- Email: [email protected]
Heparin attenuates symptoms and mast cell degranulation induced by AMP nasal provocation
Heparin attenuates symptoms and mast cell degranulation induced by AMP nasal provocation Dewan Zeng, PhD,a Gaetano Prosperini, MD,b Cristina Russo, MD,b Lucia Spicuzza, MD,b Rosella R. Cacciola, MD, PhD,c Guiseppe U. Di Maria, MD,b and Riccardo Polosa, MD, PhDb Palo Alto, Calif, and Catania, Italy Mechanisms of asthma and allergic inflammation
Background: Previous studies have shown that inhaled heparin attenuated the airway responses to allergen, exercise, and AMP bronchial provocation, possibly through an inhibition of mast cell activation. Objective: The aim of this study was to provide the evidence of in vivo inhibition of human mast cell activation by heparin in a noninvasive model. Methods: Nine atopic and 6 nonatopic subjects received placebo and unfractionated heparin sodium (5000 IU/mL) 15 minutes before an AMP nasal provocation in a double-blind crossover study design. The nasal lavage was collected from these subjects before or 3, 5, 15, or 30 minutes after the AMP nasal challenge, and concentrations of histamine and tryptase in the nasal lavage were measured. Results: AMP nasal provocation produced considerable sneezing and induced a transient increase in histamine and tryptase release, with peak values achieved at 3 to 5 minutes after the challenge in all atopic subjects. Compared with placebo, inhaled heparin significantly attenuated the release of histamine and tryptase induced by AMP challenge (P = .012 and .004, respectively). Moreover, the AMP-induced sneezing was also inhibited by pretreatment with heparin (P = .016). In nonatopic subjects, AMP did not induce a significant increase in histamine and tryptase release on placebo-treated or heparin-treated days. Conclusion: These data suggest that AMP nasal provocation and AMP bronchial provocation cause mast cell mediator release in a similar fashion. In addition, the data support the hypothesis that inhaled heparin plays a protective role against AMP provocation by inhibition of mast cell activation. (J Allergy Clin Immunol 2004;114:316-20.) Key words: Heparin, allergic rhinitis, histamine, tryptase, adenosine challenge
From athe Department of Drug Research and Pharmacological Sciences, CV Therapeutics, Inc, Palo Alto; and bDipartimento di Medicina Interna e Specialistica and cDipartimento di Scienze Biomediche—Sezione Ematologia, University of Catania. Supported in part by a research grant from the University of Catania (grant, 60%). Disclosure of potential conflict of interest: Dr Zeng is an employee of CV Therapeutics, Inc. All other authors are employed by the University of Catania and have no conflicts to declare. Received for publication January 22, 2004; revised April 30, 2004; accepted for publication May 4, 2004. Reprint requests: Riccardo Polosa, MD, PhD, Dipartimento di Medicina Interna e Specialistica, University of Catania, Via Passo Gravina, 187, 95125 Catania, Italy. E-mail: [email protected]. 0091-6749/$30.00 Ó 2004 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2004.05.026
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Abbreviation used NLF: Nasal lavage fluid
Heparin is a highly sulfated unbranched glycosaminoglycan. In addition to its well established anticoagulant properties, it exerts several nonanticoagulant properties, including modulation of various proteases,1,2 inhibition of cell growth,3,4 and attenuation of inflammatory responses.5,6 In experimental models of clinical asthma, it has been reported that heparin given by inhalation inhibits the bronchoconstrictor response to a variety of stimuli, including allergen,7 exercise,8 and adenosine.9 The mechanism of action for this protective effect of heparin is not known. Page10 has speculated that the inhibitory effect of inhaled heparin in the asthmatic airways may be a result of its suppressive action on mast cell degranulation. Lucio et al11 have shown that heparin inhibits anti–IgE-dependent histamine release from human uterine mast cells. In addition, because a major component of the bronchoconstriction produced by allergen, exercise, and adenosine in asthma is dependent on mediator release from airway mast cells,12-14 it is likely that inhaled heparin might exert its effect via an inhibitory modulation of mast cell functions. However, there is no direct in vivo evidence in human beings that heparin protects against mast cell degranulation. Adenosine is known to stimulate the cell surface A2B adenosine receptors expressed on airway mast cells with subsequent release of preformed and newly formed mediators in allergic individuals.14,15 In support of this view, we have recently shown that nasal provocation with the purine nucleotide AMP elicits a substantial increase in symptom scores in subjects with allergic rhinitis in association with an immediate rise in histamine level in their nasal lavage fluid (NLF).16 By using nasal provocation with AMP as a possible in vivo model of airway mast cell mediator release in allergic individuals, we sought to demonstrate that the protective effect of heparin is likely to be ascribed to an inhibition in mast cell degranulation by measuring histamine and tryptase levels in the NLF of individuals with allergic rhinitis who were pretreated with topically applied heparin.
METHODS Subjects We studied 9 atopic subjects (6 male subjects; age range, 18-35 years) with seasonal allergic rhinitis and 6 nonatopic subjects (4 male subjects; age range, 21-31 years). All atopic subjects reported typical hay fever symptoms in April and May and a positive skin prick test response and specific IgE of class 2 or more to either grass pollen or Parietaria judaica (Pharmacia CAP System; Pharmacia Diagnostics AB, Uppsala, Sweden). The nonatopic controls had no previous history of allergic symptoms, and skin tests were negative. None of the subjects studied was taking any form of medication at the time of the study, and none had ever received specific immunotherapy. No subject was admitted to the study within 6 weeks of a respiratory tract infection. None of the subjects studied had nasal polyposis, chronic nasal obstruction, or other serious illness. Subjects were informed about the purpose of the investigation, and all signed forms indicating their consent to participate. The study protocol was approved by the local ethics committee.
