDS Hyperosmolar-induced inflammatory mediator release
Summary Airway dehydration and subsequent hyperosmolarity of periciliary fluid are considered critical events in exercise-induced bronchoconstriction (EIB). It has been shown that an in vitro hyperosmolar stimulation of basophils and mast cells with mannitol can induce the release of histamine and leukotrienes. The aim of this study was to establish if a hyperosmolar challenge could trigger activation of eosinophils to release chemokines and lipid mediators. Peripheral blood eosinophils were isolated from seven asthmatic and six non-asthmatic subjects. Hyperosmolar stimulation of eosinophils with mannitol (0.7 M), resulted in a significant increase in LTC4 levels compared to baseline in both asthmatic (15.274.6 vs. 70.179.5; P ¼ 0:0002) and control subjects (14.374.0 vs. 55.675.6; P ¼ 0:0001). ECP levels did not increase significantly above baseline following mannitol stimulation in either group. This study shows that eosinophils can be activated by a hyperosmolar stimulus. Therefore it seems reasonable to suggest that eosinophils could contribute to EIB. r 2003 Elsevier Science Ltd. All rights reserved.
Introduction During exercise, the ventilation rate increases, thus the respiratory tract needs to condition much larger volumes of air over a much shorter time. This causes airway dehydration and subsequent exercise-induced bronchoconstriction (EIB) in a large proportion of asthmatics.1–4 The finding in 1977,1 that inhaling fully humidified air at body conditions could prevent EIB, demonstrated the importance of water loss from the airways. Airway dehydration and the subsequent hyperosmolarity of the periciliary fluid are considered critical events in *Corresponding author. Adult Intensive Care Unit, Imperial College School of Medicine at the National Heart and Lung Institute, Royal Brompton Hospital, London SW3 6NP, UK. Tel.: +44-207-351-8528; fax: +44-207-351-8524. E-mail address: [email protected] (E.D. Moloney).
exercise-induced bronchoconstriction.5,6 One hypothesis suggests that the hyperosmolar environment triggers release of bronchoconstricting mediators from inflammatory cells.7 It has been shown that an in vitro hyperosmolar stimulus with mannitol can induce the release of mediators, including histamine and leukotrienes, from basophils and mast cells.8–10 The eosinophil is an effector cell in the inflammed asthmatic airway. Eosinophils release cytotoxic granules and generate lipid meditors such as cysteinyl leukotrienes that cause bronchial epithelial damage and bronchoconstriction, respectively.11,12 Therefore, the aim of this study was to establish if a hyperosmolar challenge could trigger activation of eosinophils to release chemokines and lipid mediators, which could promote bronchoconstriction and perpetuate inflammation in the asthmatic airway.
0954-6111/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0954-6111(03)00119-7
ARTICLE IN PRESS Release of inflammatory mediators from eosinophils
Methods Subjects Seven atopic subjects who met the American Thoracic Society criteria for mild asthma, and six non-atopic, non-asthmatic control subjects volunteered to participate in this study. Subject characteristics are displayed in Table 1. Atopy was determined in subjects using a battery of skin prick test antigens with a positive response defined as a grade 2+ reaction (erythema 21–30 mm, papule 5– 10 mm), to at least one antigen. All asthmatic subjects had episodic symptoms of shortness of breath, wheeze, cough or chest tightness controlled by use of as required inhaled bronchodilator therapy. None of the asthmatic subjects had taken oral or inhaled corticosteroid therapy within the 3 months before the study, and none was taking theophylline, cromones or leukotriene modifiers. Approval for this study was obtained from the local Ethics Committee of James Connolly Memorial Hospital, and full written informed consent was obtained from each subject.
Eosinophil purification and stimulation Peripheral blood (70 ml) was collected from each subject into lithium heparinised tubes, and blood was diluted with an equal volume of ice cold phosphate buffered saline (PBS). Isotonic Percoll (15 ml) (density 1.082 g/ml) was layered under
Table 1
929
30 ml aliquots of blood, and centrifuged at 3000 rpm for 30 min at room temperature. Following centrifugation, the upper layer containing mononuclear cells, serum, and Percoll were discarded. The remaining erythrocyte pellet, containing the polymorphonuclear cells (PMN), was suspended in PBS containing 2% heat inactivated fetal calf serum (PBS/FCS). The red cells were then lysed with reverse osmosis sterile water (40 ml for 30 s), and 4 PBS was used to return the cell suspension to isotonicity. Cell lysis was repeated 2– 3 times. The PMN were incubated for 1 h at 41C in 5 ml PBS/FCS with anti-CD16 immunomagnetic microbeads (Miltenyi Biotec, England). Following this, PMN were then layered onto a magnetic cell separation column type CS (Miltenyi Biotec, England), and unlabelled eosinophils were eluted with 45 ml of ice cold PBS/FCS. This procedure resulted in total yields of 6–12 million viable eosinophils as counted with a modified Neubauer hemocytometer (Knittel, Glaser, Germany), with a purity of 98–99% as assessed by Diff-Quik staining and a viability of 99% as assessed by Trypan Blue. Purified eosinophils were suspended at 1 106 cells/ml in RPMI 1640 culture medium supplemented with penicillin (103 U/ml), streptomycin (10 mg/ml), L-glutamine (2 mM), and 10% FCS. Cells were stimulated with calcium ionophore (A23187) 2.5 mM and hyperosmolar mannitol 0.7 M for 15 min, in a humidified incubator at 371C with 5% CO2. Stimulation was then terminated by addition of ice-cold methanol to each well, and supernatants were removed and stored at –201C until analysis.
