Adoptive transfer of IL-4Rα+ macrophages is sufficient to enhance eosinophilic inflammation in a mouse model of allergic lung inflammation

Abstract

Background: The IL-4 receptor α (IL-4Rα) chain has a broad expression pattern and participates in IL-4 and IL-13 signaling, allowing it to influence several pathological components of allergic lung inflammation. We previously reported that IL-4Rα expression on both bone marrow-derived and non-bone marrow-derived cells contributed to the severity of allergic lung inflammation. There was a correlation between the number of macrophages expressing the IL-4Rα, CD11b, and IA(d), and the degree of eosinophilia in ovalbumin challenged mice. The engagement of the IL-4Rα by IL-4 or IL-13 is able to stimulate the alternative activation of macrophages (AAM). The presence of AAM has been correlated with inflammatory responses to parasites and allergens. Therefore, we hypothesized that IL-4Rα⁺ AAM play an active role in allergic lung inflammation. To directly determine the role of AAM in allergic lung inflammation, M-CSF-dependent macrophages (BMM) were prepared from the bone-marrow of IL-4Rα positive and negative mice and transferred to IL-4RαxRAG2(-/-) mice. Wild type TH2 cells were provided exogenously.

Results: Mice receiving IL-4Rα(+/+) BMM showed a marked increase in the recruitment of eosinophils to the lung after challenge with ovalbumin as compared to mice receiving IL-4Rα(-/-) BMM. As expected, the eosinophilic inflammation was dependent on the presence of TH2 cells. Furthermore, we observed an increase in cells expressing F4/80 and Mac3, and the AAM marker YM1/2 in the lungs of mice receiving IL-4Rα(+/+) BMM. The BAL fluid from these mice contained elevated levels of eotaxin-1, RANTES, and CCL2.

Conclusions: These results demonstrate that transfer of IL-4Rα + macrophages is sufficient to enhance TH2-driven, allergic inflammation. They further show that stimulation of macrophages through IL-4Rα leads to their alternative activation and positive contribution to the TH2-driven allergic inflammatory response in the lung. Since an increase in AAM and their products has been observed in patients with asthma exacerbations, these results suggest that AAM may be targeted to alleviate exacerbations.

Figure 1

AAM are present in the lungs of chimeric mice receiving IL-4Rα^+/+ bone-marrow. Balb/c IL-4RαxRAG2^-/- mice were irradiated and reconstituted with bone-marrow cells isolated from IL-4Rα^-/- or IL-4Rα^+/+ mice as previously described [16]. Six weeks later, the mice were provided with OVA specific TH2 effectors (1 × 10⁷/mouse) by tail vein injection. They were then sensitized to 100 μg chicken egg ovalbumin (OVA) adsorbed to aluminum hydroxide (Alum) via i.p. injection on day 0 and boosted with the same reagent on day 14. Mice were exposed to 1% OVA in PBS by nebulization for 20 min each day on days 19, 22, and 27. Lung sections from OVA/alum primed mice were prepared and stained with H&E, anti-F4/80, anti-YM1/2, or control rabbit IgG as indicated. Digital images from a representative mouse (n = 4) for the 2 groups are shown (20X). These results are representative of 3 independent experiments.

Figure 2

Protocol for macrophage transfer and inflammation induction. Bone-marrow derived macrophages (BMM) were derived from IL-4Rα^+/+ or IL-4Rα^-/- mice on the RAG2^-/- background as described in the Materials and Methods. BMM were washed and transferred to BALB/c IL-4RαxRAG2^-/- mice by IP injection at 5 × 10⁶ cells per mouse. The next day, in vitro differentiated OVA-specific TH2 cells were injected IV at 1 × 10⁷ per mouse. They were then sensitized to 100 μg chicken egg ovalbumin (OVA) adsorbed to aluminum hydroxide (Alum) via i.p. injection on day 0 and boosted with the same reagent on day 14. Mice were exposed to 1% OVA in PBS by nebulization for 20 min each day on days 19, 22, and 27.

Figure 3

Phenotype of macrophages pre- and post- transfer. A. BMM were prepared from RAG2^-/- and IL-4RαxRAG2^-/- mice and were stained with anti-CD11b (solid line) or control antibody (dotted line) as indicated and analyzed by FACS. B. BMM were prepared from RAG2^-/- (solid line) and IL-4RαxRAG2^-/- (dotted line) mice and were stained with anti-IL-4Rα as indicated and analyzed by FACS. C. BMM were prepared from RAG2^-/- (solid line) and IL-4RαxRAG2^-/- (dotted line) mice and transferred to BALB/c IL-4RαxRAG2^-/- mice by IP injection at 5 × 10⁶ cells per mouse. Six weeks later spleens were harvested and stained with anti-CD11b and anti-IL-4Rα and analyzed by FACS. Live spleen cells were gated on expression of CD11b; the IL-4Rα staining histograms are shown. These results are representative of 3 independent experiments. D. BMM were prepared from RAG2^-/- (IL-4Rα^+/+) and IL-4RαxRAG2^-/- (IL-4Rα^-/-) mice and transferred to IL-4RαxRAG2^-/- mice by IP injection at 5 x10⁶ cells per mouse. Six weeks later spleens were harvested and single cell suspensions were prepared. In addition, single cell suspensions were prepared from untreated BALB/c mice. Pooled spleen cells were cultured in the presence or absence of IL-4 (10 ng/ml) for 30 min as indicated. Cell lysates were prepared and immunoprecipitated with anti-STAT6. The precipitates were analyzed by western blotting with anti-phosphotyrosine antibody. The membranes were stripped and re-probed with anti-STAT6.