Study design Subjects attended on 2 separate occasions, at least 1 week apart, to receive either 5000 IU/mL (5 mL) unfractionated heparin sodium administered as an aerosol (heparin sodium; Bristol-Myers Squibb SpA, Anagni, Italy) or normal saline (placebo) before nasal challenge with AMP (6.5 mg administered as a single dose) in a randomized, double-blind, placebo-controlled study design. The dosage of heparin used in this investigation and the timing of administration before challenge were chosen on the basis of previous studies that have been shown to reduce the bronchospastic response to AMP effectively in subjects with asthma.9 Treatment solutions were freshly prepared on each visit by a technician who was blind to the study rationale and were aerosolized from a nebulizer (DeVilbiss 646; Somerset, Pa) connected to a nasal adaptor. At each study visit, sequential prechallenge lavages were performed bilaterally 3 times before administration of the study treatments to reduce the baseline level of histamine. Further nasal lavages were performed at 3, 5, 10, 15, and 30 minutes after AMP challenges to evaluate changes in histamine and tryptase levels. The numbers of sneezes were counted for as long as 3 minutes after AMP challenge. Nasal blockage and rhinorrhea could not be recorded in a reliable way because of the nasal lavage procedure. All visits to the laboratory occurred at the same time of day and outside the pollen season.
AMP nasal challenge Nasal challenges with AMP were performed in the morning, 15 minutes after nebulization of placebo or heparin. AMP (Sigma Chemicals, St Louis, Mo) was dissolved in 0.9% wt/vol normal saline to achieve a concentration of 50 mg/mL, divided into small aliquots, and frozen at ÿ208C. Saline and AMP solutions for nasal challenge were kept at room temperature for 1 hour before provocation. AMP was sprayed by using a hand-held pump spray calibrated to deliver 130 ( ± 9) lL per puff. The pump spray was placed in 1 of the nostrils while the contralateral nostril was occluded, and was activated once during quiet inspiration. The procedure was then repeated in the opposite nostril. At the concentration used, this method delivered a total dose of approximately 6.5 mg AMP equally divided between the 2 nostrils. This dose of 6.5 mg was chosen on the basis of our previous dose-response studies with AMP in subjects with rhinitis; this dose elicited nasal symptoms in all of the subjects studied.16
Nasal lavage and sample processing The lavage technique has been previously described in detail.16 In brief, each subject was instructed to tilt his neck backwards, to hold
his breath, and to refrain from swallowing. Prewarmed normal saline (2.5 mL/nostril) was then instilled into each nostril, and after 10 seconds, the subject flexed his neck and expelled the mixture of mucus and saline into a conical polypropylene tube placed on ice. Samples were immediately filtered to remove mucus and centrifuged at 400g for 10 minutes at 48C. The supernatant was separated from the cell pellet by pipetting, concentrated in centrifugal filter units (Amicon Ultra-15; Millipore, Watford, United Kingdom), divided into aliquot parts in Eppendorf tubes, and stored at ÿ708C for subsequent analysis. The recovery of NLF was consistent among subjects. The volume of original NLF was 4 mL, and the volume of concentrated NFL was 0.3 mL; thus, the concentration factor was 13-fold.
Levels of histamine and tryptase in nasal lavage fluid Histamine level in concentrated nasal lavage fluid was assayed by using a commercially available histamine ELISA kit (Immuno Biological Laboratories, Hamburg, Germany), whereas tryptase was measured by a fluorescence immunoassay method (UniCap; Pharmacia Diagnostics AB, Uppsala, Sweden). Samples were assayed according to the instructions of the manufacturers. The lowest detectable levels for histamine and tryptase were 0.5 ng/mL and 1.0 ng/mL, respectively. In addition, standard curves for both heparin and tryptase were performed in the presence and absence of heparin (5000 IU/mL); coefficients of variation between measured optical densities in the presence and absence of heparin were within 10% (within the experiment error) throughout the entire calibration curves. Thus, heparin sodium (final dose, 5000 IU/mL) did not interfere with the immunoassay measurement of histamine or tryptase in the lavage fluid.
Analyses of data Data shown are mean ± SEM values of all subjects in each group. Numbers of sneezes between allergic and nonallergic subjects were compared by using the t test. Numbers of sneezes and the basal and peak values of histamine and tryptase concentrations between placebo and heparin-treated days were compared by using the Wilcoxon signed rank test. A P value < .05 was considered significant. All statistical analyses were performed by using SigmaStat 2.03 (SAS Institute, Cary, NC).
RESULTS Sneeze count Nasal provocation with AMP produced considerable sneezing in subjects with atopic but not in nonatopic subjects (Table I). The numbers of sneezes were 4.4 ± 0.7 and 1.2 ± 0.5 for atopic and nonatopic subjects, respectively (P = .006). The AMP-induced sneezing in atopic subjects was significantly reduced by pretreatment with heparin. The numbers of sneezes were 4.4 ± 0.7 and 2.3 ± 0.6 on placebo-treated and heparin-treated days, respectively (P = .016; Table I). Histamine levels in nasal lavage fluid In atopic subjects (n = 9) treated with placebo, nasal provocation with AMP elicited a significant rise in histamine levels, with a peak response at 3 minutes (4.1fold ± 0.6-fold greater than basal in mean values; P = .004). In the same subjects, pretreatment with heparin significantly reduced the peak increase in histamine levels induced by AMP challenge (P = .012) without affecting
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Mechanisms of asthma and allergic inflammation FIG 1. A, Time course of histamine concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in atopic subjects. Data shown are means ± SEMs of histamine concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in histamine concentrations (bottom graph). B, Time course of histamine concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in nonatopic subjects. Data shown are means ± SEMs of histamine concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in histamine concentrations (bottom graph).
