Figure 1.
Purification and characterization of mouse eosinophils from IL-5 Tg mice.
(A). FACS and cytospin analysis of peripheral blood “buffy coat” cells prior to and after MACS column purification. Peripheral blood from IL-5 Tg mice was spun over Percoll, the red cells were lysed and the eosinophils were purified using a negative separation MACS column-based strategy as described [17] with modifications [18]. The purity of the eosinophil preparation was assessed by FACS double-staining for the surface markers, Gr-1 and CCR3, as well as Siglec F and by examination of cytospins following DiffQuik staining (40×). Red-stained eosinophilic granules are clearly visible in the cells at 100× magnification. (B). Eosinophils were purified as described in the Methods. Eosinophils (5 × 105 per Transwell insert) were placed into the upper chamber of a 24-well Transwell chemotaxis assay plate in duplicate with increasing concentrations of eotaxin-1 in the lower chamber and incubated at 37°C for 2 hours [18]. The total number of live cells that had migrated into the bottom chamber at each concentration was counted and divided by the number that migrated to medium alone to give the fold change above control, which was plotted against eotaxin-1 concentration. (C). Migration of mouse eosinophils to CCR1, CCR3, CCR4 agonists and BALF from Alum- and OVA/Alum-sensitized and challenged mice. Thirty nanomolar solutions of each chemokine or BALF from mice subjected to ovalbumin sensitization and challenge or alum control diluted 1∶4 [15] were used in the chemotaxis assay as described in (B) in triplicate. Migrated cells were collected after 2 hours and counted by FACS with CountBright™ beads as described in the Methods. (*P<0.05, compared to medium control or Alum-treated control).
Figure 2.
Expression of mRNA and surface protein for components of the IL-4/IL-13 receptor complex on mouse eosinophils.
(A). Expression of mRNA for the different subunits of the IL-4/IL-13 receptor complex. RNA was extracted from purified eosinophils and the mouse macrophage cell line, RAW 264.7, and cDNA was synthesized. Relative quantitation by RealTime PCR with specific primer pairs for each receptor subunit was performed, using the standard 2−ΔΔCt method, and the products were resolved on a 3% agarose gel. The amount of expressed mRNA for each subunit from mouse eosinophils (filled bars) relative to the amount in the control RAW cell line ( = 1, open bars) was graphed. (B). Cell surface expression of the IL-4/IL-13 receptor subunits on mouse eosinophils. FACS analysis using specific antibodies to each receptor subunit was performed. Representative FACS histograms from one experiment (of four to five independent experiments) are shown with staining with specific antibody (solid black line) and isotype-matched control antibody (dotted line). Receptor staining on mouse bone marrow-derived macrophages from wildtype and γC-deficient mice, generated as described [12], and to RAW 264.7 cells is shown for comparison.
Figure 3.
Migration of purified mouse eosinophils to IL-4 and IL-13 in the presence or absence of eotaxin-1.
(A). Eosinophil chemotaxis to IL-4 (left panel) and IL-13 (right panel) in the presence (filled bars) or absence (open bars) of 30 nM eotaxin-1 was measured. The indicated concentrations of IL-4 were placed in the lower chamber and 5 x 105 eosinophils were placed in the upper chamber. A representative experiment is shown. (B). Average data from multiple independent chemotaxis experiments performed as in (A) using IL-4 or IL-13 with (squares) or without (circles) eotaxin in the lower chamber (n = 2–4). For the IL-4 graph, number of migrated cells to medium = 9,733± SEM 2,468 and to eotaxin-1 = 54,444± SEM 10,926 and for the IL-13 graph, number of migrated cells to medium = 4,478± SEM 689 and to eotaxin-1 = 56,654± SEM 4,949.
Figure 4.
Pre-treatment with IL-4 but not IL-13 enhanced migration of purified mouse eosinophils to eotaxin-1 and eotaxin-2.