Subject characteristics of eosinophil donors.
Asthmatic subjects
Age (years)
Sex
Atopy/Allergen
Therapy
1 2 3 4 5 6 7
29 25 21 19 17 35 51
F M M F M F M
HDM HDM HDM HDM HDM HDM HDM
S S S S S S S
Non-asthmatic subjects
Age (years)
Sex
Atopy/Allergen
1 2 3 4 5 6
22 23 33 24 24 45
F M F M F F
2+ 3+ 3+ 2+ 3+, GRASS 2+ 2+, GRASS 2+ 3+, GRASS 3+
Hdm = house dust mite, Grass = grass mix, S = salbutamol, prn = taken as required.
(prn) (prn) (prn) (prn) (prn) (prn) (prn)
Therapy
ARTICLE IN PRESS 930
E.D. Moloney et al.
Analysis of mediators
5000
LTC 4 pg/ml
4000
Leukotriene C4 (LTC4) LTC4 was measured with a commercially available enzyme-immunoassay kit (Cayman Chemical Co, Ann Arbor, MI, USA). The lower limit of detection of this assay is 7.8 pg/ml, and at 50% maximum binding the antibody shows a cross-reactivity of 48% with LTD5, 45% with LTD4, 28% with N-acetyl LTE4, 7% with LTE5, and less than 0.01% with other eicosanoids.
3000 2000 1000 120 90 60 30 0
Unstim
(A)
Mannitol
A23187
4500 4000 3500
ECP µg/L
Eosinophil cationic protein (ECP) ECP was measured with a commercially available radio-immunoassay kit (Pharmacia-Upjohn AC, Uppsala, Sweden). The lower limit of detection of this assay is 2 mg/l, and at 50% maximum binding the antibody shows a cross-reactivity of o0.06% with EPX.
3000 2500 2000 1500 1000 500 0
Data analysis Normally distributed data (as assessed by Kolmogorov-Smirnov) are expressed as group mean 7 SE. Differences between groups were assessed using a one-way analysis of variance and students t-test. Results were considered significant if Po0:05: The statistical calculations were controlled by the use of a validated statistical software package for personal computers (Graph Pad Prism Inc, San Diego, CA, USA).
Results Baseline levels of LTC4 and ECP did not differ between the asthmatic or control group. Following stimulation with calcium ionophore, a significant increase in LTC4 levels above baseline was seen in both asthmatic (P ¼ 0:0005) and control subjects (P ¼ 0:03) (Fig. 1a). Levels of ECP did not significantly increase from baseline in either asthmatic (P ¼ 0:41) or control subjects (P ¼ 0:71) following ionophore stimulation (Fig. 1b). Hyperosmolar stimulation of eosinophils with mannitol, resulted in a significant increase in LTC4 levels compared to baseline in both asthmatic (15.274.6 vs. 70.179.5; P ¼ 0:0002) and control subjects (14.374.0 vs. 55.675.6; P ¼ 0:0001) (Fig. 1a). In contrast, ECP levels did not increase significantly above baseline following mannitol stimulation in either the asthmatic (P ¼ 0:36) or control subjects (P ¼ 0:30) (Fig. 1b).
(B)
Unstim
Mannitol
A23187
Fig. 1 (A) Mean7SE leukotriene C4 (LTC4) levels (pg/ml) from eosinophils isolated from asthmatic (open bars) and non-asthmatic control subjects (filled bars). Following a hyperosmolar stimulation of eosinophils with mannitol, a significant increase in LTC4 is seen compared to unstimulated eosinophils, in both asthmatic (P ¼ 0:0002) and control subjects (P ¼ 0:0001). There is no significant difference between asthmatic and control subjects, in LTC4 levels from unstimulated eosinophils (P=0.9), and from mannitol stimulated eosinophils (P ¼ 0:2). (B) Mean7SE eosinophil cationic protein (ECP) levels (mg/l) from eosinophils isolated from asthmatic (open bars) and non-asthmatic control subjects (filled bars). Following a hyperosmolar stimulation of eosinophils with mannitol, no significant increase in ECP levels was detected compared to unstimulated eosinophils, in both asthmatic (P ¼ 0:4) and control subjects (P ¼ 0:3).