TABLE I. Effect of pretreatment with heparin or placebo on AMP-induced sneezing in the atopic and non-atopic subjects* Atopic subjects Subject no.
1 2 3 4 5 6 7 8 9 Mean SEM
Nonatopic subjects
Placebo
Heparin
Subject no.
8 4 5 3 7 2 6 3 2 4.4 0.7
4 3 2 0 5 2 3 0 2 2.3 0.6
1 2 3 4 5 6
Mean SEM
Placebo
Heparin
2 0 2 3 0 0
0 2 2 2 0 0
1.2 0.5
1.0 0.4
*Data shown are numbers of sneezes after AMP nasal challenge.
the basal histamine levels (Fig 1, A). The baseline histamine levels were 5.0 ± 1.0 ng/mL and 4.1 ± 0.7 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). The peak histamine levels at 3 minutes after nasal challenge were 20.3 ± 4.5 ng/mL and
7.5 ± 1.0 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). In contrast with atopic subjects, in nonatopic subjects (n = 6), AMP nasal challenge did not affect the histamine levels in nasal lavage fluid on placebo-treated or heparin-treated days (Fig 1, B).
Tryptase levels in nasal lavage fluid Similarly, in atopic subjects treated with placebo, nasal provocation with AMP elicited a significant rise in tryptase levels, with a peak response at 3 minutes (3.7fold ± 0.7-fold greater than basal in mean values; P = .004). In the same subjects, pretreatment with heparin significantly reduced the peak increase in tryptase levels induced by AMP challenge (P = .004) without affecting the basal tryptase levels (Fig 2, A). The baseline tryptase levels were 2.3 ± 03 ng/mL and 1.9 ± 0.2 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). The peak tryptase levels at 3 minutes after nasal challenge were 7.9 ± 1.3 ng/mL and 2.8 ± 0.3 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). In contrast with atopic subjects, in nonatopic subjects (n = 6), AMP nasal challenge did not affect the tryptase levels in nasal lavage fluid on placebo-treated or heparin-treated days (Fig 2, B).
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FIG 2. A, Time course of tryptase concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in atopic subjects. Data shown are means ± SEMs of tryptase concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in tryptase concentrations (bottom graph). B, Time course of tryptase concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in nonatopic subjects. Data shown are means ± SEMs of tryptase concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in tryptase concentrations (bottom graph).
DISCUSSION In the current study, we have extended our previous observations that nasal provocation with AMP provokes nasal symptoms and mast cell degranulation in subjects with allergic rhinitis.16,17 We have also shown that administration of topical heparin reduces sneezing and inhibits the release of histamine and tryptase in the nasal lavage fluid of allergic subjects challenged with AMP. Topical heparin reduced AMP-induced sneezing in almost all of the atopic subjects studied. This finding is in agreement with a recent study in which allergen-induced nasal symptoms were significantly attenuated when patients received topical heparin.18 Taken together, these findings indicate that aerosol heparin and related molecules may have beneficial effects in alleviating symptoms of allergic rhinitis, but larger studies are needed to confirm our observations and to define the characteristics of the patients who would benefit most from such a therapeutic approach. Although the mode of action of AMP in provoking symptoms in allergic rhinitis is not known with certainty, stimulation of specific cell surface A2B adenosine receptors expressed on airway mast cells with subsequent degranulation is a possibility.14,15 In vitro, adenosine is
known to enhance mediator release from immunologically primed human airway mast cells.19,20 These in vitro data are compatible with the results of the current study in vivo, which clearly show that adenosine induced a marked increase in histamine and tryptase levels in all of the atopic subjects studied. These changes in mediator levels were associated with considerable sneezing. Likewise, the airways of allergic asthmatics are known to respond to endobronchial instillation of AMP with bronchial narrowing, and this is coupled with a rise in histamine levels in the bronchoalveolar lavage fluid.21 Thus, potentiation of mast cell mediator release may account for the nasal symptoms provoked by nasal challenge with AMP in allergic rhinitis. Previous studies in allergic sheep model indicated that fractionated low-molecular-weight heparins attenuate antigen-induced acute bronchoconstrictor response and airway hyperresponsiveness, and there is an inverse relationship between the antiallergic activity of heparin fractions and molecular weight.22 On the other hand, inhaled unfractionated heparin and low-molecular-weight heparins inhibited antigen-induced histamine release in bronchoalveolar lavage fluid by 81% and 75%; thus, the inhibitory effects of unfractionated heparin and fractionated heparins on histamine release are not strictly
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molecular weight–dependent.23 In our current study, the unfractionated heparin was used mainly because we have shown previously that inhaled unfractionated heparin attenuated the airway response to AMP bronchial provocation.9 The mechanism by which heparin attenuates the nasal response to AMP and allergen is unclear and requires additional studies. In asthma, inhaled heparin seems to exert a preferential protective effect against stimuli such as allergen,7 exercise,8 and AMP9 that are known to cause bronchoconstriction mainly by releasing spasmogenic mediators from airway mast cells. Hence, the antiallergic actions of heparin may be related to the inhibition of mediator release from mast cells. In support of this view, we have demonstrated for the first time that administration of topical heparin inhibited the release of histamine and tryptase in the nasal lavage fluid of almost all of the allergic subjects challenged with AMP. Indeed, the ability of heparin to prevent the release of mast cell mediators such as histamine and tryptase might explain the substantial reduction in the number of sneezes observed immediately after AMP challenge. Endogenous heparin, a highly anionic molecule, has the ability to bind mast cell mediators, and thus it is possible that heparin administered by inhalation may cause a reduction in the detected levels of histamine and tryptase in the nasal washes merely because of its negative charge density. However, our pilot experiments demonstrated that heparin did not interfere with the immunoassay measurement of spiked histamine and tryptase in lavage fluid samples, and this suggested that direct binding between heparin and mediators is unlikely to provide an explanation for the protective effect of heparin. The precise cellular mechanism of action for the inhibitory effect of heparin on mast cell mediator release is not completely understood. It is not clear whether the effect of heparin is caused by its binding to receptors or adhesion molecules on the surface of mast cells directly or by its binding to cytokines or chemokines or other inflammatory cells in the airway. Future studies using purified mast cells and other inflammatory cells from human airways may help advance our understanding of the cellular mechanism of action for inhaled heparin. In conclusion, topical heparin has been shown to inhibit AMP-induced responses in allergic rhinitis. Taking into account the notion of airway mast cell mediators contributing to the nasal response of AMP, the current findings support the hypothesis that inhaled heparin plays a protective role, possibly by inhibition of mast cell activation and subsequent release of mast cell mediators. Further work is required to clarify the molecular mechanism by which heparin produces this novel anti-inflammatory activity and to determine whether this has any relevance to a possible therapeutic benefit in allergic rhinitis. REFERENCES 1. Schwartz LB, Bradford TR. Regulation of tryptase from human lung mast cells by heparin. J Biol Chem 1986;261:7372-9.
