Subpollen particles: Carriers of allergenic proteins and oxidases

Attila Bacsi

Barun Choudhury

Nilesh Dharajiya

Sanjiv Sur

Istvan Boldogh

^{Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Tex.}

^{Division of Allergy, Pulmonary, Immunology, Critical Care and Sleep, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Tex.}

Reprint requests: Istvan Boldogh, DM&B, PhD, Department of Microbiology and Immunology, University of Texas Medical Branch, 3.170 Medical Research Building, 301 University Blvd, Galveston, TX 77555. ude.bmtu@hgodlobs.

^{Dr Bacsi is currently affiliated with the Institute of Immunology, University of Debrecen, Debrecen, Hungary.}

Abstract

Background

Pollen is known to induce allergic asthma in atopic individuals, although only a few inhaled pollen grains penetrate into the lower respiratory tract.

Objective

We sought to provide evidence that subpollen particles (SPPs) of respirable size, possessing both antigenic and redox properties, are released from weed pollen grains and to test their role in allergic airway inflammation.

Methods

The release of SPPs was analyzed by means of microscopic imaging and flow cytometry. The redox properties of SPPs and the SPP-mediated oxidative effect on epithelial cells were determined by using redox-sensitive probes and specific inhibitors. Western blotting and amino acid sequence analysis were used to examine the protein components of the SPP. The allergenic properties of the SPP were determined in a murine model of experimental asthma.

Results

Ragweed pollen grains released 0.5 to 4.5 μm of SPPs on hydration. These contained Amb a 1, along with other allergenic proteins of ragweed pollen, and possessed nicotinamide adenine dinucleotide (reduced) or nicotinamide adenine dinucleotide phosphate (reduced) [NAD(P)H] oxidase activity. The SPPs significantly increased the levels of reactive oxygen species (ROS) in cultured cells and induced allergic airway inflammation in the experimental animals. Pretreatment of the SPPs with NAD(P)H oxidase inhibitors attenuated their capacity to increase ROS levels in the airway epithelial cells and subsequent airway inflammation.

Conclusions

The allergenic potency of SPPs released from ragweed pollen grains is mediated in tandem by ROS generated by intrinsic NAD(P)H oxidases and antigenic proteins.

Clinical implications

Severe clinical symptoms associated with seasonal asthma might be explained by immune responses to inhaled SPPs carrying allergenic proteins and ROS-producing NAD(P)H oxidases.

Keywords: Subpollen particles, oxidative stress, inflammation

Abstract

Pollen allergens are a major cause of allergic reactions in the skin, eyes, and upper and lower respiratory tracts during the flowering season.¹4 How pollen-carried allergens contribute to the development of inflammation in the lower airways of the lungs has remained a puzzle because only a small percentage of pollen grains deposit in the peripheral airways. It has been shown that hydration in rainwater, high humidity conditions, and moisture can cause the expulsion of pollen grains from grasses and trees, leading to the release of allergen-containing micronic and submicronic particles (0.12-5 μm).⁵8 These findings correlate with previous reports showing that significant allergen exposure can occur in the absence of identifiable pollen grains in the air.⁹ Similarly, allergen levels are increased in the environment in the flowering season, especially after heavy rainfall, which correlates with the increased frequency of hospital admissions of atopic individuals.¹⁰

The pollen of short ragweed (Ambrosia artemisiifolia) is one of the most abundant aeroallergens causing severe seasonal allergic symptoms in the United States and Canada. This wind-pollinated flowering plant produces large amounts of pollen. Exposure to ragweed pollen antigens has been shown to elicit allergic responses.¹¹ The molecular characterization of ragweed pollen revealed Amb a 1 to be the predominant allergen.¹² More then 90% of ragweed-sensitive subjects have antibodies against Amb a 1.¹² However, there is a poor correlation between pollen counts and the abundance of airborne allergens, suggesting that allergen exposure might occur in the absence of pollen grains.⁹13 Indeed, it has been proposed that submicronic particles that easily penetrate the lower airways represent the major allergen source and are the cause of severe asthma associated with the pollen season.³1416 In support, ragweed allergens have been identified in the form of fine particles with sizes ranging from 0.2 to 5.25 μm from outdoor air samples analyzed by a nonviable cascade impactor.¹³ Yet the generation of subpollen particles (SPPs) has not been observed for ragweed or other weed species.