Figure 4

Transfer of IL-4Rα^+/+ BMM enhances the TH2-driven, OVA-induced eosinophilic inflammation in the lungs and airways of mice. BMM were prepared from IL-4Rα^+/+ or IL-4Rα^-/- mice as described above. BMM (5 × 10⁶) were transfered by IP injection to IL-4RαxRAG2^-/- mice on day -1. On day 0 mice were injected with PBS alone or with TH2 cells derived from D011.10 mice as indicated (1 × 10⁷). The mice were immunized with OVA/alum on day 1 as indicated, followed by boost and challenge as described in Figure 2. Lung sections from OVA/alum primed mice were prepared and stained with H&E. A. Several digital images (A-F) from a representative mouse from each group (n = 4) are shown at 10 and 20X. B. The slides from individual mice were evaluated for the degree of pathology (0-3) as described in Materials and Methods. The mean score for each group is represented in the form of bar graphs ± SEM. In most cases the SEM was too small to show on the graph. Significant differences between the two groups noted were determined using the Student's T-test. ** (p < 0.01) C. BMM were prepared as described above and transfered by IP injection to IL-4RαxRAG2^-/- mice on day -1. On day 0 all mice were injected with TH2 cells derived from D011.10 mice (1 × 10⁷). The mice were immunized with alum or OVA/alum on day 1 as indicated, followed by boost and challenge as described in Figure 2. The BAL from individual mice (n = 3-4) was evaluated by differential counting after cytospin. Total numbers of cells present in the BAL are represented in the form of bar graphs ± SEM (T, total cells; M, macrophages; E, eosinophils; L, lymphocytes; and P, polymorphonuclear leukocytes). ** Significant differences between the two groups noted were determined using the Student's T-test (p < 0.01). D. The percentage of eosinophils in the BAL isolated from the individual mice is shown with the mean of the values represented by the bar. Significant differences between groups were determined using the Student's T-test. * (p < 0.05) These results are representative of 3 independent experiments.

Figure 5

Phenotype of macrophages in the lungs of recipient mice. BMM were prepared from IL-4Rα^+/+ or IL-4Rα^-/- mice as described above. Where indicated BMM (5 × 10⁶) were transfered by IP injection to IL-4RαxRAG2^-/- mice on day -1. On day 0 all mice were injected with TH2 cells derived from D011.10 mice (1 × 10⁷). The mice were immunized with OVA/alum on day 1 as indicated, followed by boost and challenge as described in Figure 2. Lung digests were prepared from mice (n = 4) as described in Materials and Methods, pooled, and counted. Lung cells were stained with anti-CD11b, and anti-F4/80, along with anti-IL-4Rα or control IgG. A. Live cells were gated on forward by side scatter and analyzed for F4/80 by CD11b staining. B. The cells were gated for high expression of CD11b and F4/80 and analyzed for expression of IL-4Rα. The histograms comparing control staining (dotted line) to anti-IL-4Rα staining (solid line) of the gated cells is shown. C. The numbers of cells expressing the markers were calculated using the percentages of cells in each quadrant in Panel (A) and the total lung cells recovered. These results are representative of 2 independent experiments.

Figure 6

Transfer of IL-4Rα^+/+ BMM leads to the presence of AAM in the lungs of mice. A. Lung sections from the OVA/alum primed mice shown in Figure 4 were stained with anti-F4/80, anti-YM1/2, or anti-CD3 as indicated. Images from a representative mouse from each group are shown at 10X or 20X as indicated. CD3+ cells appear brown. B. The CD3+ cells in each 20X field were counted (n = 5). The average number of CD3+ cells per field were calculated and graphed. Data represented as cell counts ± SEM. These results are representative of 3 independent experiments.

Figure 7

YM1/2 and Mac3 staining coincide in the allergic lung. A. Serial sections of lungs from a representative OVA/alum-primed mouse that received IL-4Rα^+/+ BMM were stained with H&E, anti-F4/80, anti-Mac3, or anti-YM1/2 as shown. Digital images were taken from the same region of the lung at 40X and 100X. Arrow heads highlight the staining of representative macrophages in the airways, while the asterisk highlights staining of macrophages surrounding blood vessels. B. Co-expression by immunofluorescence microscopy. A representative lung section was stained with rat anti-Mac3 and rabbit anti-YM1/2 (or control serum), followed by goat anti-Rat-PE and goat anti-rabbit-FITC. Nuclei were stained with DAPI. Digital images were captured for each color and processed as described in Materials and Methods. The 3 color images were merged into a single image. Appearance of yellow indicates expression of both Mac3 and YM1/2. C. Five fields were analyzed for the total number of nuclei, total number of cells staining red only, number of cells staining green only, and number of cells with both colors (yellow). The percentage of cells in each category was calculated by dividing each number by the total number of nuclei per field. These results are representative of 2 independent experiments.

Figure 8

Transfer of IL-4Rα⁺ BMM alters cytokine and chemokine levels in the bronchalveolar lavage fluid (BAL). BMM were prepared from IL-4Rα^+/+ or IL-4Rα^-/- mice as described above. BMM (5 × 10⁶) were transfered by IP injection to IL-4RαxRAG2^-/- mice on day -1. On day 0 all mice were injected with TH2 cells derived from D011.10 mice (1 × 10⁷). The mice were immunized with OVA/alum on day 1 as indicated, followed by boost and challenge as described in Figure 2. The BAL fluid from individual mice (n = 3-4) was evaluated for cytokine and chemokine levels using Pierce Searchlight. The mean +/- SEM is shown. These results are representative of 3 independent experiments. Significant differences between groups was determined using the Student's T-test.