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2. Sayama S, Iozzo RV, Lazarus GS, Schecter NM. Human skin chymotrypsin like proteinase chymase: subcellular localization to mast cell granules and interaction with heparin and other glycosaminoglycans. J Biol Chem 1987;262:6808-15. 3. Castellot JJ, Cochran DL, Karnovsky MM. Effect of heparin on vascular smooth muscle cell, I: cell metabolism. J Cell Physiol 1985;124: 21-8. 4. Wright TC, Cochran DL, Karnovsky MJ. Inhibition of rat cervical epithelial cell growth by heparin and its reversal by EGF. J Cell Physiol 1985;125:499-506. 5. Roemisch J, Gray E, Hoffmann JN, Wiedermann CJ. Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagul Fibrinolysis 2002;13:657-70. 6. Okajima K. Regulation of inflammatory responses by natural anticoagulants. Immunol Rev 2001;184:258-74. 7. Bowler SD, Smith SM, Lavercombe PS. Heparin inhibits the immediate response to antigen in the skin and lungs of allergic subjects. Am Rev Respir Dis 1993;147:160-3. 8. Ahmed T, Garrigo J, Danta I. Preventing bronchoconstriction in exercise-induced asthma with inhaled heparin. N Engl J Med 1993; 329:90-5. 9. Polosa R, Magri S, Vancheri C, Armato F, Santonocito G, Mistretta A, et al. Time course of changes in adenosine 59-monophosphate airway responsiveness with inhaled heparin in allergic asthma. J Allergy Clin Immunol 1997;99:338-44. 10. Page C. The role of proteoglycans in the regulation of airways inflammation and airways remodelling. J Allergy Clin Immunol 2000;105: S518-21. 11. Lucio J, D’Brot J, Guo CB, Abraham WB, Lichtenstein LM, Kagey-Sobotka A, et al. Immunologic mast cell-mediated responses and histamine release are attenuated by heparin. J Appl Physiol 1992;73: 1093-101. 12. Lee TH, Assoufi BK, Kay AB. The link between exercise, respiratory heat exchange and the mast cell in bronchial asthma. Lancet 1983;1: 520-2. 13. Howarth PH, Durham SR, Lee TH, Kay AB, Church MK, Holgate ST. Influence of albuterol, cromolyn sodium and ipratropium bromide on the airways and circulating mediator responses to antigen bronchial provocation in asthma. Am Rev Respir Dis 1985;132:986-92. 14. Spicuzza L, Bonfiglio C, Polosa R. Research applications and implications of adenosine in diseased airways. Trends Pharmacol Sci 2003;24:409-13. 15. Polosa R. Adenosine-receptor subtypes: their relevance to adenosinemediated responses in asthma and chronic obstructive pulmonary disease. Eur Respir J 2002;20:488-96. 16. Polosa R, Pagano C, Prosperini G, Low JL, Dokic D, Church MK, et al. Histamine release upon adenosine 59-monophosphate (AMP) nasal provocation in allergic subjects. Thorax 1999;54:230-3. 17. Crimi N, Polosa R, Mistretta A. Mechanisms of the effector function of adenosine in asthma and rhinitis. Drug Dev Res 1993;28:322-7. 18. Vancheri C, Mastruzzo C, Armato F, Tomaselli V, Magri S, Pistorio MP, et al. Intranasal heparin reduces eosinophil recruitment after nasal allergen challenge in patients with allergic rhinitis. J Allergy Clin Immunol 2001;108:703-8. 19. Hughes PJ, Holgate ST, Church MK. Adenosine inhibits and potentiates IgE-dependent histamine release from human lung mast cells by an A2-purinoceptor mediated mechanism. Biochem Pharmacol 1984;33: 3847-52. 20. Peachell PT, Columbo M, Kagey-Sobotka A, Lichtenstein LM, Marone G. Adenosine potentiates mediator release from human lung mast cells. Am Rev Respir Dis 1988;138:1143-51. 21. Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK, et al. Release of mast cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med 1995;151:624-9. 22. Martinez-Salas J, Mendelssohn R, Abraham WM, Hsiao B, Ahmed T. Inhibition of allergic airway responses by inhaled low-molecularweight heparins: molecular-weight dependence. J Appl Physiol 1998; 84:222-8. 23. Molinari JF, Campo C, Shakir S, Ahmed T. Inhibition of antigeninduced airway hyperresponsiveness by ultralow molecular-weight heparin. Am J Respir Crit Care Med 1998;157:887-93.
