Murine lung eosinophil activation and chemokine production in allergic airway inflammation

Abstract

Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1alpha, MIP-1gamma, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1gamma in inflamed lungs. Eosinophil production of C10 and MIP-1gamma correlated with the marked influx of CD11b(high) lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation.

Figure 1

Comparison of lung eosinophil, macrophage and DC surface markers in naive mice. Lung digests were prepared from naive mice as described in the ‘Materials and methods' section and stained with FITC-conjugated F4/80 for eosinophil enrichment by anti-FITC conjugated magnetic microbeads. (A) F4/80⁺ cells were resolved into three populations identified as Eos, Mac and DCs. The surface markers of the gated Eos and Mac populations are shown in (C). (B) The F4/80⁻ cells in the flow-through after anti-F4/80 magnetic bead selection were further selected by anti-CD11c-conjugated magnetic beads and stained with mAbs against I-A, CD11c and surface markers shown in (C). The surface markers of the gated DC population are shown in (C), bottom panel. The marker expression of F4/80⁺ and F4/80⁻ macrophages in panels A and B respectively was similar. The results are representative of four independent experiments. The percentages of marker positive cells are shown in the zebra plots. DC, dendritic cell; Eos, eosinophils; mAb, monoclonal antibody; Mac, macrophages.

Figure 2

Lung and splenic eosinophil surface costimulation and adhesion molecules in naive mice. Lung and splenic single cell suspensions were clear of CD11c⁺ DCs and macrophages by anti-CD11c-magnetic beads followed by F4/80 selection as described in Figure 1. The cleared CD11c⁺ cells contained no eosinophils. Live cells gated on eosinophils as shown in (d) were analyzed for costimulation and adhesion molecules shown in (a–c). The experiment has been repeated three times. DC, dendritic cell.

Figure 3

Stimulation of T-cell proliferation by lung eosinophils and DCs. Lung eosinophils and DCs from naive mice were sorted to 98% purity as described in the ‘Materials and methods' section and added to cell cultures in the numbers per well as indicated. CD4⁺CD25⁻ splenic T cells were obtained from DO11.10 mice by magnetic microbead depletion as described in the ‘Materials and methods' section and used at 1×10⁵/well. OVA_323−339 peptide and anti-CD3 mAbs were added to a final concentration of 25 µM and 8 µg/ml respectively and incubated for 5 d. [³H]-thymidine was added for the last 8 h incubation. The experiment was repeated twice with similar results. APC, antigen presenting cell; DC, dendritic cell; Eos, eosinophils; mAb, monoclonal antibody; OVA, ovalbumin.

Figure 4

Blood and lung eosinophils. (A) Quantitation of blood eosinophils. Blood leukocytes after red blood cell lysis were stained by 7-amino-actinomycin D (7-AAD) and mAbs against I-A, CCR3, Siglec-F and CD11b and live cells were successively gated and analyzed as shown in the figure. Eosinophil numbers in (d) were quantitated with Caltag calibration beads. Four such determinations of blood cells from multiple mice in each experiment have been performed. (B) Lung eosinophil staining. Cells from total lung digests of naive mice (a) were selected by anti-F4/80 followed by magnetic microbeads, stained with fluorochrome-conjugated mAbs against I-A, Siglec-F, CD11b, CCR3 and GR-1, and sorted (c). Total lung cells from mice immunized with OVA (b) were similarly stained and sorted (d) without preselection with F4/80 magnetic microbead. Cytospin preparations of total lung cells (a, b) and sorted eosinophils (c, d) were stained with Diff-Quik. This experiment has been repeated twice. mAb, monoclonal antibody; OVA, ovalbumin.

Figure 5

Allergic airway inflammation in mice. BALB/c mice were immunized as described in the ‘Materials and methods' section and in (A). (B) Airway resistance was measured and expressed as APTI. A total of eight mice were used per group. Bronchoalveolar lavage cell number (C) and differential (D) were determined as described. OVA-specific IgG were determined by ELISA and expressed as units against a reference standard (E). In (F), hematoxylin and eosin staining of control and immunuized lung sections are shown in ×2 magnification. The experiments have been repeated three times. Bars show SD and significance of differences between groups are shown as: *P<0.05; ***P<0.005. AHR, airway hyper-responsiveness; APTI, airway pressure time index; ar, arterioles; br, bronchioles; Eos, eosinophils; Ig, immunoglobulin; Mac, macrophages; OVA, ovalbumin; PMN, neutrophils.