Eosinophils were purified as described in the Methods. (A). Eosinophils were then incubated with various concentrations of IL-4 or IL-13 at 37°C for 30 minutes. The treated cells were washed and then placed (5×105) into the upper chamber insert of the Transwell plate; the lower chamber contained either 0 or 30 nM eotaxin-1. The chemotaxis assay was performed and the number of migrated cells was counted as described in Figure 1. A representative experiment is shown. (B) Average data from three independent chemotaxis experiments (n = 3; *P<0.05, compared to migration to eotaxin-1 alone). For the IL-4 graph, number of migrated cells to medium = 11,614± SEM 3,257 and to eotaxin-1 = 102,435± SEM 27,964 and for the IL-13 graph, number of migrated cells to medium = 8,581± SEM 2,163 and to eotaxin-1 = 75,651± SEM 20,436. (C, left panel) Time of IL-4 pre-treatment of eosinophils prior to chemotaxis. Eosinophils were pre-treated with 10 ng/mL IL-4 for the indicated times, subjected to chemotaxis to 30 nM eotaxin-1 and counting as in (A). P<0.05. (C, right panel) Eosinophils were pre-treated with 10 ng/mL IL-4 for the indicated time, subjected to chemotaxis to 30 nM eotaxin-2 and counting as in (A). P<0.05.
Figure 5.
Signaling responses to IL-4 and IL-13 in mouse eosinophils.
(A) Eosinophils were purified as described in the Methods. The cells were serum-starved for 2 hours prior to stimulation with 20 ng/mL IL-4 (“4”) or IL-13 (“13”) for 15 mins or no stimulation (“–”). Eosinophil cell lysates were prepared as described in the Methods and subjected to SDS-PAGE and Western blotting with antibodies specific for the phosphorylated form of the particular signaling protein. Blots were stripped and re-probed with antibodies specific for the unphosphorylated form of the protein. Films from one representative experiment are shown. (B). Densitometric analysis of Western blot films. Films from independent experiments were scanned and the amount of phosphoprotein/total target protein was quantitated by densitometry. This ratio was normalized for loading (to α-tubulin) and graphed with the amount of phosphoprotein in unstimulated cells equal to 1). Average data from three to four independent experiments are shown (*P<0.05).
Figure 6.
Effect of pre-treatment with IL-4 and IL-13 on CCR3 expression and eotaxin-1 signaling in mouse eosinophils.
(A). Eosinophils were treated with (heavy solid line) or without (light solid line) 100 ng/mL IL-4 (left panel) or IL-13 (right panel) for 30 min and FACS analysis with specific antibodies to CCR3 (solid lines), an isotype control (dotted) or unstained (dashed line) was performed as described (Figure 1). (B) The cells were serum-starved for 2 hours prior to stimulation with 20 ng/mL IL-4 (“4”) or IL-13 (“13”) for 15 mins or 30 nM eotaxin-1 (“eot”) for 1 minute or no stimulation (“–”) as indicated. Cell lysates were prepared and analyzed by Western blotting as Figure 5.
Figure 7.
Migration of bone marrow-derived eosinophils (bmEos) from γC+/+ and γC−/− mice to eotaxin-1.
Eosinophils were differentiated from the bone marrow of γC+/+ (black bars) or γC−/− (open bars) mice in vitro as described in the Methods. The cells were either untreated or primed with 10 ng/mL IL-4 for 30 min prior to placement into the upper chamber of the Transwell chemotaxis assay. Eotaxin-1 (30 nM) was placed in the lower chamber. Migrated eosinophils from triplicate wells were counted as described in Figure 1. The average data from two experiments performed in triplicate are shown (* P<0.05, compared to medium control). For γC+/+, mean number of cells migrated to medium = 44,567± SEM 10,947; to eotaxin-1 = 155,817± SEM 34,864 and IL-4 pre-treated cells to eotaxin-1 = 217,043± SEM 24,531. For γC−/−, mean number of cells migrated to medium = 51,640± SEM 12,840; to eotaxin-1 = 149,862± SEM 13,133 and IL-4 pre-treated cells to eotaxin-1 = 145,768± SEM 19,289.
Figure 8.
Proposed mechanism of IL-4 enhancement of mouse eosinophil chemotaxis to eotaxin.
Our data showed that the STAT6 and IRS-2 pathways are activated in mouse eosinophils in response to IL-4 (black oval) but to a lesser degree by IL-13. Engagement of CCR3 by eotaxin-1 (white oval) activates G-proteins leading to activation of PI3K isoforms and other signaling pathways. The two pathways may intersect to enhance migration in the activation of Rho guanine nucleotide exchange factors (GEFs), which leads to cytoskeletal rearrangement and enhanced chemotaxis. IL-4/STAT6 has been implicated in spreading/movement of B-cells via Rho GEFs [63]. IL-4 is also known to upregulate adhesion molecules involved in extravasation/chemotaxis – this may be due to activation of STAT6-mediated transcription or activation of the AKT pathway downstream of IRS-2.