Background: Previous studies have shown that inhaled heparin attenuated the airway responses to allergen, exercise, and AMP bronchial provocation, possibly through an inhibition of mast cell activation. Objective: The aim of this study was to provide the evidence of in vivo inhibition of human mast cell activation by heparin in a noninvasive model. Methods: Nine atopic and 6 nonatopic subjects received placebo and unfractionated heparin sodium (5000 IU/mL) 15 minutes before an AMP nasal provocation in a double-blind crossover study design. The nasal lavage was collected from these subjects before or 3, 5, 15, or 30 minutes after the AMP nasal challenge, and concentrations of histamine and tryptase in the nasal lavage were measured. Results: AMP nasal provocation produced considerable sneezing and induced a transient increase in histamine and tryptase release, with peak values achieved at 3 to 5 minutes after the challenge in all atopic subjects. Compared with placebo, inhaled heparin significantly attenuated the release of histamine and tryptase induced by AMP challenge (P = .012 and .004, respectively). Moreover, the AMP-induced sneezing was also inhibited by pretreatment with heparin (P = .016). In nonatopic subjects, AMP did not induce a significant increase in histamine and tryptase release on placebo-treated or heparin-treated days. Conclusion: These data suggest that AMP nasal provocation and AMP bronchial provocation cause mast cell mediator release in a similar fashion. In addition, the data support the hypothesis that inhaled heparin plays a protective role against AMP provocation by inhibition of mast cell activation. (J Allergy Clin Immunol 2004;114:316-20.) Key words: Heparin, allergic rhinitis, histamine, tryptase, adenosine challenge
From athe Department of Drug Research and Pharmacological Sciences, CV Therapeutics, Inc, Palo Alto; and bDipartimento di Medicina Interna e Specialistica and cDipartimento di Scienze Biomediche—Sezione Ematologia, University of Catania. Supported in part by a research grant from the University of Catania (grant, 60%). Disclosure of potential conflict of interest: Dr Zeng is an employee of CV Therapeutics, Inc. All other authors are employed by the University of Catania and have no conflicts to declare. Received for publication January 22, 2004; revised April 30, 2004; accepted for publication May 4, 2004. Reprint requests: Riccardo Polosa, MD, PhD, Dipartimento di Medicina Interna e Specialistica, University of Catania, Via Passo Gravina, 187, 95125 Catania, Italy. E-mail: [email protected]. 0091-6749/$30.00 Ó 2004 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2004.05.026
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Abbreviation used NLF: Nasal lavage fluid
Heparin is a highly sulfated unbranched glycosaminoglycan. In addition to its well established anticoagulant properties, it exerts several nonanticoagulant properties, including modulation of various proteases,1,2 inhibition of cell growth,3,4 and attenuation of inflammatory responses.5,6 In experimental models of clinical asthma, it has been reported that heparin given by inhalation inhibits the bronchoconstrictor response to a variety of stimuli, including allergen,7 exercise,8 and adenosine.9 The mechanism of action for this protective effect of heparin is not known. Page10 has speculated that the inhibitory effect of inhaled heparin in the asthmatic airways may be a result of its suppressive action on mast cell degranulation. Lucio et al11 have shown that heparin inhibits anti–IgE-dependent histamine release from human uterine mast cells. In addition, because a major component of the bronchoconstriction produced by allergen, exercise, and adenosine in asthma is dependent on mediator release from airway mast cells,12-14 it is likely that inhaled heparin might exert its effect via an inhibitory modulation of mast cell functions. However, there is no direct in vivo evidence in human beings that heparin protects against mast cell degranulation. Adenosine is known to stimulate the cell surface A2B adenosine receptors expressed on airway mast cells with subsequent release of preformed and newly formed mediators in allergic individuals.14,15 In support of this view, we have recently shown that nasal provocation with the purine nucleotide AMP elicits a substantial increase in symptom scores in subjects with allergic rhinitis in association with an immediate rise in histamine level in their nasal lavage fluid (NLF).16 By using nasal provocation with AMP as a possible in vivo model of airway mast cell mediator release in allergic individuals, we sought to demonstrate that the protective effect of heparin is likely to be ascribed to an inhibition in mast cell degranulation by measuring histamine and tryptase levels in the NLF of individuals with allergic rhinitis who were pretreated with topically applied heparin.
METHODS Subjects We studied 9 atopic subjects (6 male subjects; age range, 18-35 years) with seasonal allergic rhinitis and 6 nonatopic subjects (4 male subjects; age range, 21-31 years). All atopic subjects reported typical hay fever symptoms in April and May and a positive skin prick test response and specific IgE of class 2 or more to either grass pollen or Parietaria judaica (Pharmacia CAP System; Pharmacia Diagnostics AB, Uppsala, Sweden). The nonatopic controls had no previous history of allergic symptoms, and skin tests were negative. None of the subjects studied was taking any form of medication at the time of the study, and none had ever received specific immunotherapy. No subject was admitted to the study within 6 weeks of a respiratory tract infection. None of the subjects studied had nasal polyposis, chronic nasal obstruction, or other serious illness. Subjects were informed about the purpose of the investigation, and all signed forms indicating their consent to participate. The study protocol was approved by the local ethics committee.