We recently reported that, in addition to their allergenic proteins, ragweed pollen grains contain intrinsic NAD(P)H oxidases, which generate oxidative stress (“signal 1”) in the airway epithelium within minutes of exposure. This process is required for antigen (“signal 2”)–mediated robust inflammation in the lower airways.¹⁷18 Because of their size, the relevance of ragweed pollen grains to lung inflammation has remained unclear. Here we demonstrate that on hydration, the viable ragweed pollen produced SPPs through biochemically active processes. The SPPs retained pollen grains’ NAD(P)H oxidases, Amb a 1, and other antigenic components and induced robust inflammation in the lower airways. These findings might explain the increased allergen levels in the absence of identifiable pollen grains and the severe asthma symptoms associated with the pollen season.

Abbreviations used

BAL	Bronchoalveolar lavage
DCF	Dichlorofluorescein
DPI	Diphenyleneiodonium
H₂DCF-DA	2′-7′-dihydro-dichlorofluorescein diacetate
MAPK	Mitogen-activated protein kinase
NADH	Nicotinamide adenine dinucleotide, reduced
NADPH	Nicotinamide adenine dinucleotide phosphate, reduced
NAD(P)H	NADH or NADPH
NBT	Nitroblue tetrazolium
NHBE	Normal human bronchial epithelial cell
QA	Quinacrine
ROS	Reactive oxygen species
RWE	Ragweed pollen extract
SPP	Subpollen particle

