Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells - PubMed (original) (raw)

Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells

Hai-Bin Wang et al. J Immunol. 2007.

Abstract

The capacity of airway eosinophils, potentially pertinent to allergic diseases of the upper and lower airways, to function as professional APCs, those specifically able to elicit responses from unprimed, Ag-naive CD4(+) T cells has been uncertain. We investigated whether airway eosinophils are capable of initiating naive T cell responses in vivo. Eosinophils, isolated free of other APCs from the spleens of IL-5 transgenic mice, following culture with GM-CSF expressed MHC class II and the costimulatory proteins, CD40, CD80, and CD86. Eosinophils, incubated with OVA Ag in vitro, were instilled intratracheally into wild-type recipient mice that adoptively received i.v. infusions of OVA Ag-specific CD4(+) T cells from OVA TCR transgenic mice. OVA-exposed eosinophils elicited activation (CD69 expression), proliferation (BrdU incorporation), and IL-4, but not IFN-gamma, cytokine production by OVA-specific CD4(+) T cells in paratracheal lymph nodes (LN). Exposure of eosinophils to lysosomotropic NH(4)Cl, which inhibits Ag processing, blocked each of these eosinophil-mediated activation responses of CD4(+) T cells. By three-color fluorescence microscopy, OVA Ag-loaded eosinophil APCs were physically interacting with naive OVA-specific CD4(+) T cells in paratracheal LN after eosinophil airway instillation. Thus, recruited luminal airway eosinophils are distinct allergic "inflammatory" professional APCs able to activate primary CD4(+) T cell responses in regional LNs.

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Conflict of interest statement

Disclosures The authors have no financial conflict of interest.

Figures

Figure 1

Figure 1

Isolated eosinophils are pure and free of CD11chigh DCs. A, Splenocytes from IL-5 Tg mice were stained with Hema 3 before and after isolation and purification of esoinophils, as described in Materials and Methods. Eosinophils were identified by their donut-shaped nucleus and eosinophilic cytoplasmic staining. B, To exclude contamination with CD11c+ DCs, purified eosinophils, stained with FITC-labeled (HL-3) or PE-labeled (N418) (inset) anti-mouse CD11c, were analyzed by flow cytometry and CD11chigh cells were isolated by flow cytometric cell sorting. The sorted CD11chigh population stained with Hema 3 was morphologically and tinctorially eosinophils.

Figure 2

Figure 2

Effect of GM-CSF on expression of MHC II and costimulatory molecules by eosinophils isolated from spleens of IL-5 Tg mice. Fresh eosinophils (green) or eosinophils incubated with GM-CSF (10 ng/ml) for 24 h (red) were stained with FITC-labeled anti-MHC II, anti-CD40, anti-CD80 anti-CD86, or isotype control IgG (gray) mAbs. Flow cytometric data, with cell counts on the _y_-axis and fluorescence intensity on the _x_-axis, are representative of three experiments.

Figure 3

Figure 3

Airway eosinophils present OVA Ag to naive OVA-specific CD4+ T cells. Eosinophils, exposed to 0.2% NaCl or NH4Cl during RBC lysis, incubated with GM-CSF for 24 h, pulsed with OVA (middle and right rows) or without OVA (left row) for 1 h, were instilled i.t. into WT mice that received an i.v. infusion of OVA-specific TCR Tg CD4+ T cells 24 h earlier. Seventy-two hours after eosinophil transfer, pLN cells were analyzed and stained with PE-KJ1–26 mAb specific for the OVA TCR. Responses of KJ1–26+ OVA-specific T cells were evaluated for activation (CD69 expression) (A), proliferation (BrdU incorporation) (B), and intracellular cytokine expression for IL-4 (C) and IFN-γ (D), as detailed in Materials and Methods. In contrast to OVA-free control eosinophils (left column), airway eosinophils exposed to OVA Ag (middle column) elicited in OVA-specific naive T cells: activation (A), proliferation (B), and IL-4 (C) but not IFN-γ production (D). OVA-pulsed eosinophils exposed to NH4Cl (right column) failed to elicit activation (A), proliferation (B), or IL-4 production by OVA-specific T cells (C). Noted percentages indicate percentages of KJ1–26+ OVA TCR-specific T cells responding. Histograms represent analyses of pooled cells obtained from four mice per group. Data are representative of three (A, C, D) and two (B) experiments.

Figure 4

Figure 4

OVA Ag-loaded, but not Ag-free, eosinophils from the airways interact with naive OVA Ag-specific CD4+ T cells in regional pLNs. A, OVA-free (green) or blue fluorescent bead OVA-loaded (B, green and blue) GFP Tg eosinophils, as indicated by arrows, were i.t. instilled into the mice that received i.v. red fluorescent dye loaded OVA TCR-specific CD4+ T cells 24 h earlier. Twenty-four hours after eosinophil transfer, pLNs were harvested, frozen, sectioned, and observed by fluorescence microscopy. OVA-specific CD4+ T cells exhibited greater proximity to OVA-bearing eosinophils (B) than to OVA-free (A) eosinophils in pLNs. In addition, physical interactions between OVA-loaded blue/green eosinophils and red OVA-specific CD4+ T cells formed yellow-colored clusters of colocalized eosinophils and T cells by 12 h (C) and to a greater extent by 24 h (D) after i.t. transfer of eosinophils, as indicated by the arrowheads. Scale bars: B, 100 μm; D, 50 μm.

Figure 5

Figure 5

Imaging of OVA-Ag presenting eosinophil APCs with OVA Ag-specific CD4+ T cell interactions in situ. pLNs were harvested from mice 24 h after i.t. administration of blue fluorescent bead OVA-loaded green GFP eosinophils and 48 h after i.v. injection of red fluorescent dye labeled OVA TCR CD4+ T cells. A, Blue OVA Ag-loaded green eosinophils interacted with red OVA-specific CD4+ T cells in pLNs. Selected areas in A were further sectioned at 0.5 μm intervals and B images were imported into software Volocity 3.6. Following deconvolution using theoretical Point Spread Function for each individual channel, images of the framed areas in B were rendered either in 2-D (C) or 3-D (D), demonstrating intimate cell-cell interactions between blue/green OVA-presenting eosinophils and red OVA-specific CD4+ T cells.

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