Study design Subjects attended on 2 separate occasions, at least 1 week apart, to receive either 5000 IU/mL (5 mL) unfractionated heparin sodium administered as an aerosol (heparin sodium; Bristol-Myers Squibb SpA, Anagni, Italy) or normal saline (placebo) before nasal challenge with AMP (6.5 mg administered as a single dose) in a randomized, double-blind, placebo-controlled study design. The dosage of heparin used in this investigation and the timing of administration before challenge were chosen on the basis of previous studies that have been shown to reduce the bronchospastic response to AMP effectively in subjects with asthma.9 Treatment solutions were freshly prepared on each visit by a technician who was blind to the study rationale and were aerosolized from a nebulizer (DeVilbiss 646; Somerset, Pa) connected to a nasal adaptor. At each study visit, sequential prechallenge lavages were performed bilaterally 3 times before administration of the study treatments to reduce the baseline level of histamine. Further nasal lavages were performed at 3, 5, 10, 15, and 30 minutes after AMP challenges to evaluate changes in histamine and tryptase levels. The numbers of sneezes were counted for as long as 3 minutes after AMP challenge. Nasal blockage and rhinorrhea could not be recorded in a reliable way because of the nasal lavage procedure. All visits to the laboratory occurred at the same time of day and outside the pollen season.
AMP nasal challenge Nasal challenges with AMP were performed in the morning, 15 minutes after nebulization of placebo or heparin. AMP (Sigma Chemicals, St Louis, Mo) was dissolved in 0.9% wt/vol normal saline to achieve a concentration of 50 mg/mL, divided into small aliquots, and frozen at ÿ208C. Saline and AMP solutions for nasal challenge were kept at room temperature for 1 hour before provocation. AMP was sprayed by using a hand-held pump spray calibrated to deliver 130 ( ± 9) lL per puff. The pump spray was placed in 1 of the nostrils while the contralateral nostril was occluded, and was activated once during quiet inspiration. The procedure was then repeated in the opposite nostril. At the concentration used, this method delivered a total dose of approximately 6.5 mg AMP equally divided between the 2 nostrils. This dose of 6.5 mg was chosen on the basis of our previous dose-response studies with AMP in subjects with rhinitis; this dose elicited nasal symptoms in all of the subjects studied.16
Nasal lavage and sample processing The lavage technique has been previously described in detail.16 In brief, each subject was instructed to tilt his neck backwards, to hold
his breath, and to refrain from swallowing. Prewarmed normal saline (2.5 mL/nostril) was then instilled into each nostril, and after 10 seconds, the subject flexed his neck and expelled the mixture of mucus and saline into a conical polypropylene tube placed on ice. Samples were immediately filtered to remove mucus and centrifuged at 400g for 10 minutes at 48C. The supernatant was separated from the cell pellet by pipetting, concentrated in centrifugal filter units (Amicon Ultra-15; Millipore, Watford, United Kingdom), divided into aliquot parts in Eppendorf tubes, and stored at ÿ708C for subsequent analysis. The recovery of NLF was consistent among subjects. The volume of original NLF was 4 mL, and the volume of concentrated NFL was 0.3 mL; thus, the concentration factor was 13-fold.
Levels of histamine and tryptase in nasal lavage fluid Histamine level in concentrated nasal lavage fluid was assayed by using a commercially available histamine ELISA kit (Immuno Biological Laboratories, Hamburg, Germany), whereas tryptase was measured by a fluorescence immunoassay method (UniCap; Pharmacia Diagnostics AB, Uppsala, Sweden). Samples were assayed according to the instructions of the manufacturers. The lowest detectable levels for histamine and tryptase were 0.5 ng/mL and 1.0 ng/mL, respectively. In addition, standard curves for both heparin and tryptase were performed in the presence and absence of heparin (5000 IU/mL); coefficients of variation between measured optical densities in the presence and absence of heparin were within 10% (within the experiment error) throughout the entire calibration curves. Thus, heparin sodium (final dose, 5000 IU/mL) did not interfere with the immunoassay measurement of histamine or tryptase in the lavage fluid.
Analyses of data Data shown are mean ± SEM values of all subjects in each group. Numbers of sneezes between allergic and nonallergic subjects were compared by using the t test. Numbers of sneezes and the basal and peak values of histamine and tryptase concentrations between placebo and heparin-treated days were compared by using the Wilcoxon signed rank test. A P value < .05 was considered significant. All statistical analyses were performed by using SigmaStat 2.03 (SAS Institute, Cary, NC).
RESULTS Sneeze count Nasal provocation with AMP produced considerable sneezing in subjects with atopic but not in nonatopic subjects (Table I). The numbers of sneezes were 4.4 ± 0.7 and 1.2 ± 0.5 for atopic and nonatopic subjects, respectively (P = .006). The AMP-induced sneezing in atopic subjects was significantly reduced by pretreatment with heparin. The numbers of sneezes were 4.4 ± 0.7 and 2.3 ± 0.6 on placebo-treated and heparin-treated days, respectively (P = .016; Table I). Histamine levels in nasal lavage fluid In atopic subjects (n = 9) treated with placebo, nasal provocation with AMP elicited a significant rise in histamine levels, with a peak response at 3 minutes (4.1fold ± 0.6-fold greater than basal in mean values; P = .004). In the same subjects, pretreatment with heparin significantly reduced the peak increase in histamine levels induced by AMP challenge (P = .012) without affecting
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Mechanisms of asthma and allergic inflammation FIG 1. A, Time course of histamine concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in atopic subjects. Data shown are means ± SEMs of histamine concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in histamine concentrations (bottom graph). B, Time course of histamine concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in nonatopic subjects. Data shown are means ± SEMs of histamine concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in histamine concentrations (bottom graph).
TABLE I. Effect of pretreatment with heparin or placebo on AMP-induced sneezing in the atopic and non-atopic subjects* Atopic subjects Subject no.
1 2 3 4 5 6 7 8 9 Mean SEM
Nonatopic subjects
Placebo
Heparin
Subject no.
