Subpollen particles: carriers of allergenic proteins and oxidases

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 microm 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.

FIG 1

Release of SPPs from ragweed pollen grains. A, Real-time microscopic images of SPPs release from ragweed pollen grains. Left to right, Vesicle-like formations on the pollen surface (magnification ×100); the release of 0.5 to 4.5 μM of SPPs (5, 10, and 15 minutes). B, Size distributions of ragweed pollen grains 5 minutes (shaded panel) and 30 minutes (nonshaded panel) after hydration. FSC, Forward scatter parameter.

FIG 2

Release of SPPs requires NAD(P)H oxidase activity. A, Formazan formation at the site of SPP release (ragweed pollen). B and C, Ragweed (Fig 2, B) and pigweed (Fig 2, C) pollen grains oxidize H₂DCF-DA into fluorescent DCF. D, Percentage of the DCF-positive pollen particles (open columns) releasing SPPs (solid columns). E, Inhibition of SPP formation. Open columns, Ragweed; solid columns, pigweed. All results are presented as means ± SEM (n = 4-7). **P < .01; ***P < .001; ****P < .0001 versus control. MT, Mock treated.

FIG 3

SPPs possess redox activity. A, Ragweed SPPs showing DCF fluorescence (magnification ×196). B, SPP-mediated O2.– generation is inhibited by NAD(P)H oxidase inhibitors or heat inactivation. SOD, Superoxide dismutase; CAT, catalase; GO, glucose oxidase. All results are presented as means ± SEM (n = 4-7). C, In-gel NBT reduction by proteins in SPPs after nondenaturing PAGE. Lane 1, SPP; lane 2, heat-treated SPP; lane 3, RWE; lane 4, heat-treated RWE. ****P < .0001 versus control.

FIG 4

SPPs from ragweed pollen grains contain Amb a 1. A, SDS-PAGE analysis of RWE and lysates of SPPs. The SPPs were released from the pollen grains and then lysed and subjected to SDS-PAGE. Fractionated proteins were stained with Coomassie brilliant blue G250. B, Amb a 1 in SPPs shown by means of Western blot analysis.

FIG 5

SPPs increase levels of ROS in airway epithelial cells. A, NHBE cells grown in submerged (solid columns) or air-liquid interphase cultures (open columns) treated with ragweed SPP. B, A549 cells exposed to ragweed or pigweed SPPs. Glucose oxidase (GO) was used as a positive control. MT, Mock treated; NAC, N-acetyl-L-cysteine. All results are presented as means ± SEM of 4 to 6 measurements. ****P < .0001 versus control.

FIG 6

A and B, SPPs induce airway inflammation. Sensitized mice were challenged with PBS, Amb a 1, or SPPs with (solid columns) or without (open columns) QA. Eosinophils (Fig 6, A) and total inflammatory cells (Fig 6, B) in BAL fluids are shown (± SEM; n = 3-5). C, Inflammatory cell infiltration in the peribronchial area (upper row) and goblet cell metaplasia (lower row). Representative images of serial lung sections are shown (n = 7-9). ***P < .001; ****P < .0001 versus control.