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. 2006 Jan 1;173(1):56-63.
doi: 10.1164/rccm.200503-361OC. Epub 2005 Sep 28.

Inhibition of spleen tyrosine kinase prevents mast cell activation and airway hyperresponsiveness

Shigeki Matsubara  1 , Guiming Li, Katsuyuki Takeda, Joan E Loader, Polly Pine, Esteban S Masuda, Nobuaki Miyahara, Satoko Miyahara, Joseph J Lucas, Azzeddine Dakhama, Erwin W Gelfand
Affiliations

Inhibition of spleen tyrosine kinase prevents mast cell activation and airway hyperresponsiveness

Shigeki Matsubara et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling.

Objective: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo.

Methods: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen.

Results: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge.

Conclusion: This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.

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Figures

<b>Figure 1.</b>
Figure 1.
R406 selectively inhibits spleen tyrosine kinase (Syk) in bone marrow mast cells (BMMCs). BMMCs were sensitized with IgE and then incubated with different concentrations of R406 for 1 h before adding antigen. Phosphorylation of Syk and linker for activation of T-cell tyrosine residue Y191 (LAT) was monitored by Western blot using anti–phosphotyrosine-specific antibodies.
<b>Figure 2.</b>
Figure 2.
Effect of R406 on antigen-induced degranulation of BMMCs and LTB4 release. IgE-sensitized BMMCs were preincubated for 1 h with different concentrations of the inhibitor before adding ovalbumin (OVA). (A) Serotonin release was measured 30 min after addition of OVA. (B) LTB4 release was measured 1 hr after addition of OVA. Each column represents mean ± SEM of three experiments performed in duplicate. ND = none detected. ++p < 0.01 compared with spontaneous (spon) release; *p < 0.05 and **p < 0.01 compared with vehicle-treated cells.
<b>Figure 2.</b>
Figure 2.
Effect of R406 on antigen-induced degranulation of BMMCs and LTB4 release. IgE-sensitized BMMCs were preincubated for 1 h with different concentrations of the inhibitor before adding ovalbumin (OVA). (A) Serotonin release was measured 30 min after addition of OVA. (B) LTB4 release was measured 1 hr after addition of OVA. Each column represents mean ± SEM of three experiments performed in duplicate. ND = none detected. ++p < 0.01 compared with spontaneous (spon) release; *p < 0.05 and **p < 0.01 compared with vehicle-treated cells.
<b>Figure 3.</b>
Figure 3.
Inhibitory effect of R406 on antigen-induced cytokine production in BMMCs. Passively sensitized BMMCs were incubated with different concentrations of the inhibitor. Supernates were recovered and assayed for cytokine by ELISA. Each column represents the mean ± SEM of three experiments performed in duplicate. ND = none detected. ++p < 0.01 compared with spontaneous (no OVA) release; *p < 0.05 and **p < 0.01 compared with vehicle-treated cells.
<b>Figure 4.</b>
Figure 4.
Effect of R406 on mitogen-activated protein kinase signaling downstream of Syk. Passively sensitized BMMCs were incubated with different concentrations of the inhibitor. Lysed samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with antibodies recognizing ERK1/2, JNK1/2, p38, and ERK5. A representative immunoblot from one of three similar experiments is shown.
<b>Figure 5.</b>
Figure 5.
Effect of R406 on allergen-induced airway hyperresponsiveness (AHR) after 10-d OVA exposure. BALB/c mice were exposed to either 10-d saline or 10-d OVA. Electric field stimulation (EFS) was performed 48 h after the last saline or OVA challenge and ES50 was calculated as described in METHODS. Each column represents the mean ± SEM of three separate experiments (n = 9). +p < 0.05 compared with 10-d saline group; *p < 0.05 compared with 10-d OVA-exposed, vehicle-treated group.
<b>Figure 6.</b>
Figure 6.