Discussion This study shows that hyperosmolar stimulation of eosinophils resulted in the release of LTC4. In the trachea the osmolarity of secretions has been demonstrated to be 360 mOsm/kg H2O.9 Anderson et al.13 suggested that after exercise bronchial secretions may result in an osmolarity of 900 mOsm/kg H2O. Basophils and lung mast cells are activated by hyperosmolar mannitol, maximal histamine release from basophils was achieved at 1050 mOsm/kg, whereas mast cells released maximally at 700–750 mOsm/kg. In the current study, purified eosinophils were stimulated with
ARTICLE IN PRESS Release of inflammatory mediators from eosinophils
hyperosmolar (0.7 M) mannitol (equivalent to approx. 950 mOsm/kg) to try to mimic the conditions in vitro, that may occur in vivo after exercise. Epithelial cells have previously been stimulated with similar molar concentrations of mannitol, without any evidence of cytotoxicity to cells as assessed by LDH expression.14 Cells were stimulated for 15 min to avoid hyperosmolar-induced cytotoxicity, which might have occurred with a longer incubation period. We found that LTC4 generation following hyperosmolar stimulation was not a unique characteristic of eosinophils from asthmatic subjects only. Hodges et al.15 previously showed that eosinophils from asthmatic subjects, when stimulated with calcium ionophore, have a decreased capacity for LTC4 synthesis compared with eosinophils of similar density isolated from normal subjects. It is generally accepted that the magnitude of EIB is strongly correlated with the underlying degree of airway hyperresponsiveness.16 Therefore, it is tempting to speculate that the underlying airway hyperresponsiveness of asthmatic subjects causes them to be more ‘‘primed’’ to the effects of LTC4 and other bronchoconstricting mediators, compared to normal subjects. One of the consequences of airway inflammation in asthma is thought to be desquamation of airway epithelial cells resulting from the action of inflammatory mediators such as eosinophil granule proteins.17 However, it has previously been suggested that ECP is not related to bronchial hyperreactivity.18 It is possible that we may have missed the peak effect of ECP release at 15 min, as it has previously been shown that eosinophil degranulation peaks after 10 min incubation with calcium ionophore.19 Moreover, Takafugi and colleagues demonstrated that neither TNF or GM-CSF could induce ECP release from human eosinophils,20 and eosinophils from atopic asthmatics did not release ECP after incubation with specific allergen or with anti-IgE monoclonal antibodies.21 To the best of our knowledge purified eosinophils have not been subjected to a hyperosmolar challenge. This is surprising given that eosinophils are one of the primary sources of the potent bronchoconstricting cysteinyl-leukotrienes.22 Togias and colleagues have shown increased LTC4 in nasal lavage following a cold dry air nasal challenge.10 The osmolarity of the nasal secretions was increased in those subjects sensitive to cold dry air. Nasal lavage collected from 15 healthy volunteers following a nasal challenge with hyperosmolar mannitol (869 mOsm/kg) contained significantly higher quantities, as compared to baseline, of immunoreactive leukotrienes LTD4/LTE4.23 In vitro
931
work with hyperosmolar stimulated basophils and mast cells have yielded confounding results. Findlay et al. investigated basophil cell supernatants for production of cysteinyl leukotrienes following stimulation with hyperosmolar solutions, but could not document an increase in the cysteinyl leukotriene production.8 It should be noted however, that guinea pig ileal contractions were used to assay the cysteinyl leukotrienes with a comparatively poor sensitivity compared to present day methodology. A study using the more sensitive radioimmunoassay did detect a non-significant doubling in the production of LTC4/LTD4 from isolated basophils stimulated with hyperosmolar mannitol.24 In the same study, the investigators did not however observe an increase in LTC4/LTD4 from human lung mast cells following hyperosmolar challenge. Cells from chopped nasal polyps containing epithelial and mononuclear cells as well as eosinophils and neutrophils were stimulated to produce increased levels of 15-hydroxyeicosatetranoic acid and leukotriene B4 following incubation with hyperosmolar buffer solution.14 Unfortunately, levels of LTC4 were not assayed. In summary, our study demonstrates that eosinophils can be activated by a hyperosmolar stimulus to release LTC4. Therefore it seems reasonable to suggest that eosinophils could contribute to EIB.
