Allergy Asthma Immunol Res.  2014 Nov;6(6):558-566. 10.4168/aair.2014.6.6.558.

The Serine Protease Inhibitor, 4-(2-aminoethyl) Benzene Sulfonyl Fluoride Hydrochloride, Reduces Allergic Inflammation in a House Dust Mite Allergic Rhinitis Mouse Model

• Kim BY
• Park HR
• Shin JH
• Kim SW
• Cho JH
• Park YJ
• Kim SW

• KMID: 2260179
• DOI: http://doi.org/10.4168/aair.2014.6.6.558

Abstract

PURPOSE
Serine protease inhibitors are involved in immune development, anti-inflammatory mechanisms, and tissue repair. In the present study, the serine protease inhibitor 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was evaluated for its prophylactic and therapeutic applications in a mouse model of allergic rhinitis (AR).
METHODS
BALB/c mice were divided into 5 groups: contol (CON), Dermatophagoides farinae (Derf), AR mice treated with AEBSF before sensitization (S), AR mice treated with AEBSF after challenge (C), and steroid groups. Derf was used as an allergen. AEBSF was administered before S or after C. Allergic symptom scores, eosinophil counts, proteolytic activity, interferon-gamma, interleukin (IL)-10 levels and serum Derf-specific IgE levels were measured. T-bet, GATA-3, Foxp3, IL-13, and transforming growth factor (TGF)-beta mRNA levels were determined using real-time polymerase chain reaction. CD4+CD25+Foxp3+ T cells were assessed using flow cytometry.
RESULTS
Symptom scores, serum Derf-specific IgE levels, GATA-3 mRNA levels, IL-13 mRNA levels, and tissue eosinophil counts decreased in both the S and C groups (P<0.05). Additionally, the percentage of CD4+CD25+Foxp3+ T cells, IL-10 levels, and Foxp3 mRNA levels increased in the S and C groups compared with those in the Derf group (P<0.05). AEBSF treatment decreased the proteolytic activity in the S and C groups (P<0.05).
CONCLUSIONS
Prophylactic and therapeutic treatment with AEBSF significantly reduces allergic airway inflammation and can induce regulatory T cells in a murine model of AR.

Keyword

Serine protease inhibitor; allergic rhinitis; regulatory T cells; animal model; 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride; house dust mite

MeSH Terms

Animals
Benzene*
Dermatophagoides farinae
Eosinophils
Flow Cytometry
Fluorides*
Immunoglobulin E
Inflammation*
Interferon-gamma
Interleukin-10
Interleukin-13
Interleukins
Mice*
Models, Animal
Pyroglyphidae*
Real-Time Polymerase Chain Reaction
Rhinitis*
RNA, Messenger
Serine Proteases*
Serine Proteinase Inhibitors
T-Lymphocytes
T-Lymphocytes, Regulatory
Transforming Growth Factors
Benzene
Fluorides
Immunoglobulin E
Interferon-gamma
Interleukin-10
Interleukin-13
Interleukins
RNA, Messenger
Serine Proteases
Serine Proteinase Inhibitors
Transforming Growth Factors

Figure

• 

  Fig. 1 Data are expressed as days. Schematic representation of the experimental allergic rhinitis model and treatment protocol. Mice in the steroid group were treated intranasally with 20 µg of ciclesonide on days 26-28. AEBSF, 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride; S, AEBSF before sensitization; C, AEBSF after challenge; Derf, Dermatophagoides farinae; IP, intraperitoneal administration; IN, intranasal administration.

• 

  Fig. 2 Nasal symptom score. Rubbing (A) and sneezing (B). Error bars represent standard deviations. *P<0.05 vs the Derf group; **P<0.05 vs the control group.

• 

  Fig. 3 Serum Derf-specific immunoglobulin E. Error bars represent standard deviations. *P<0.05 vs the Derf group; **P<0.05 vs the control group.

• 

  Fig. 4 Infiltration of eosinophils (arrows) in the nasal mucosa of BALB/c mice: (A) control group, (B) Derf group, (C) S group, and (D) C group (E) steroid group (hematoxylin and eosin staining; original magnification, ×400).

• 

  Fig. 5 Eosinophil counts in the nasal mucosa of each study group. Error bars represent standard deviations. *P<0.05 vs the Derf group; **P<0.05 vs the control group.

• 

  Fig. 6 Cytokine levels in the nasal lavage fluid of each study group: (A) interferon- (B) interleukin-10. Error bars represent standard deviations. *P<0.05 vs the Derf group; **P<0.05 vs the control group.

• 

  Fig. 7 Quantitative analysis of T-bet (A), GATA-3 (B), and Foxp3 (C) mRNA levels in splenic mononuclear cells by real-time PCR. IL-13 (D) and TGF-β (E) mRNA levels in nasal mucosa. The results were normalized to GAPDH expression. Error bars represent standard deviations. *P<0.05 vs the Derf group; **P<0.05 vs the control group.

• 

  Fig. 8 (A) Flow cytometric analysis of CD4+CD25+Foxp3+ T cell subsets. Representative fluorescence-activated cell sorting analysis in each group. RU, upper right quadrant, which represents CD4+CD25+Foxp3+ T cells. (B) The percentage of splenic mononuclear cells that were CD4+CD25+Foxp3+ T cells. Error bars represent standard deviations. *P<0.05 vs the Derf group; **P<0.05 vs the control group.

• 

  Fig. 9 Proteolytic activity in terms of RFU/min in Derf-challenged and AEBSR/steroid-treated mice. Error bars represent standard deviations. *P<0.05 vs the Derf group; **P<0.05 vs the control group.

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