The role of protease-activated receptor-2 on pulmonary neutrophils in the innate immune response to cockroach allergen

Abstract

Background: Serine proteases in German cockroach (GC) have been shown to mediate allergic airway inflammation through the activation of protease activated receptor (PAR)-2. Neutrophils play an important role in regulating the innate immune response, and are recruited into the airways following GC frass exposure. As such, we investigated the role of PAR-2 in airway neutrophil recruitment, activation and cytokine production following allergen exposure.

Methods: Wild type and PAR-2-deficient mice were administered a single intratracheal instillation of PBS or GC frass and neutrophil recruitment, expression of PAR-2, CD80, CD86, and MHC class II were assessed by flow cytometry and levels of tumor necrosis factor (TNF)α was assessed by ELISA. Uptake of AlexaFluor 405-labeled GC frass by neutrophils was performed by flow cytometry.

Results: Neutrophil recruitment in the lung and airways following GC frass exposure was significantly decreased in PAR-2-deficient mice compared to wild type mice. GC frass exposure increased the level of PAR-2 on pulmonary neutrophils and increased numbers of PAR-2-positive neutrophils were found in the lungs; however PAR-2 did not play a role in meditating allergen uptake. Comparing wild type and PAR-2-deficient mice, we found that a single exposure to GC frass increased levels of CD80 and CD86 on pulmonary neutrophils, an effect which was independent of PAR-2 expression. Neutrophils isolated from the whole lungs of naïve PAR-2-deficient mice treated ex vivo with GC frass produced significantly less TNFα than in similarly treated wild type neutrophils. Lastly, neutrophils were isolated from the bronchoalveolar lavage fluid of wild type and PAR-2-deficient mice following a single intratracheal exposure to GC frass. Airway neutrophils from PAR-2-deficient mice released substantially decreased levels of TNFα, suggesting a role for PAR-2 in neutrophil-derived cytokine production.

Conclusions: Together these data suggest PAR-2 expression can be upregulated on lung neutrophils following allergen exposure and the consequence is altered release of TNFα which could drive the early innate immune response.

Figure 1

Neutrophil recruitment into the lung and BAL fluid following allergen challenge. Naïve BALB/c mice were administered a single intratracheal instillation of PBS or GC frass (40 μg/40 μl) and 20 h later, mice were either lavaged to isolate BAL cells or whole lungs were digested and analyzed for neutrophil content by flow cytometry. A. BAL fluid was harvested and differential cell counts performed. Data represent 8 mice per group and are expressed as total cell number mean ± SEM. Statistical significance was determined by ANOVA (*p < 0.001). B. Cells were dissociated from whole lungs and stained for flow cytometry analysis. CD11c + cells were subsequently analyzed for CD11b and Gr1 expression. Data are from a single mouse per group but are representative of 6 mice in each group.

Figure 2

GC frass increased PAR-2 expression on pulmonary neutrophils. Lungs from PBS or GC frass-treated mice were harvested 20 h post exposure and stained for flow cytometric analysis of PAR-2 expression. A. Representative histogram showing PAR-2 expression on gated pulmonary neutrophils from PBS-treated (solid line) or GC frass-treated (broken line) mice. Solid grey histogram depicts staining with isotype control antibody. B. Average PAR-2 MFI. C. Percentage of PAR-2-positive neutrophils. In both cases (B + C), data are expressed as mean ± SEM (n = 8 mice per group) and statistical significance was determined by Student’s t-test (*p < 0.001).

Figure 3

PAR-2 expression does not alter allergen uptake in pulmonary neutrophils. AF405-labeled GC frass was instilled in the airways of wild type and PAR-2-deficient mice and 20 h post exposure, lungs were isolated and stained for flow cytometry. The percentage of AF405-positive pulmonary neutrophils (gated on CD11c+, CD11b+, Gr1+ cells) in the lung are shown and the data are expressed as the mean ± SEM (n = 8 mice per group).

Figure 4

Co-stimulatory molecule expression on pulmonary neutrophils in wild type and PAR-2-deficient mice following exposure to GC frass. BALB/c and PAR-2-deficient mice were administered a single intratracheal instillation of PBS or GC frass and whole lungs were isolated 20 h later. Cells were dissociated from the lungs, and stained for flow cytometry analysis. Neutrophil (gated on CD11c+, CD11b+, Gr1+) populations were analysed for CD86 (A + B), CD80 (C + D), and MHC class II (E + F). MFI is shown in the left panel and the frequency of parent is shown on the right panel. Values are means ± SEM, n = 8 mice per group and statistical analysis was performed by ANOVA (*p < 0.001).

Figure 5

TNFα release is diminished in pulmonary neutrophils from PAR-2-deficient mice compared to wild type mice. A. Whole lungs from naïve BALB/c and PAR-2 mice were dissociated and neutrophils were isolated by Percoll gradient. Neutrophils (1 × 10⁶) were then treated ex vivo with PBS or GC frass and 18 h later, the supernatants were harvested, clarified, and analyzed for TNFα levels by ELISA. Data are expressed as means ± SEM (n = 3 separate experiments run in duplicate) and statistical significance was analyzed by ANOVA (*p < 0.001). B. BALB/c and PAR-2-deficient mice were administered a single intratracheal instillation of GC frass and 18 h later BAL fluid was harvested. Neutrophils (5 × 10⁵) were isolated by Percoll gradient and were cultured without additional treatment for 6 h. Supernatants were then harvested, clarified and analyzed for TNFα levels by ELISA. Data are expressed as means ± SEM (n = 5 mice per group) and statistical significance was analyzed by Student’s t-test (*p = 0.002).