8 4 5 3 7 2 6 3 2 4.4 0.7
4 3 2 0 5 2 3 0 2 2.3 0.6
1 2 3 4 5 6
Mean SEM
Placebo
Heparin
2 0 2 3 0 0
0 2 2 2 0 0
1.2 0.5
1.0 0.4
*Data shown are numbers of sneezes after AMP nasal challenge.
the basal histamine levels (Fig 1, A). The baseline histamine levels were 5.0 ± 1.0 ng/mL and 4.1 ± 0.7 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). The peak histamine levels at 3 minutes after nasal challenge were 20.3 ± 4.5 ng/mL and
7.5 ± 1.0 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). In contrast with atopic subjects, in nonatopic subjects (n = 6), AMP nasal challenge did not affect the histamine levels in nasal lavage fluid on placebo-treated or heparin-treated days (Fig 1, B).
Tryptase levels in nasal lavage fluid Similarly, in atopic subjects treated with placebo, nasal provocation with AMP elicited a significant rise in tryptase levels, with a peak response at 3 minutes (3.7fold ± 0.7-fold greater than basal in mean values; P = .004). In the same subjects, pretreatment with heparin significantly reduced the peak increase in tryptase levels induced by AMP challenge (P = .004) without affecting the basal tryptase levels (Fig 2, A). The baseline tryptase levels were 2.3 ± 03 ng/mL and 1.9 ± 0.2 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). The peak tryptase levels at 3 minutes after nasal challenge were 7.9 ± 1.3 ng/mL and 2.8 ± 0.3 ng/mL on placebo-treated and heparin-treated days, respectively (n = 9). In contrast with atopic subjects, in nonatopic subjects (n = 6), AMP nasal challenge did not affect the tryptase levels in nasal lavage fluid on placebo-treated or heparin-treated days (Fig 2, B).
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FIG 2. A, Time course of tryptase concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in atopic subjects. Data shown are means ± SEMs of tryptase concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in tryptase concentrations (bottom graph). B, Time course of tryptase concentrations in NLF after nasal challenge with AMP in placebo (open symbol) or heparin-treated (closed symbol) days in nonatopic subjects. Data shown are means ± SEMs of tryptase concentrations in the concentrated NLF with a concentration factor of 13 (top graph) or the fold increase in tryptase concentrations (bottom graph).
DISCUSSION In the current study, we have extended our previous observations that nasal provocation with AMP provokes nasal symptoms and mast cell degranulation in subjects with allergic rhinitis.16,17 We have also shown that administration of topical heparin reduces sneezing and inhibits the release of histamine and tryptase in the nasal lavage fluid of allergic subjects challenged with AMP. Topical heparin reduced AMP-induced sneezing in almost all of the atopic subjects studied. This finding is in agreement with a recent study in which allergen-induced nasal symptoms were significantly attenuated when patients received topical heparin.18 Taken together, these findings indicate that aerosol heparin and related molecules may have beneficial effects in alleviating symptoms of allergic rhinitis, but larger studies are needed to confirm our observations and to define the characteristics of the patients who would benefit most from such a therapeutic approach. Although the mode of action of AMP in provoking symptoms in allergic rhinitis is not known with certainty, stimulation of specific cell surface A2B adenosine receptors expressed on airway mast cells with subsequent degranulation is a possibility.14,15 In vitro, adenosine is
known to enhance mediator release from immunologically primed human airway mast cells.19,20 These in vitro data are compatible with the results of the current study in vivo, which clearly show that adenosine induced a marked increase in histamine and tryptase levels in all of the atopic subjects studied. These changes in mediator levels were associated with considerable sneezing. Likewise, the airways of allergic asthmatics are known to respond to endobronchial instillation of AMP with bronchial narrowing, and this is coupled with a rise in histamine levels in the bronchoalveolar lavage fluid.21 Thus, potentiation of mast cell mediator release may account for the nasal symptoms provoked by nasal challenge with AMP in allergic rhinitis. Previous studies in allergic sheep model indicated that fractionated low-molecular-weight heparins attenuate antigen-induced acute bronchoconstrictor response and airway hyperresponsiveness, and there is an inverse relationship between the antiallergic activity of heparin fractions and molecular weight.22 On the other hand, inhaled unfractionated heparin and low-molecular-weight heparins inhibited antigen-induced histamine release in bronchoalveolar lavage fluid by 81% and 75%; thus, the inhibitory effects of unfractionated heparin and fractionated heparins on histamine release are not strictly
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molecular weight–dependent.23 In our current study, the unfractionated heparin was used mainly because we have shown previously that inhaled unfractionated heparin attenuated the airway response to AMP bronchial provocation.9 The mechanism by which heparin attenuates the nasal response to AMP and allergen is unclear and requires additional studies. In asthma, inhaled heparin seems to exert a preferential protective effect against stimuli such as allergen,7 exercise,8 and AMP9 that are known to cause bronchoconstriction mainly by releasing spasmogenic mediators from airway mast cells. Hence, the antiallergic actions of heparin may be related to the inhibition of mediator release from mast cells. In support of this view, we have demonstrated for the first time that administration of topical heparin inhibited the release of histamine and tryptase in the nasal lavage fluid of almost all of the allergic subjects challenged with AMP. Indeed, the ability of heparin to prevent the release of mast cell mediators such as histamine and tryptase might explain the substantial reduction in the number of sneezes observed immediately after AMP challenge. Endogenous heparin, a highly anionic molecule, has the ability to bind mast cell mediators, and thus it is possible that heparin administered by inhalation may cause a reduction in the detected levels of histamine and tryptase in the nasal washes merely because of its negative charge density. However, our pilot experiments demonstrated that heparin did not interfere with the immunoassay measurement of spiked histamine and tryptase in lavage fluid samples, and this suggested that direct binding between heparin and mediators is unlikely to provide an explanation for the protective effect of heparin. The precise cellular mechanism of action for the inhibitory effect of heparin on mast cell mediator release is not completely understood. It is not clear whether the effect of heparin is caused by its binding to receptors or adhesion molecules on the surface of mast cells directly or by its binding to cytokines or chemokines or other inflammatory cells in the airway. Future studies using purified mast cells and other inflammatory cells from human airways may help advance our understanding of the cellular mechanism of action for inhaled heparin. In conclusion, topical heparin has been shown to inhibit AMP-induced responses in allergic rhinitis. Taking into account the notion of airway mast cell mediators contributing to the nasal response of AMP, the current findings support the hypothesis that inhaled heparin plays a protective role, possibly by inhibition of mast cell activation and subsequent release of mast cell mediators. Further work is required to clarify the molecular mechanism by which heparin produces this novel anti-inflammatory activity and to determine whether this has any relevance to a possible therapeutic benefit in allergic rhinitis. REFERENCES 1. Schwartz LB, Bradford TR. Regulation of tryptase from human lung mast cells by heparin. J Biol Chem 1986;261:7372-9.
