Phospholipase cε, an effector of ras and rap small GTPases, is required for airway inflammatory response in a mouse model of bronchial asthma

Abstract

Background: Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma.

Methods: We prepared a mouse model of allergic asthma using PLCε+/+ mice and PLCεΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells.

Results: After OVA challenge, the OVA-immunized PLCεΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCεΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCεΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency.

Conclusions: PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.

Figure 1. Attenuated asthmatic response in PLCε ^ΔX/ΔX mice.

(A) AHR to methacholine. AHR was assessed in OVA-sensitized PLCε ^+/+ (filled symbols) and PLCε ^ΔX/ΔX (open symbols) mice 1 day after the last challenge with the aerosol containing OVA (squares) or with vehicle alone (circles). Resistance is expressed as an increase over the baseline set at 1 cmH₂O/ml/s. Data are shown as the mean ± SD obtained with 3 or 4 mice of each group. *, p<0.05 between the OVA-challenged PLCε ^+/+ and PLCε ^ΔX/ΔX mice. (B) H&E staining of airway sections. Airway sections were prepared from the OVA-sensitized mice of the indicated PLCε genotype 1 day after the last challenge either with OVA-containing aerosol or with vehicle alone as indicated. OVA enlarged show the enlargement of the boxed areas in OVA. Bars, 100 µm. (C, D) Frequency of PAS⁺ cells. Airway sections prepared as in (B) were subjected to PAS staining to vitalize mucus-producing cells. Nuclei were counterstained with hematoxylin. Representative sections are shown in (D), where OVA enlarged show the enlargement of the boxed areas in OVA and asterisks denote PAS⁺ cells. PAS⁺ bronchial epithelial cells and total epithelial cells were counted on the specimens prepared from 3 or 5 mice of each group, and the percentage of PAS⁺ epithelial cells was determined as 100×(PAS⁺ cell number)/(total epithelial cell number) (%). Data in (C) are expressed as the mean ± SD. *, p<0.05 between the OVA-challenged two PLCε genotypes. Bars in (D), 100 µm.

Figure 2. Reduced Th2 response in the respiratory system of PLCε ^ΔX/ΔX mice.

(A) Total and differential leukocyte counts. BALF was collected 1 day after the last challenge of the OVA-sensitized PLCε ^+/+ (filled bars) and PLCε ^ΔX/ΔX (open bars) mice with the aerosol containing OVA or with vehicle alone as indicated. For differential leukocyte counting, leukocytes were pelleted from the collected BALF and stained with Diff-Quick. Data are expressed as the mean ± SD obtained with 6 to 10 mice of each group. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., statistically not significant. (B) Cytokine content in BALF. The supernatant of the centrifugation obtained in A was subjected to the determination of the BALF cytokine levels by ELISA. Data are expressed as the mean ± SD. *, p<0.05; ***, p<0.001.

Figure 3. Reduced number of Th2 cells in the thoracic lymph nodes of PLCε ^ΔX/ΔX mice.

(A) Total leukocyte count of the thoracic lymph nodes. All visible thoracic lymph nodes were collected from the sensitized mice with the indicated PLCε genotype 1 day after the last challenge with vehicle alone or OVA (6 to 15 mice of each group), and subjected to isolation of leukocytes. Leukocyte number was determined using a hematocytometer. Data are expressed as the mean ± SD. ***, p<0.001. (B) Flowcytometric analysis of leukocytes. Collected leukocytes in (A) were further analyzed flowcytometrically for the expression of the indicated cell surface antigens. Data are expressed as the mean ± SD. *, p<0.05; **, p<0.01. n.s., statistically not significant.

Figure 4. No effect of the PLCε genotype on OVA-specific IgE and IgGs.

Serums were prepared from the mice with the indicated PLCε genotype 7 days after the last injection of OVA (OVA) or vehicle alone (Sham), and they were subjected to the determination of the indicated Ig levels using ELISA. Lines indicate the mean, and each symbol represents an individual mouse of PLCε ^+/+ (filled symbols) or PLCε ^ΔX/ΔX (open symbols). n.s., statistically not significant.