References 1. Chen WY, Horton DJ. Heat and water loss from the airways and exercise-induced asthma. Respiration 1977;34:305–13. 2. Strauss RH, McFadden Jr. ER, Ingram RH, Jaeger JJ. Enhancement of exercise-induced asthma by cold air. N Engl J Med 1977;297:743–7. 3. Deal Jr. EC, McFadden Jr. ER, Ingram Jr. RH, Jaeger JJ. Hyperpnea and heat flux: initial reaction sequence in exercise-induced asthma. J Appl Physiol 1979;46:476–83. 4. Anderson SD, Schoeffel RE, Follet R, Perry CP, Daviskas E, Kendall M. Sensitivity to heat and water loss at rest and during exercise in asthmatic patients. Eur J Respir Dis 1982;63:459–71. 5. Smith CM, Anderson SD. Hyperosmolarity as the stimulus to asthma induced by hyperventilation. J Allergy Clin Immunol 1986;77:729–36. 6. Anderson SD, Daviskas E, Smith CM. Exercise-induced asthma: a difference in opinion regarding stimulus. Allergy Proc 1989;10:215–26. 7. Anderson SD. Is there a unifying hypothesis for exerciseinduced asthma? J Allergy Clin Immunol 1984;73:660–5. 8. Findlay SR, Dvorak AM, Kagey-Sobotka A, Lichtenstein LM. Hyperosmolar triggering of histamine release from human basophils. J Clin Invest 1981;67:1604–13. 9. Eggleston PA, Kagey-Sobotka A, Schleimer RP, Lichtenstein LM. Interaction between hyperosmolar and IgE-mediated histamine release from basophils and mast cells. Am Rev Respir Dis 1984;130:86–91.
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10. Togias AG, Naclerio RM, Proud D, et al. Nasal challenge with cold, dry air results in release of inflammatory mediators: possible mast cell involvement. J Clin Invest 1985;76:1375– 81. 11. Motojima S, Frigas E, Loegering DA, Gleich GJ. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am Rev Respir Dis 1989;139:801–5. 12. Dahlen SE, Hedqvist P, Hammarstrom S, Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature 1980;288:484–6. 13. Anderson SD, Daviskas E. The airway microvasculature and exercise-induced asthma. Thorax 1992;47:748–52. 14. Souques F, Crampette L, Mondain M, et al. Stimulation of dispersed nasal polyp cells by hyperosmolar solutions. J Allergy Clin Immunol 1995;96:980–5. 15. Hodges MK, Weller PF, Gerard NP, Ackerman SJ, Drazen JM. Heterogeneity of leukotriene C4 production by eosinophils from asthmatic and from normal subjects. Am Rev Respir Dis 1988;138:799–804. 16. Anderton RC, Cuff MT, Frith PA, et al. Bronchial responsiveness to inhaled histamine and exercise. J Allergy Clin Immunol 1979;63:315–20. 17. Frigas E, Motojima S, Gleich GJ. The eosinophilic injury to the mucosa of the airways in the pathogenesis of bronchial asthma. Eur Respir J 1991;13:123s–35s.
E.D. Moloney et al.
18. Ferdousi HA, Dreborg S. Asthma, bronchial hyperreactivity and mediator release in children with birch pollinosis. ECP and EPX levels are not related to bronchial hyperreactivity. Clin Exp Allergy 1997;27:530–9. 19. Kroegel C, Yukawa T, Dent G, Venge P, Chung KF, Barnes PJ. Stimulation of degranulation from human eosinophils by platlet-activating factor. J Immunol 1989;142:3518–26. 20. Takafugi S, Tadokoro K, Ito K, Nakagawa T. Release of granule proteins from human eosinophils stimulated with mast cell mediators. Allergy 1998;53:951–6. 21. Tomassini M, Tsicopoulos A, Tai PC, et al. Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation. J Allergy Clin Immunol 1991;88:365–75. 22. Laviolette M, Ferland C, Comtois JF, Champagne K, Bosse M, Boulet LP. Blood eosinophil leukotriene C4 production in asthma of different severities. Eur Respir J 1995;8:1465–72. 23. Silber G, Proud D, Warner J, et al. In vivo release of inflammatory mediators by hyperosmolar solutions. Am Rev Respir Dis 1988;137:606–12. 24. Eggleston PA, Kagey-Sobotka A, Proud D, Adkinson Jr. NF, Lichtenstein LM. Dissociation of the release of histamine and arachidonic acid metabolites from osmotically activated basophils and human lung mast cells. Am Rev Respir Dis 1990;141:960–4.
