S100A4 Is Critical for a Mouse Model of Allergic Asthma by Impacting Mast Cell Activation

Abstract

Background: The calcium-binding protein S100A4 demonstrates important regulatory roles in many biological processes including tumorigenesis and inflammatory disorders such as allergy. However, the specific mechanism of the contribution of S100A4 to allergic diseases awaits further clarification.

Objective: To address the effect of S100A4 on the regulation of mast cell activation and its impact on allergy.

Methods: Bone marrow-derived cultured mast cells (BMMCs) were derived from wild-type (WT) or S100A4^-/- mice for in vitro investigation. WT and S100A4^-/- mice were induced to develop a passive cutaneous anaphylaxis (PCA) model, a passive systemic anaphylaxis (PSA) model, and an ovalbumin (OVA)-mediated mouse asthma model.

Results: Following OVA/alum-based sensitization and provocation, S100A4^-/- mice demonstrated overall suppressed levels of serum anti-OVA IgE and IgG antibodies and proinflammatory cytokines in serum, bronchoalveolar lavage fluid (BALF), and lung exudates. S100A4^-/- mice exhibited less severe asthma signs which included inflammatory cell infiltration in the lung tissue and BALF, and suppressed mast cell recruitment in the lungs. Reduced levels of antigen reencounter-induced splenocyte proliferation in vitro were recorded in splenocytes from OVA-sensitized and challenged mice that lacked S100A4^-/-. Furthermore, deficiency in the S100A4 gene could dampen mast cell activation both in vitro and in vivo, evidenced by reduced β-hexosaminidase release and compromised PCA and PSA reaction. We also provided evidence supporting the expression of S100A4 by mast cells.

Conclusion: S100A4 is required for mast cell functional activation, and S100A4 may participate in the regulation of allergic responses at least partly through regulating the activation of mast cells.

Keywords: S100A4; airway inflammation; allergic asthma; allergy; mast cell.

Figure 1

S100A4^-/- mice exhibit suppressed antigen-specific antibody and proinflammatory cytokine responses following asthmatic sensitization and provocation. Mice of wild-type (WT) and S100A4–/– strains were sensitized with 20 μg OVA adsorbed to 1 mg alum 4 times i.p. with a 1-week interval. Starting from day 28, mice were challenged with a daily exposure to aerosol of 2% OVA (w/v) for 30 minutes for 7 consecutive days. Control mice were administered with PBS on both occasions as mock immunization and provocation. Mice were killed one day after the last aerosol challenge, and serum was collected for analysis. (A) OVA-specific IgE, IgG, and IgG subclasses were measured using ELISA. (B) Relevant cytokines were analyzed using cytometric bead array analysis. Data are plotted where each dot represents the value of an individual mouse and the horizontal bars represent the mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, using the two-way ANOVY with Tukey multiple comparisons test for statistical significance.

Figure 2

S100A4^-/- mice demonstrate reduced cellular infiltration and cytokine secretion in bronchoalveolar lavage fluid (BALF) following asthmatic sensitization and provocation. Mice of wild-type (WT) and S100A4^–/– strains were induced to develop experimental asthma as described in Figure 1 and BALF was collected. (A) Total BALF cell number enumeration as well as cell type discrimination was carried out by Wright’s staining and microscopy. Data are presented as the mean ± SEM (n = 6). (B) Selected cytokines in BALF were analyzed by cytometric bead array analysis. Data are plotted where each dot represents the value of an individual mouse and the horizontal bars represent the mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, using the two-way ANOVY with Tukey multiple comparisons test for statistical significance.

Figure 3

Cellular infiltration and cytokine secretion in lung tissues are suppressed in S100A4^-/- mice following asthmatic sensitization and provocation. Wild-type (WT) and S100A4^–/– strains of mice were induced to develop experimental asthma as described in Figure 1 and lung tissue exudates were prepared. (A) Haematoxylin-eosin staining was performed and inflammation intensity was scored. (B) Periodic acid-Schiff (PAS) staining was used to reveal goblet cells and relative mucin production based on frequencies of goblet cells was estimated employing a semi-quantitative scoring system. (C) Mast cells were revealed by toluidine blue staining and counted (at least three random fields per tissue section). (D) Single cell suspensions from lung tissues were prepared for the assessment of infiltration of eosinophils (SiglecF⁺CD11c^–), dendritic cells (SiglecF^–CD11c⁺), neutrophils (Gr-1⁺CD11c^– SiglecF^–), and alveolar macrophages (SiglecF⁺CD11c⁺F4/80⁺) by flow cytometric analysis. (E) Selected cytokines in lung tissue exudates were measured using cytometric bead array analysis. Data in (A–D) are presented as the mean ± SEM (n = 6). Data in (E) are plotted where each dot represents the value of an individual mouse and the horizontal bars represent the mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, using the two-way ANOVY with Tukey multiple comparisons test for statistical significance.