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2. Sayama S, Iozzo RV, Lazarus GS, Schecter NM. Human skin chymotrypsin like proteinase chymase: subcellular localization to mast cell granules and interaction with heparin and other glycosaminoglycans. J Biol Chem 1987;262:6808-15. 3. Castellot JJ, Cochran DL, Karnovsky MM. Effect of heparin on vascular smooth muscle cell, I: cell metabolism. J Cell Physiol 1985;124: 21-8. 4. Wright TC, Cochran DL, Karnovsky MJ. Inhibition of rat cervical epithelial cell growth by heparin and its reversal by EGF. J Cell Physiol 1985;125:499-506. 5. Roemisch J, Gray E, Hoffmann JN, Wiedermann CJ. Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagul Fibrinolysis 2002;13:657-70. 6. Okajima K. Regulation of inflammatory responses by natural anticoagulants. Immunol Rev 2001;184:258-74. 7. Bowler SD, Smith SM, Lavercombe PS. Heparin inhibits the immediate response to antigen in the skin and lungs of allergic subjects. Am Rev Respir Dis 1993;147:160-3. 8. Ahmed T, Garrigo J, Danta I. Preventing bronchoconstriction in exercise-induced asthma with inhaled heparin. N Engl J Med 1993; 329:90-5. 9. Polosa R, Magri S, Vancheri C, Armato F, Santonocito G, Mistretta A, et al. Time course of changes in adenosine 59-monophosphate airway responsiveness with inhaled heparin in allergic asthma. J Allergy Clin Immunol 1997;99:338-44. 10. Page C. The role of proteoglycans in the regulation of airways inflammation and airways remodelling. J Allergy Clin Immunol 2000;105: S518-21. 11. Lucio J, D’Brot J, Guo CB, Abraham WB, Lichtenstein LM, Kagey-Sobotka A, et al. Immunologic mast cell-mediated responses and histamine release are attenuated by heparin. J Appl Physiol 1992;73: 1093-101. 12. Lee TH, Assoufi BK, Kay AB. The link between exercise, respiratory heat exchange and the mast cell in bronchial asthma. Lancet 1983;1: 520-2. 13. Howarth PH, Durham SR, Lee TH, Kay AB, Church MK, Holgate ST. Influence of albuterol, cromolyn sodium and ipratropium bromide on the airways and circulating mediator responses to antigen bronchial provocation in asthma. Am Rev Respir Dis 1985;132:986-92. 14. Spicuzza L, Bonfiglio C, Polosa R. Research applications and implications of adenosine in diseased airways. Trends Pharmacol Sci 2003;24:409-13. 15. Polosa R. Adenosine-receptor subtypes: their relevance to adenosinemediated responses in asthma and chronic obstructive pulmonary disease. Eur Respir J 2002;20:488-96. 16. Polosa R, Pagano C, Prosperini G, Low JL, Dokic D, Church MK, et al. Histamine release upon adenosine 59-monophosphate (AMP) nasal provocation in allergic subjects. Thorax 1999;54:230-3. 17. Crimi N, Polosa R, Mistretta A. Mechanisms of the effector function of adenosine in asthma and rhinitis. Drug Dev Res 1993;28:322-7. 18. Vancheri C, Mastruzzo C, Armato F, Tomaselli V, Magri S, Pistorio MP, et al. Intranasal heparin reduces eosinophil recruitment after nasal allergen challenge in patients with allergic rhinitis. J Allergy Clin Immunol 2001;108:703-8. 19. Hughes PJ, Holgate ST, Church MK. Adenosine inhibits and potentiates IgE-dependent histamine release from human lung mast cells by an A2-purinoceptor mediated mechanism. Biochem Pharmacol 1984;33: 3847-52. 20. Peachell PT, Columbo M, Kagey-Sobotka A, Lichtenstein LM, Marone G. Adenosine potentiates mediator release from human lung mast cells. Am Rev Respir Dis 1988;138:1143-51. 21. Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK, et al. Release of mast cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med 1995;151:624-9. 22. Martinez-Salas J, Mendelssohn R, Abraham WM, Hsiao B, Ahmed T. Inhibition of allergic airway responses by inhaled low-molecularweight heparins: molecular-weight dependence. J Appl Physiol 1998; 84:222-8. 23. Molinari JF, Campo C, Shakir S, Ahmed T. Inhibition of antigeninduced airway hyperresponsiveness by ultralow molecular-weight heparin. Am J Respir Crit Care Med 1998;157:887-93.