Figure 5. No effect of the PLCε genotype on the antigen transport by CD11c⁺ dendritic cells.

Fluorescein-conjugated OVA (green) was instilled into the OVA-sensitized PLCε ^+/+ (upper) and PLCε ^ΔX/ΔX (lower) mice. Twenty-four hours later, the thoracic lymph nodes were sampled for staining for CD11c (red) and fluorescence-microscopically observed. Cells doubly positive for fluorescein-conjugated OVA and CD11c (yellow in Merged) were identified as CD11c⁺ dendritic cells carrying OVA. Bars, 100 µm.

Figure 6. Attenuation of inflammatory cytokine production by PLCε deficiency.

(A) Comparison of cytokine mRNA levels of the whole lungs of the OVA-challenged PLCε ^+/+ and PLCε ^ΔX/ΔX mice. OVA-sensitized mice were challenged with OVA, and 1 day later, their whole lungs were collected for RNA preparation. RNA was pooled from the lungs of 6 animals of each group and subjected to qRT-PCR to determine the relative cytokine mRNA levels. Fold difference was obtained by dividing the mRNA level in OVA-challenged PLCε ^+/+ mice with that in OVA-challenged PLCε ^ΔX/ΔX mice. p values shown were derived from the comparisons between the OVA-challenged two PLCε genotypes. (B) Immunostaining for cytokines and PLCε. OVA-sensitized mice of the indicated PLCε genotype were challenged with vehicle alone or OVA, and 1 day later the airway sections were prepared for immunostaining for Ccl2 (red in upper) and Cxcl2 (red in lower) as well as PLCε (green). The lipase-dead mutant PLCε in PLCε ^ΔX/ΔX mice could also be detected by the anti-PLCε antibody against the C-terminus of PLCε. Nuclei were visualized by 4′,6-Diamidino-2-Phenylindole (DAPI) staining (blue). Enlarged show the enlargement of the boxed areas in Merged. Bars, 100 µm.

Figure 7. Inhibition of TNF-α-induced cytokine gene activation by PLCε deficiency in primary-cultured bronchial epithelial cells.

(A) Assessment of PLCε expression in primary-cultured bronchial epithelial cells was by RT-PCR. Primary cultures of bronchial epithelial cells were prepared from naïve adult PLCε ^+/+ mice. RNA was prepared for the first-strand preparation with (+) or without (−) reverse transcriptase (RTase) as indicated. (B) Effects of the PLCε genotype on Ccl2 and Cxcl2 expression induced by TNF-α. Primary cultures of bronchial epithelial cells prepared from naïve adult PLCε ^+/+ (filled bars) and PLCε ^ΔX/ΔX (open bars) mice were treated without (−) or with (+) 10 ng/ml TNF-α for 3 h. RNA was purified and subjected to qRT-PCR to determine the mRNA levels of Ccl2 and Cxcl2. Data are representative of three independent experiments and expressed as the mean ± SD obtained by triplicate determinations. *, p<0.05; **, p<0.01; ***, p<0.001 between PLCε ^+/+ (filled bars) and PLCε ^ΔX/ΔX (open bars) cells stimulated with TNF-α. (C) Effects of intracellular signaling inhibitors on Ccl2 expression induced by TNF-α stimulation. Primary-cultured PLCε ^+/+ bronchial epithelial cells prepared as in (B) were pretreated with dimethyl sulfoxide vehicle alone (−), 3 µM IKK inhibitor III (IKK), or 5 µM U73122 (U) for 10 min. Subsequently, they were stimulated with 10 ng/ml TNF-α for 3 h. QRT-PCR was carried out to determine the Ccl2 mRNA level, and data are presented as in (B). ***, p<0.001; n.s., statistically not significant.