Release of inflammatory mediators from eosinophils following a hyperosmolar stimulus E.D. Moloneya,*, S. Griffinb, C.M. Burkea, L.W. Poulterc, S. O’Sullivanc a
Department of Respiratory Medicine, James Connolly Memorial Hospital, Dublin, Ireland Department of Respiratory Research, Beaumont Hospital, Dublin, Ireland c Department of Clinical Immunology, Royal Free University Hospital, London, UK b
KEYWORDS Hyperosmolar-induced inflammatory mediator release
Summary Airway dehydration and subsequent hyperosmolarity of periciliary fluid are considered critical events in exercise-induced bronchoconstriction (EIB). It has been shown that an in vitro hyperosmolar stimulation of basophils and mast cells with mannitol can induce the release of histamine and leukotrienes. The aim of this study was to establish if a hyperosmolar challenge could trigger activation of eosinophils to release chemokines and lipid mediators. Peripheral blood eosinophils were isolated from seven asthmatic and six non-asthmatic subjects. Hyperosmolar stimulation of eosinophils with mannitol (0.7 M), resulted in a significant increase in LTC4 levels compared to baseline in both asthmatic (15.274.6 vs. 70.179.5; P ¼ 0:0002) and control subjects (14.374.0 vs. 55.675.6; P ¼ 0:0001). ECP levels did not increase significantly above baseline following mannitol stimulation in either group. This study shows that eosinophils can be activated by a hyperosmolar stimulus. Therefore it seems reasonable to suggest that eosinophils could contribute to EIB. r 2003 Elsevier Science Ltd. All rights reserved.
Introduction During exercise, the ventilation rate increases, thus the respiratory tract needs to condition much larger volumes of air over a much shorter time. This causes airway dehydration and subsequent exercise-induced bronchoconstriction (EIB) in a large proportion of asthmatics.1–4 The finding in 1977,1 that inhaling fully humidified air at body conditions could prevent EIB, demonstrated the importance of water loss from the airways. Airway dehydration and the subsequent hyperosmolarity of the periciliary fluid are considered critical events in *Corresponding author. Adult Intensive Care Unit, Imperial College School of Medicine at the National Heart and Lung Institute, Royal Brompton Hospital, London SW3 6NP, UK. Tel.: +44-207-351-8528; fax: +44-207-351-8524. E-mail address: [email protected] (E.D. Moloney).
exercise-induced bronchoconstriction.5,6 One hypothesis suggests that the hyperosmolar environment triggers release of bronchoconstricting mediators from inflammatory cells.7 It has been shown that an in vitro hyperosmolar stimulus with mannitol can induce the release of mediators, including histamine and leukotrienes, from basophils and mast cells.8–10 The eosinophil is an effector cell in the inflammed asthmatic airway. Eosinophils release cytotoxic granules and generate lipid meditors such as cysteinyl leukotrienes that cause bronchial epithelial damage and bronchoconstriction, respectively.11,12 Therefore, the aim of this study was to establish if a hyperosmolar challenge could trigger activation of eosinophils to release chemokines and lipid mediators, which could promote bronchoconstriction and perpetuate inflammation in the asthmatic airway.
0954-6111/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0954-6111(03)00119-7
ARTICLE IN PRESS Release of inflammatory mediators from eosinophils
Methods Subjects Seven atopic subjects who met the American Thoracic Society criteria for mild asthma, and six non-atopic, non-asthmatic control subjects volunteered to participate in this study. Subject characteristics are displayed in Table 1. Atopy was determined in subjects using a battery of skin prick test antigens with a positive response defined as a grade 2+ reaction (erythema 21–30 mm, papule 5– 10 mm), to at least one antigen. All asthmatic subjects had episodic symptoms of shortness of breath, wheeze, cough or chest tightness controlled by use of as required inhaled bronchodilator therapy. None of the asthmatic subjects had taken oral or inhaled corticosteroid therapy within the 3 months before the study, and none was taking theophylline, cromones or leukotriene modifiers. Approval for this study was obtained from the local Ethics Committee of James Connolly Memorial Hospital, and full written informed consent was obtained from each subject.
