<b><i>Dermatophagoides farinae</i></b> Upregulates the Effector Functions of Eosinophils through αMβ₂-Integrin and Protease-Activated Receptor-2

Abstract

Background: Even in subjects who are not sensitized to house dust mite (HDM), allergic symptoms can be aggravated by exposure to dust, suggesting that innate immune responses may be involved in these processes. Since eosinophils express pattern recognition receptors, HDM may directly upregulate eosinophil functions through these re ceptors. The objective of this study was to examine whether Dermatophagoides farinae (Df), a representative HDM, or Der f 1, a major allergen of Df, modifies the effector functions of eosinophils. Methods: Eosinophils isolated from the blood of healthy donors or allergic patients were stimulated with Df extract or Der f 1, and their adhesion to recombinant human intercellular adhesion molecule (ICAM)-1 was measured using eosinophil peroxidase assays. Generation of the eosinophil superoxide anion (O₂^–) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) concentrations in cell media were measured by ELISA as a marker of degranulation. Results: Df extract or Der f 1 directly induced eosinophil adhesion to ICAM-1, O₂^– generation, and EDN release. Anti-αM- or anti-β₂-integrin antibodies or protease-activated receptor (PAR)-2 antagonists suppressed the eosinophil adhesion, O₂^– generation, and EDN release induced by Df extract or Der f 1. Eosinophils from allergic patients showed higher adhesion to ICAM-1 than those from healthy donors. Conclusions: These findings suggested that Df extract and Der f 1 directly activate eosinophil functions through αMβ₂-integrin and PAR-2. Eosinophil activation by HDM may play roles in the aggravation of allergic symptoms, not only in HDM-sensitized patients, but also in nonsensitized patients.

Japanese cedar pollen upregulates the effector functions of eosinophils

“…This study was approved by the Ethical Committee of Saitama Medical University (Institutional Review Board permission number: 781-III), and written informed consent was obtained from the subjects before the collection of each blood sample. Eosinophils were isolated by the combination of Percoll density gradient centrifugation and negative selection using anti-CD16 Ab-coated magnetic beads (Miltenyi Biotec, Auburn, CA, USA), as previously described [ 13 14 15 16 17 18 ]. Over 98% of the cells were eosinophils, as determined by morphological criteria using May-Grünwald-Giemsa staining.…”

“…The effects of JCP and Cry j 1 on eosinophil adhesion to recombinant human (rh) intercellular adhesion molecule (ICAM)-1-coated plates were assessed based on the residual eosinophil peroxidase (EPO) activity of the adherent eosinophils, as previously described [ 13 14 15 16 17 18 ]. Briefly, eosinophils (100 μL of 1 × 10 5 cells/mL in HBSS/gel) from nonallergic volunteers were incubated in the presence or absence of JCP (0.1 to 10 μg/mL) or Cry j 1 (1 to 100 pg/mL) in rh-ICAM-1 (10 μg/mL; R&D Systems, Minneapolis, MN, USA)-coated Costar cell culture plates (Corning, Inc., Corning, NY, USA) at 37°C for 20 minutes.…”

“…The reaction was stopped by adding 20 μL of 4 M H 2 SO 4 , and the absorbance was measured at 490 nm. In some experiments, suspended eosinophils were pre-incubated with an isotype-matched control mouse IgG1 (3 μg/mL; clone MOPC-21, Becton Dickinson, Franklin Lakes, NJ, USA), anti-αM integrin monoclonal Ab (mAb; 3 μg/mL; clone 2LPM19c, Pierce, Rockford, IL, USA), anti-β2 integrin mAb (3 μg/mL; clone L130, Becton Dickinson), or a PAR-2 antagonist (10 μM FSLLRY-NH2 [ 13 20 21 22 ], R&D Systems, or 10 μM ENMD-1068 [ 13 23 24 25 ], Enzo Life Sciences, Farmingdale, NY, USA) for 20 minutes before addition to the wells. Each experiment was performed in quadruplicate using eosinophils from a single donor, and the percentage of eosinophil adhesion was determined from the mean values that were calculated from log-dose response curves.…”

“…Eosinophil O 2 − generation was measured in 96-well enzyme-linked immunosorbent assay (ELISA) plates, as previously described, based on the superoxide dismutase (SOD)-inhibitable reduction of cytochrome C [ 13 14 15 16 17 18 ]. We initially added SOD (0.2 mg/mL in HBSS/gel; 20 μL) to SOD control wells, then added HBSS/gel to all wells of the rh-ICAM-1-coated plates (10 μg/mL) to bring the final volume to 100 μl/well.…”

“…PAR-2 is associated with the inflammatory response to some infections and microbial proteases [ 11 ]. We previously reported that HDM directly activates the functions of eosinophils obtained from the peripheral blood of nonallergic healthy subjects partly through PAR-2 [ 13 ]. Therefore, similar mechanisms may play roles in the JCP-related exacerbation of allergic diseases.…”