Figure 4

S100A4^-/- mice exhibit attenuated immune recall response following asthmatic sensitization and provocation. Wild-type (WT) and S100A4^–/– strains of mice were induced to develop experimental asthma as described in Figure 1 . Splenocytes were harvested and incubated with 1mg/mL OVA for 3 days, and the CCK-8 reagent was added for the last 4 h. (A) Cell proliferation was measured using a spectrophotometry-based CCK-8 assay. Data are presented as the mean ± SEM (n = 6). (B) Supernatants were collected for the measurement of selected cytokines using cytometric bead array analysis. Data are plotted where each dot represents the value of an individual mouse and the horizontal bars represent the mean. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, using the two-way ANOVY with Tukey multiple comparisons test for statistical significance.

Figure 5

S100A4^-/- mast cells demonstrate compromised degranulation and cytokine production after IgE-mediated activation. BMMCs were cultured from wild-type (WT) or S100A4^-/- mice. Cells were pre-treated with 1 µg/ml anti-DNP IgE or vehicle control (PBS) overnight, followed by treatment with 100 µg/ml DNP-HSA. (A) Cell culture supernatant (serum-free) was collected 30 minutes after the addition of DNP-HSA. β-hexosaminidase release was measured using a colorimetric method and the results were shown as OD values. (B) Cell culture supernatant was collected 3 hours after the addition of DNP-HSA. Selected cytokines were analyzed by cytometric bead array analysis. Shown is one representative set of data of 3 experiments. Data are presented as the mean ± SEM (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 using the two-way ANOVY with Tukey multiple comparisons test for statistical significance.

Figure 6

S100A4^-/- mice exhibit reduced passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). (A–D) Wild-type (WT) or S100A4^-/- mice were injected in the ear with anti-DNP IgE (right) or PBS (left) followed by a tail vein injection of DNP-HSA mixed with the Evans blue dye to induce the PCA reaction. The ears were collected 30 minutes later for the quantification of the tissue-extravasated dye, or sectioned to reveal mast cell morphology by toluidine blue staining. PCA reaction is demonstrated visually by dye accumulation in the ears (A) and quantification of the extravasated Evans blue dye in the ear by spectrophotometry (B). Ear mast cell morphology was revealed by toluidine blue staining (C) and the numbers were quantified (D). Red arrows, resting mast cells; green arrows, degranulating mast cells. (E) The WT and S100A4^-/- mice were sensitized intravenously with 200 µL of PBS alone or containing 10 µg anti-DNP IgE followed by intravenously administration with 100 µg DNP-HSA 24 h later, and core body temperature was recorded. Data in (B, E) are presented as the mean ± SEM (n = 6). ***P < 0.001, ****P ≤ 0.0001, respectively using the two-way ANOVY with Tukey multiple comparisons test for statistical significance.

Figure 7

Mast cells express S100A4 and its deficiency does not affect mast cell differentiation. (A) BMMCs were cultured from non-GFP-containing normal C57BL/6 mice or S100A4^+/+.GFP mice. Expression of S100A4 by BMMCs was indicated by GFP expression after 3 weeks of culture. (B) Peritoneal cells obtained from normal C57BL/6 mice or S100A4^+/+.GFP transgenic mice were stained for FcεRI and cKit. FcεRI⁺cKit⁺ mast cells were gated for the analysis of GFP expression which indicates S100A4 expression using histograms. A population of FSC^lowSSC^low cells, which were negative for S100A4, were also gated for analysis as a negative control. (C) S100A4^+/+.GFP mice were sensitized with OVA/alum followed by challenge with OVA aerosol as explained in Figure 1 . Lung tissue sections were prepared followed by staining with cKit and DAPI to show S100A4-expressing (GFP positive) mast cells (magnification, × 400; scale bar, 100 µm).