Eosinophil purification and stimulation Peripheral blood (70 ml) was collected from each subject into lithium heparinised tubes, and blood was diluted with an equal volume of ice cold phosphate buffered saline (PBS). Isotonic Percoll (15 ml) (density 1.082 g/ml) was layered under
Table 1
929
30 ml aliquots of blood, and centrifuged at 3000 rpm for 30 min at room temperature. Following centrifugation, the upper layer containing mononuclear cells, serum, and Percoll were discarded. The remaining erythrocyte pellet, containing the polymorphonuclear cells (PMN), was suspended in PBS containing 2% heat inactivated fetal calf serum (PBS/FCS). The red cells were then lysed with reverse osmosis sterile water (40 ml for 30 s), and 4 PBS was used to return the cell suspension to isotonicity. Cell lysis was repeated 2– 3 times. The PMN were incubated for 1 h at 41C in 5 ml PBS/FCS with anti-CD16 immunomagnetic microbeads (Miltenyi Biotec, England). Following this, PMN were then layered onto a magnetic cell separation column type CS (Miltenyi Biotec, England), and unlabelled eosinophils were eluted with 45 ml of ice cold PBS/FCS. This procedure resulted in total yields of 6–12 million viable eosinophils as counted with a modified Neubauer hemocytometer (Knittel, Glaser, Germany), with a purity of 98–99% as assessed by Diff-Quik staining and a viability of 99% as assessed by Trypan Blue. Purified eosinophils were suspended at 1 106 cells/ml in RPMI 1640 culture medium supplemented with penicillin (103 U/ml), streptomycin (10 mg/ml), L-glutamine (2 mM), and 10% FCS. Cells were stimulated with calcium ionophore (A23187) 2.5 mM and hyperosmolar mannitol 0.7 M for 15 min, in a humidified incubator at 371C with 5% CO2. Stimulation was then terminated by addition of ice-cold methanol to each well, and supernatants were removed and stored at –201C until analysis.
Subject characteristics of eosinophil donors.
Asthmatic subjects
Age (years)
Sex
Atopy/Allergen
Therapy
1 2 3 4 5 6 7
29 25 21 19 17 35 51
F M M F M F M
HDM HDM HDM HDM HDM HDM HDM
S S S S S S S
Non-asthmatic subjects
Age (years)
Sex
Atopy/Allergen
1 2 3 4 5 6
22 23 33 24 24 45
F M F M F F
2+ 3+ 3+ 2+ 3+, GRASS 2+ 2+, GRASS 2+ 3+, GRASS 3+
Hdm = house dust mite, Grass = grass mix, S = salbutamol, prn = taken as required.
(prn) (prn) (prn) (prn) (prn) (prn) (prn)
Therapy
ARTICLE IN PRESS 930
E.D. Moloney et al.
Analysis of mediators
5000
LTC 4 pg/ml
4000
Leukotriene C4 (LTC4) LTC4 was measured with a commercially available enzyme-immunoassay kit (Cayman Chemical Co, Ann Arbor, MI, USA). The lower limit of detection of this assay is 7.8 pg/ml, and at 50% maximum binding the antibody shows a cross-reactivity of 48% with LTD5, 45% with LTD4, 28% with N-acetyl LTE4, 7% with LTE5, and less than 0.01% with other eicosanoids.
3000 2000 1000 120 90 60 30 0
Unstim
(A)
Mannitol
A23187
4500 4000 3500
ECP µg/L
Eosinophil cationic protein (ECP) ECP was measured with a commercially available radio-immunoassay kit (Pharmacia-Upjohn AC, Uppsala, Sweden). The lower limit of detection of this assay is 2 mg/l, and at 50% maximum binding the antibody shows a cross-reactivity of o0.06% with EPX.
3000 2500 2000 1500 1000 500 0
Data analysis Normally distributed data (as assessed by Kolmogorov-Smirnov) are expressed as group mean 7 SE. Differences between groups were assessed using a one-way analysis of variance and students t-test. Results were considered significant if Po0:05: The statistical calculations were controlled by the use of a validated statistical software package for personal computers (Graph Pad Prism Inc, San Diego, CA, USA).
Results Baseline levels of LTC4 and ECP did not differ between the asthmatic or control group. Following stimulation with calcium ionophore, a significant increase in LTC4 levels above baseline was seen in both asthmatic (P ¼ 0:0005) and control subjects (P ¼ 0:03) (Fig. 1a). Levels of ECP did not significantly increase from baseline in either asthmatic (P ¼ 0:41) or control subjects (P ¼ 0:71) following ionophore stimulation (Fig. 1b). Hyperosmolar stimulation of eosinophils with mannitol, resulted in a significant increase in LTC4 levels compared to baseline in both asthmatic (15.274.6 vs. 70.179.5; P ¼ 0:0002) and control subjects (14.374.0 vs. 55.675.6; P ¼ 0:0001) (Fig. 1a). In contrast, ECP levels did not increase significantly above baseline following mannitol stimulation in either the asthmatic (P ¼ 0:36) or control subjects (P ¼ 0:30) (Fig. 1b).