R406 prevents pulmonary eosinophilia after 10-d OVA exposure. Peribronchial eosinophil inflammation was detected using an antibody to major basic protein (MBP). (A) a: 10-d saline exposure; b: 10-d OVA exposure control; c: 10-d OVA exposure with vehicle treatment; d: 10-d OVA exposure after treatment with 3 mg/kg R406; e: 10-d OVA exposure after treatment with 30 mg/kg R406. Arrows indicate typical eosinophils. (B) R406 prevents pulmonary eosinophilia after 10-d OVA exposure. Peribronchial eosinophil inflammation was quantified in lung tissue stained with an antibody to MBP. Results are expressed as the number of MBP-positive cells per millimeter of basement membrane (BM). Each column represents the mean ± SEM of three separate experiments (n = 9). ++p < 0.01 compared with 10-d saline group; *p < 0.05 and **p < 0.01 compared with 10-d OVA-exposed, vehicle-treated group.
<b>Figure 6.</b>
Figure 6.
R406 prevents pulmonary eosinophilia after 10-d OVA exposure. Peribronchial eosinophil inflammation was detected using an antibody to major basic protein (MBP). (A) a: 10-d saline exposure; b: 10-d OVA exposure control; c: 10-d OVA exposure with vehicle treatment; d: 10-d OVA exposure after treatment with 3 mg/kg R406; e: 10-d OVA exposure after treatment with 30 mg/kg R406. Arrows indicate typical eosinophils. (B) R406 prevents pulmonary eosinophilia after 10-d OVA exposure. Peribronchial eosinophil inflammation was quantified in lung tissue stained with an antibody to MBP. Results are expressed as the number of MBP-positive cells per millimeter of basement membrane (BM). Each column represents the mean ± SEM of three separate experiments (n = 9). ++p < 0.01 compared with 10-d saline group; *p < 0.05 and **p < 0.01 compared with 10-d OVA-exposed, vehicle-treated group.
<b>Figure 7.</b>
Figure 7.
Effect of R406 on goblet cell metaplasia after 10-d OVA exposure. Goblet cell metaplasia was detected by Periodic acid-Schiff (PAS) staining 48 h after saline or OVA exposure. (A) a: 10-d saline exposure; b: 10-d OVA exposure control; c: 10-d OVA exposure after vehicle treatment; d: 10-d OVA exposure after treatment with 3 mg/kg R406; e: 10-d OVA exposure and after treatment with 30 mg/kg R406. (B) Effect of R406 on goblet cell metaplasia after 10-d OVA exposure. Goblet cell metaplasia was quantified in lung tissue stained with PAS. Results are expressed as the number of PAS-positive cells per millimeter of BM. Each column represents the mean ± SEM of three separate experiments (n = 9). ++p < 0.01 compared with 10-d saline group; *p < 0.05 and **p < 0.01 compared with 10-d OVA-exposed, vehicle-treated group.
<b>Figure 7.</b>
Figure 7.
Effect of R406 on goblet cell metaplasia after 10-d OVA exposure. Goblet cell metaplasia was detected by Periodic acid-Schiff (PAS) staining 48 h after saline or OVA exposure. (A) a: 10-d saline exposure; b: 10-d OVA exposure control; c: 10-d OVA exposure after vehicle treatment; d: 10-d OVA exposure after treatment with 3 mg/kg R406; e: 10-d OVA exposure and after treatment with 30 mg/kg R406. (B) Effect of R406 on goblet cell metaplasia after 10-d OVA exposure. Goblet cell metaplasia was quantified in lung tissue stained with PAS. Results are expressed as the number of PAS-positive cells per millimeter of BM. Each column represents the mean ± SEM of three separate experiments (n = 9). ++p < 0.01 compared with 10-d saline group; *p < 0.05 and **p < 0.01 compared with 10-d OVA-exposed, vehicle-treated group.
<b>Figure 8.</b>
Figure 8.
Effect of R406 on AHR in passively sensitized mice. Anti-OVA IgE was administered intravenously, followed by OVA exposure via the airways on 2 consecutive d. Tracheas were isolated and ES50 values were calculated. Each column represents the mean ± SEM of three separate experiments (n = 9). ++p < 0.01 compared with nonsensitized recipients receiving vehicle treatment; *p < 0.05 compared with sensitized and vehicle-treated group.
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