Figure 6

Surface marker, costimulation molecule, and adhesion molecule expression on eosinophils from immunized mice. Mice were immunized as in Figure 5A and eosinophils gated as in (A-a) and 7-AAD⁻ live cells were analyzed for marker expression shown in (A). These cells were further gated on Siglec-F⁺ and CD11b^high cells and assessed for their expression of costimulation and adhesion molecules in (B). A representative experiment of three trials is shown. APC, antigen presenting cell; IgG, immunoglobulin G.

Figure 7

Expression of chemokine mRNA and protein by eosinophils from naive and OVA-immunized mice. (A) Eosinophils from naive or OVA-immunized mice were sorted to 98% purity as described in the ‘Materials and methods' section and lysed in RNeasy for total RNA preparation. cDNA were prepared and amplified by real-time PCR with primers described previously. The PCR products at the end of 40 cycles were run on a 1.8% agarose gel to confirm amplification of the correct product. The lanes were: -, no cDNA; C, eosinophil cDNA from control naive mice; O, eosinophil cDNA from OVA-immunized mice. (B, C) Intracellular chemokine expression in eosinophils from naive mice (B) or OVA-immunized mice (C). Intracellular chemokine staining was performed as described in the ‘Materials and methods' section. Eosinophils were enriched by anti-F4/80 mAbs plus magnetic microbeads for control lung cells (B) but not for lung cells from OVA-immunized mice (C). Cells were either stained for eosinophil markers followed by fixation immediately after cell isolation (0 h) or incubated in medium with monensin for 4 h (4 h) before staining and fixation. (D) Chemokine production by sorted eosinophils from naive mice or OVA-immunized mice. Eosinophils were sorted as described in (A) and cultured at 0.5×10⁶/ml as described in Materials and methods. Supernatants were assayed for chemokines by ELISA. Error bars show the standard deviation of triplicates. Significance in the differences of chemokine production are shown as: *P<0.05; **P<0.01; ***P<0.005. (A–C) were repeated three times and (D) twice. b.p., base pairs; ELC, Epstein–Barr virus-induced molecule-1 ligand chemokine; IgG, immunoglobulin G; mAb, monoclonal antibody; MCP, monocyte chemoattractant protein; MEC, mucosa-associated epithelial chemokine; MIP, macrophage inflammatory protein; OVA, ovalbumin; TCA, thymus-derived chemotactic agent.

Figure 8

Eosinophil production of chemokines in mice with allergic lung inflammation. Mice were immunized as shown in Figure 5A, fixed in paraformaldehyde, equilibrated in sucrose and sectioned. In (a–e), sections were stained with mouse anti-I-A-FITC mAbs, rabbit antichemokine Abs, and rat anti-mMBP mAbs followed by anti-FITC-Alexa dye 488, antirabbit IgG-Alexa dye 555, and antirat IgG-Alexa dye 647. In (f), anti-CD11b-FITC instead of anti-I-A-FITC was used. Confocal microscopy was performed as described. Arrows indicate eosinophils and arrowheads show non-eosinophil cells. Bars in the left column represent 50 µm and those in the right column are 5 µm. The staining has been repeated three times on different tissue blocks. Ab, antibody; ar, arterioles; br, bronchioles; ELC, Epstein–Barr virus-induced molecule-1 ligand chemokine; IgG, immunoglobulin G; mAb, monoclonal antibody; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; mMBP; murine major basic protein.

Figure 9

Lung DC accumulation during allergic airway inflammation and chemokine receptor expression. (a) Lung CD11b^high DCs (gray bars) and CD103⁺ DCs (black bars) numbers from control saline-injected mice and OVA-immunized mice were determined by flow cytometry as described in the ‘Materials and methods' section. The mean and SD from 11 mice in each group are shown. (b) Chemokine receptor mRNA levels of CD11b^high DCs and CD103⁺ DCs were determined by Affymetrix microarrays and the mean and SD of three determinations are shown. ***P<0.005; ****P<0.001. DC, dendritic cell; OVA, ovalbumin.