(B)
Unstim
Mannitol
A23187
Fig. 1 (A) Mean7SE leukotriene C4 (LTC4) levels (pg/ml) from eosinophils isolated from asthmatic (open bars) and non-asthmatic control subjects (filled bars). Following a hyperosmolar stimulation of eosinophils with mannitol, a significant increase in LTC4 is seen compared to unstimulated eosinophils, in both asthmatic (P ¼ 0:0002) and control subjects (P ¼ 0:0001). There is no significant difference between asthmatic and control subjects, in LTC4 levels from unstimulated eosinophils (P=0.9), and from mannitol stimulated eosinophils (P ¼ 0:2). (B) Mean7SE eosinophil cationic protein (ECP) levels (mg/l) from eosinophils isolated from asthmatic (open bars) and non-asthmatic control subjects (filled bars). Following a hyperosmolar stimulation of eosinophils with mannitol, no significant increase in ECP levels was detected compared to unstimulated eosinophils, in both asthmatic (P ¼ 0:4) and control subjects (P ¼ 0:3).
Discussion This study shows that hyperosmolar stimulation of eosinophils resulted in the release of LTC4. In the trachea the osmolarity of secretions has been demonstrated to be 360 mOsm/kg H2O.9 Anderson et al.13 suggested that after exercise bronchial secretions may result in an osmolarity of 900 mOsm/kg H2O. Basophils and lung mast cells are activated by hyperosmolar mannitol, maximal histamine release from basophils was achieved at 1050 mOsm/kg, whereas mast cells released maximally at 700–750 mOsm/kg. In the current study, purified eosinophils were stimulated with
ARTICLE IN PRESS Release of inflammatory mediators from eosinophils
hyperosmolar (0.7 M) mannitol (equivalent to approx. 950 mOsm/kg) to try to mimic the conditions in vitro, that may occur in vivo after exercise. Epithelial cells have previously been stimulated with similar molar concentrations of mannitol, without any evidence of cytotoxicity to cells as assessed by LDH expression.14 Cells were stimulated for 15 min to avoid hyperosmolar-induced cytotoxicity, which might have occurred with a longer incubation period. We found that LTC4 generation following hyperosmolar stimulation was not a unique characteristic of eosinophils from asthmatic subjects only. Hodges et al.15 previously showed that eosinophils from asthmatic subjects, when stimulated with calcium ionophore, have a decreased capacity for LTC4 synthesis compared with eosinophils of similar density isolated from normal subjects. It is generally accepted that the magnitude of EIB is strongly correlated with the underlying degree of airway hyperresponsiveness.16 Therefore, it is tempting to speculate that the underlying airway hyperresponsiveness of asthmatic subjects causes them to be more ‘‘primed’’ to the effects of LTC4 and other bronchoconstricting mediators, compared to normal subjects. One of the consequences of airway inflammation in asthma is thought to be desquamation of airway epithelial cells resulting from the action of inflammatory mediators such as eosinophil granule proteins.17 However, it has previously been suggested that ECP is not related to bronchial hyperreactivity.18 It is possible that we may have missed the peak effect of ECP release at 15 min, as it has previously been shown that eosinophil degranulation peaks after 10 min incubation with calcium ionophore.19 Moreover, Takafugi and colleagues demonstrated that neither TNF or GM-CSF could induce ECP release from human eosinophils,20 and eosinophils from atopic asthmatics did not release ECP after incubation with specific allergen or with anti-IgE monoclonal antibodies.21 To the best of our knowledge purified eosinophils have not been subjected to a hyperosmolar challenge. This is surprising given that eosinophils are one of the primary sources of the potent bronchoconstricting cysteinyl-leukotrienes.22 Togias and colleagues have shown increased LTC4 in nasal lavage following a cold dry air nasal challenge.10 The osmolarity of the nasal secretions was increased in those subjects sensitive to cold dry air. Nasal lavage collected from 15 healthy volunteers following a nasal challenge with hyperosmolar mannitol (869 mOsm/kg) contained significantly higher quantities, as compared to baseline, of immunoreactive leukotrienes LTD4/LTE4.23 In vitro
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work with hyperosmolar stimulated basophils and mast cells have yielded confounding results. Findlay et al. investigated basophil cell supernatants for production of cysteinyl leukotrienes following stimulation with hyperosmolar solutions, but could not document an increase in the cysteinyl leukotriene production.8 It should be noted however, that guinea pig ileal contractions were used to assay the cysteinyl leukotrienes with a comparatively poor sensitivity compared to present day methodology. A study using the more sensitive radioimmunoassay did detect a non-significant doubling in the production of LTC4/LTD4 from isolated basophils stimulated with hyperosmolar mannitol.24 In the same study, the investigators did not however observe an increase in LTC4/LTD4 from human lung mast cells following hyperosmolar challenge. Cells from chopped nasal polyps containing epithelial and mononuclear cells as well as eosinophils and neutrophils were stimulated to produce increased levels of 15-hydroxyeicosatetranoic acid and leukotriene B4 following incubation with hyperosmolar buffer solution.14 Unfortunately, levels of LTC4 were not assayed. In summary, our study demonstrates that eosinophils can be activated by a hyperosmolar stimulus to release LTC4. Therefore it seems reasonable to suggest that eosinophils could contribute to EIB.