Abbreviations used

Footnotes

Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

Footnotes

REFERENCES

References

1. Grote MIn situ localization of pollen allergens by immunogold electron microscopy: allergens at unexpected sites. Int Arch Allergy Immunol. 1999;118:1–6.[PubMed][Google Scholar]
2. Vrtala S, Grote M, Duchene M, van Ree R, Kraft D, Scheiner O, et al Properties of tree and grass pollen allergens: reinvestigation of the linkage between solubility and allergenicity. Int Arch Allergy Immunol. 1993;102:160–9.[PubMed][Google Scholar]
3. Suphioglu C, Singh MB, Taylor P, Bellomo R, Holmes P, Puy R, et al Mechanism of grass-pollen-induced asthma. Lancet. 1992;339:569–72.[PubMed][Google Scholar]
4. Roberts G, Hurley C, Bush A, Lack GLongitudinal study of grass pollen exposure, symptoms, and exhaled nitric oxide in childhood seasonal allergic asthma. Thorax. 2004;59:752–6.[Google Scholar]
5. Schappi GF, Taylor PE, Staff IA, Rolland JM, Suphioglu CImmunologic significance of respirable atmospheric starch granules containing major birch allergen Bet v 1. Allergy. 1999;54:478–83.[PubMed][Google Scholar]
6. Grote M, Vrtala S, Niederberger V, Valenta R, Reichelt RExpulsion of allergen-containing materials from hydrated rye grass (Lolium perenne) pollen revealed by using immunogold field emission scanning and transmission electron microscopy. J Allergy Clin Immunol. 2000;105:1140–5.[PubMed][Google Scholar]
7. Burge HAAn update on pollen and fungal spore aerobiology. J Allergy Clin Immunol. 2002;110:544–52.[PubMed][Google Scholar]
8. Taylor PE, Flagan RC, Miguel AG, Valenta R, Glovsky MMBirch pollen rupture and the release of aerosols of respirable allergens. Clin Exp Allergy. 2004;34:1591–6.[PubMed][Google Scholar]
9. Barnes C, Schreiber K, Pacheco F, Landuyt J, Hu F, Portnoy JComparison of outdoor allergenic particles and allergen levels. Ann Allergy Asthma Immunol. 2000;84:47–54.[PubMed][Google Scholar]
10. Wark PA, Simpson J, Hensley MJ, Gibson PGAirway inflammation in thunderstorm asthma. Clin Exp Allergy. 2002;32:1750–6.[PubMed][Google Scholar]
11. King TPChemical and biological properties of some atopic allergens. Adv Immunol. 1976;23:77–105.[PubMed][Google Scholar]
12. Rafnar T, Griffith IJ, Kuo MC, Bond JF, Rogers BL, Klapper DGCloning of Amb a I (antigen E), the major allergen family of short ragweed pollen. J Biol Chem. 1991;266:1229–36.[PubMed][Google Scholar]
13. Menetrez MY, Foarde KK, Ensor DSAn analytical method for the measurement of nonviable bioaerosols. J Air Waste Manag Assoc. 2001;51:1436–42.[PubMed][Google Scholar]
14. Schappi GF, Suphioglu C, Taylor PE, Knox RBConcentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere. J Allergy Clin Immunol. 1997;100:656–61.[PubMed][Google Scholar]
15. Grote M, Valenta R, Reichelt RAbortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy. J Allergy Clin Immunol. 2003;111:1017–23.[PubMed][Google Scholar]
16. Knox RB, Suphioglu C, Taylor P, Desai R, Watson HC, Peng JL, et al Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution. Clin Exp Allergy. 1997;27:246–51.[PubMed][Google Scholar]
17. Bacsi A, Dharajiya N, Choudhury BK, Sur S, Boldogh IEffect of pollen-mediated oxidative stress on immediate hypersensitivity reactions and late-phase inflammation in allergic conjunctivitis. J Allergy Clin Immunol. 2005;116:836–43.[Google Scholar]
18. Boldogh I, Bacsi A, Choudhury BK, Dharajiya N, Alam R, Hazra TK, et al ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation. J Clin Invest. 2005;115:1–13.[Google Scholar]
19. Day JP, Kell DB, Griffith GWDifferentiation of Phytophthora infestans sporangia from other airborne biological particles by flow cytometry. Appl Environ Microbiol. 2002;68:37–45.[Google Scholar]
20. Liochev SI, Fridovich ISuperoxide from glucose oxidase or from nitroblue tetrazolium? Arch Biochem Biophys. 1995;318:408–10.[PubMed][Google Scholar]
21. Van Gestelen P, Asard H, Caubergs RJSolubilization and separation of a plant plasma membrane NADPH-O2- synthase from other NAD(P)H oxidoreductases. Plant Physiol. 1997;115:543–50.[Google Scholar]
22. Zhang S, Klessig DFMAPK cascades in plant defense signaling. Trends Plant Sci. 2001;6:520–7.[PubMed][Google Scholar]
23. Powell CS, Wright MM, Jackson RMp38mapk and MEK1/2 inhibition contribute to cellular oxidant injury after hypoxia. Am J Physiol Lung Cell Mol Physiol. 2004;286:L826–33.[PubMed][Google Scholar]
24. Das A, Hazra TK, Boldogh I, Mitra S, Bhakat KKInduction of the human oxidized base-specific DNA glycosylase NEIL1 by reactive oxygen species. J Biol Chem. 2005;280:35272–80.[PubMed][Google Scholar]
25. van Zandwijk NN-acetylcysteine (NAC) and glutathione (GSH): antioxidant and chemopreventive properties, with special reference to lung cancer. J Cell Biochem Suppl. 1995;22:24–32.[PubMed][Google Scholar]
26. Kawasaki T, Koita H, Nakatsubo T, Hasegawa K, Wakabayashi K, Takahashi H, et al Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice. Proc Natl Acad Sci U S A. 2006;103:230–5.[Google Scholar]
27. Liszkay A, Kenk B, Schopfer PEvidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth. Planta. 2003;217:658–67.[PubMed][Google Scholar]
28. Edinger AL, Thompson CBDeath by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol. 2004;16:663–9.[PubMed][Google Scholar]
29. Lam E, Kato N, Lawton MProgrammed cell death, mitochondria and the plant hypersensitive response. Nature. 2001;411:848–53.[PubMed][Google Scholar]
30. Frahry G, Schopfer PNADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay. Planta. 2001;212:175–83.[PubMed][Google Scholar]
31. Henricks PA, Nijkamp FPReactive oxygen species as mediators in asthma. Pulm Pharmacol Ther. 2001;14:409–20.[PubMed][Google Scholar]
32. Rahman IOxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J Biochem Mol Biol. 2003;36:95–109.[PubMed][Google Scholar]
33. Thannickal VJ, Fanburg BLReactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol. 2000;279:L1005–28.[PubMed][Google Scholar]