Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-TAT

Abstract

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Deltap85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85alpha, which was fused to HIV-TAT (TAT-Deltap85). Intraperitoneal administration of TAT-Deltap85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3-10 mg/kg of TAT-Deltap85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Deltap85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.

Figure 1.

Purified proteins used in this study. (a) Structure of TAT-Δp85 fusion protein. Six His residues and the 11–amino acid TAT peptide precede the NH₂-terminal of the Δp85 protein. The 11 amino acids of TAT are the protein transduction domain (PTD). (b) Control Δp85 protein lacking the PTD. (c) TAT-GFP, a control protein lacking Δp85.

Figure 2.

Kinetics of TAT-Δp85 transduction into whole-blood leukocytes in mice measured by flow cytometry. Whole-blood leukocytes were isolated at 0, 2, 6, 12, and 24 h after i.p. injection of FITC-labeled TAT-Δp85 to determine time-course and specificity of transmembrane uptake of TAT-Δp85. Nonspecific cell-surface fluorescence was quenched by resuspension in pH 6.8 HBSS. (a) Conjugated TAT-Δp85 demonstrates maximal transcellular uptake at 2 h and is progressively diminished over 24 h. (b) FITC-conjugated control Δp85 protein, which lacks the HIV-TAT domain, demonstrates no change in fluorescence versus after resuspension in pH 6.8 HBSS.

Figure 3.

Western blot analysis of the uptake of TAT-Δp85 into lung tissue and the efficacy of TAT-Δp85 protein transduction on FMLP-induced PKB phosphorylation in lung tissues. (A) Transduction of TAT-Δp85 into the lung. Lungs were excised from mice at indicated times after i.p. administration of TAT-Δp85 (10 mg/kg), and lung tissue extracts were mixed with loading buffer and separated by SDS-PAGE, and probed by anti-His antibody. (B) Western blot analysis of the effect of TAT-Δp85 on FMLP-induced PKB phosphorylation in whole-lung tissue. FMLP was instilled intranasally at 0, 2, 4, 6, 12, and 24 h after administration of TAT-Δp85. Whole-lung cell extracts were prepared and analyzed by Western blot with antiphosphorylation-specific PKB Ab (top). Equivalency of loading was established for each lane with anti-PKB Ab, which measures total PKB (phosphorylated and nonphosphorylated) (bottom). Inhibition of PKB phosphorylation is maximal at 2–6 h, corresponding to fluorescence data from Fig. 2. By 12 h, there is diminished inhibition of PKB phosphorylation by TAT-Δp85. The result shown is representative of three different experiments.

Figure 4.

Effect of IP administration of TAT-Δp85 on airway inflammation after OVA sensitization and challenge. Analysis of inflammatory cells from BAL fluid 24 h after last OVA challenge. Positive (▪) and negative (□) controls are OVA and saline challenged animals, respectively. These animals received no other treatment. OVA challenge caused increased total cell count, which is largely composed of eosinophils. Increase in lymphocytes and neutrophils are recorded on a different scale (see ordinate). There was no effect on macrophage number. Migration of eosinophils and lymphocytes is blocked progressively with 3–10 mg/kg TAT-Δp85 but unaffected by 10 mg/kg Δp85 lacking the TAT domain. TAT-Δp85 has no effect on the migration of neutrophils, which is minimal. Each bar represents the mean ± SEM of 4–6 mice. *P < 0.05 and **P < 0.01 compared with positive control group. ND, not detectable.

Figure 5.

Effects of TAT-Δp85 on eosinophil accumulation in BAL fluid 12 h after intranasal administration of IL-5. Cell counts were obtained from animals receiving IL-5 + either control Δp85 (lacking TAT domain) or TAT-Δp85. Eosinophils were not detectable in airways of animals before challenge with IL-5. TAT-Δp85 blocked progressively the eosinophil migration caused by IL-5. Δp85 lacking the TAT domain was not different from IL-5 alone. Accordingly, TAT-Δp85 blocks both IL-5 synthesis (Fig. 6) as well as the chemotactic effects of IL-5 on eosinophils. Each bar represents the mean ± SEM of 4–6 mice. *P < 0.05 and **P < 0.01 compared with positive control group. ND, not detectable.

Figure 6.

Effect of TAT-Δp85 on Th1 and Th2 cytokine secretion after four OVA challenges on days 21, 22, 23, and 24. BAL fluids were collected 24 h after the last OVA challenge. Positive (▪) and negative (□) controls are OVA and saline challenged animals, respectively, without any pretreatment. TAT-Δp85 reduces the level of Th2 cytokines, IL-4 and IL-5, but has no effect on the Th1 cytokine IFNγ. Note difference in scale on the ordinate. Each bar represents the mean ± SEM of 4–6 mice. **P < 0.01 compared with positive control group. ND, not detectable.

Figure 7.

Histologic examination of lung tissues. (A) Representative sections of the lungs. The lung sections were obtained from saline- (1, 4) or OVA- (2, 3, 5, 6) challenged mice in the absence (1, 2, 4, 5) or presence (3, 6) of TAT-Δp85 (10 mg/kg). Sections are stained with haematoxylin and eosin (1, 2, 3) for morphological analysis of inflammation and with alcian blue/PAS (4, 5, 6) for analysis of mucin-containing cells. Tissue was examined by light microscopy (original magnification 400×). Substantial inflammation and mucin production caused by OVA (2, 5) are blocked by TAT-Δp85 (3, 6); compare with saline control (1, 4). (B) Semiquantitative analysis of the severity of peribronchial inflammation and the abundance of PAS-positive mucus-containing cells. Total lung inflammation and PAS-positive cells were defined as the average of the scores as described in Materials and Methods. *P < 0.05 and **P < 0.01 compared with positive control group.

Figure 8.

Effect of TAT-Δp85 on antigen-induced airway hyperresponsiveness to methacholine in immunized mice following four OVA challenges on days 21, 22, 23 and 24. Airway responsiveness was measured 24 h after the last OVA challenge. Positive (□) and negative (Δ) controls are OVA and saline challenged animals, respectively, without any pretreatment. Δp85 lacking the TAT domain has no attenuating effect on antigen challenge in immune sensitized mice (□). By contrast, 10 mg/kg TAT-Δp85 (▪) blocked methacholine responsiveness in sensitized animals to control level (Δ), i.e., the response was comparable to that of sensitized, saline-challenged animals. Each bar represents the mean ± SEM of 4–6 mice. *P < 0.05 and **P < 0.01 compared with positive control group.