References 1. Chen WY, Horton DJ. Heat and water loss from the airways and exercise-induced asthma. Respiration 1977;34:305–13. 2. Strauss RH, McFadden Jr. ER, Ingram RH, Jaeger JJ. Enhancement of exercise-induced asthma by cold air. N Engl J Med 1977;297:743–7. 3. Deal Jr. EC, McFadden Jr. ER, Ingram Jr. RH, Jaeger JJ. Hyperpnea and heat flux: initial reaction sequence in exercise-induced asthma. J Appl Physiol 1979;46:476–83. 4. Anderson SD, Schoeffel RE, Follet R, Perry CP, Daviskas E, Kendall M. Sensitivity to heat and water loss at rest and during exercise in asthmatic patients. Eur J Respir Dis 1982;63:459–71. 5. Smith CM, Anderson SD. Hyperosmolarity as the stimulus to asthma induced by hyperventilation. J Allergy Clin Immunol 1986;77:729–36. 6. Anderson SD, Daviskas E, Smith CM. Exercise-induced asthma: a difference in opinion regarding stimulus. Allergy Proc 1989;10:215–26. 7. Anderson SD. Is there a unifying hypothesis for exerciseinduced asthma? J Allergy Clin Immunol 1984;73:660–5. 8. Findlay SR, Dvorak AM, Kagey-Sobotka A, Lichtenstein LM. Hyperosmolar triggering of histamine release from human basophils. J Clin Invest 1981;67:1604–13. 9. Eggleston PA, Kagey-Sobotka A, Schleimer RP, Lichtenstein LM. Interaction between hyperosmolar and IgE-mediated histamine release from basophils and mast cells. Am Rev Respir Dis 1984;130:86–91.
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10. Togias AG, Naclerio RM, Proud D, et al. Nasal challenge with cold, dry air results in release of inflammatory mediators: possible mast cell involvement. J Clin Invest 1985;76:1375– 81. 11. Motojima S, Frigas E, Loegering DA, Gleich GJ. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am Rev Respir Dis 1989;139:801–5. 12. Dahlen SE, Hedqvist P, Hammarstrom S, Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature 1980;288:484–6. 13. Anderson SD, Daviskas E. The airway microvasculature and exercise-induced asthma. Thorax 1992;47:748–52. 14. Souques F, Crampette L, Mondain M, et al. Stimulation of dispersed nasal polyp cells by hyperosmolar solutions. J Allergy Clin Immunol 1995;96:980–5. 15. Hodges MK, Weller PF, Gerard NP, Ackerman SJ, Drazen JM. Heterogeneity of leukotriene C4 production by eosinophils from asthmatic and from normal subjects. Am Rev Respir Dis 1988;138:799–804. 16. Anderton RC, Cuff MT, Frith PA, et al. Bronchial responsiveness to inhaled histamine and exercise. J Allergy Clin Immunol 1979;63:315–20. 17. Frigas E, Motojima S, Gleich GJ. The eosinophilic injury to the mucosa of the airways in the pathogenesis of bronchial asthma. Eur Respir J 1991;13:123s–35s.
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18. Ferdousi HA, Dreborg S. Asthma, bronchial hyperreactivity and mediator release in children with birch pollinosis. ECP and EPX levels are not related to bronchial hyperreactivity. Clin Exp Allergy 1997;27:530–9. 19. Kroegel C, Yukawa T, Dent G, Venge P, Chung KF, Barnes PJ. Stimulation of degranulation from human eosinophils by platlet-activating factor. J Immunol 1989;142:3518–26. 20. Takafugi S, Tadokoro K, Ito K, Nakagawa T. Release of granule proteins from human eosinophils stimulated with mast cell mediators. Allergy 1998;53:951–6. 21. Tomassini M, Tsicopoulos A, Tai PC, et al. Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation. J Allergy Clin Immunol 1991;88:365–75. 22. Laviolette M, Ferland C, Comtois JF, Champagne K, Bosse M, Boulet LP. Blood eosinophil leukotriene C4 production in asthma of different severities. Eur Respir J 1995;8:1465–72. 23. Silber G, Proud D, Warner J, et al. In vivo release of inflammatory mediators by hyperosmolar solutions. Am Rev Respir Dis 1988;137:606–12. 24. Eggleston PA, Kagey-Sobotka A, Proud D, Adkinson Jr. NF, Lichtenstein LM. Dissociation of the release of histamine and arachidonic acid metabolites from osmotically activated basophils and human lung mast cells. Am Rev Respir Dis 1990;141:960–4.