Paired immunoglobulin-like receptor A is an intrinsic, self-limiting suppressor of IL-5-induced eosinophil development

Abstract

Eosinophilia is a hallmark characteristic of T helper type 2 (TH2) cell-associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of β₂-microglobulin (β₂M) molecules, PIR-B served as a permissive checkpoint for IL-5-induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B-deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb(-/-) mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5-induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.

Figure 1. Pirb^−/− low-density bone marrow-derived cells fail to differentiate into mature eosinophils in vitro

Low-density bone marrow cells were obtained from wild-type (WT) and Pirb^−/− mice and differentiated in vitro into eosinophils. Representative photomicrograph images at the indicated time points of stained cytospins (a) and day 14 total cell counts (b) are shown. Filled and broken black arrows (a) indicate single nucleated and multi-nucleated myeloid cells, respectively. CCR3 surface expression (c), eosinophil major basic protein (Mbp, d), cell size and granularity (e), Gata1 (f) and Gata2 (g) expression were assessed in LDBM WT and Pirb^−/− cells. All qPCR analyses were normalized to the house keeping gene hypoxanthine-guanine phosphoribosyltransferase (Hprt). In (h), total eosinophil numbers as determined by CCR3⁺Siglec-F⁺ cells is depicted. Assessment of CD45⁺CD34⁺Lin⁻Sca-1⁻C-Kit^intIL-5Rα⁺ eosinophil progenitors (EoPs) (i-j) and IL-5 receptor (R) α expression in EoPs (k) is shown. ns-non significant, *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of at least five independent experiments conducted in triplicates (a-e); mean and s.e.m. of cultures obtained from three (f-h) or five (j-k) mice per group.

Figure 2. PIR-B regulates eosinophil apoptosis during low-density bone marrow-derived eosinophil differentiation

Low-density bone marrow (LDBM) cells were obtained from wild-type (WT) and Pirb^−/− mice and differentiated in vitro. Representative photomicrograph images at the indicated time points of apoptotic eosinophils (a) as well as engulfed eosinophils (b) are shown. Representative density plot analysis (c) and a summary of annexin-V propidium idodide (PI) stained cells (d) is depicted. Numbers in each quadrant indicates the percentage of cells corresponding with this quadrant. Representative density plots of anti-Siglec F and annexin-V stained cells (e) and analysis of the percentage and total cell counts of Siglec-F⁺annexin V⁺ cells and Siglec-F⁺annexin V⁻ (f, g, h, i) is shown. LDBM cells were stained with anti-Siglec-F, annexin-V and anti-active caspase 3 (j), anti-Bim (l), or anti-GATA-1 (m) and expression was assessed using flow cytometry. Bim expression was assessed in cDNA obtained from LDBM cells by qPCR analysis (k) and normalized to the house keeping gene hypoxanthine-guanine phosphoribosyltransferase (Hprt). *P < 0.05, **P < 0.01,***P < 0.001. Data are representative of at least five independent experiments conducted in triplicates (a-m); mean and s.e.m. of data obtained from three mice per group (d,f,g-I,k) or representative histograms from the respective experiments (j, i, m).

Figure 3. PIR-B regulates IL-5- but not IL-3/GM-CSF-induced colony formation

Low-density bone marrow cells were obtained from wild-type (WT) and Pirb^−/− mice and subjected to colony forming unit (CFU) assays in response to IL-5 (a, b, c), IL-3 (d, e) and GM-CSF (f, g). Total CFU counts (a, d, f) and relative cell counts per CFU (b, e, g) were assessed. In addition, IL-5-induced CFUs from WT and Pirb^−/− cultures were obtained and stained with anti-Siglec-F and annexin-V. Thereafter, Siglec-F⁺ cells were assessed for annexin-V expression (c); ns-non significant, **P < 0.01, ***P < 0.001. Data are mean and s.e.m. of three independent experiments of three mice per group (a-g).

Figure 4. PIR-B regulates eosinophil apoptosis in-vivo even in the presence of increased IL-5

Bone marrow cells were obtained from naïve wild-type (WT) and Pirb^−/− mice. Siglec-F⁺CCR3^int and Siglec-F⁺CCR3^hi cells (a) were gated (R1 and R2, respectively) and assessed for annexin-V expression (b, c). BM neutrophils and monocytes were gated (d) and assessed for annexin-V expression (d). PB was obtained from naïve WT and Pirb^−/− mice and assessed for circulating eosinophil levels using anti-Siglec-F and anti-CCR3 staining (e, f). WT and Pirb^−/− mice were treated with anti-IL-5 (TRFK5) or isotype control antibodies (Iso). Thereafter, bone marrow cells were stained with anti-Siglec-F and anti-CCR3 and the levels of Siglec-F⁺CCR3^int (g) and Siglec-F⁺CCR3^hi (h) cells as well as PB eosinophils (i) were assessed. In addition, the expression of PIRs was assessed on the surface of blood eosinophils (j). IL-5 was administered daily for 5 consecutive days and PB and BM were obtained. BM cells were stained with anti-Siglec-F, anti-CCR3 and bone marrow Annexin-VSiglec-F⁺CCR3^int and Annexin-VSiglec-F⁺CCR3^hi cells were gated (R1 and R2, respectively) and assessed for annexin-V expression (k, l). The levels of PB eosinophils were assessed (m). Expression of PIRs in BM (n, o) and PB (p) eosinophils is shown; ns-non significant, *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative histograms, dot-plots and mean and s.e.m. of at least fifteen mice (a-f); or mean and s.e.m of two independent experiments with five to seven mice per group (g-p).

Figure 5. Increased eosinophil development and decreased apoptosis in the absence of MHC-I expression

Low-density bone marrow cells were obtained from wild-type (WT) and B2m^−/− mice and subjected to colony forming unit (CFU) assays in response to IL-5 (a-c). Thereafter, total CFU counts (a) and relative cell counts per CFU (b) were assessed. In addition, IL-5-induced CFUs from WT and B2m^−/− cell cultures were stained with anti-Siglec-F and annexin-V for assessment of apoptosis (c). Bone marrow cells were obtained from naïve WT and B2m^−/− mice. Cells were stained with anti-Siglec-F, anti-CCR3 and annexin-V. Thereafter, Siglec-F⁺CCR3^int and Siglec-F⁺CCR3^hi cells were gated and assessed for annexin-V expression (d, e); ns-non significant, *P < 0.05. Data are mean and s.e.m of three independent experiments using three (a-c) and seven (d, e) mice.

Figure 6. Neutralization of PIR-A in Pirb^−/− eosinophil cultures, attenuates eosinophil apoptosis

The expression of PIR-A throughout the low-density bone marrow (LDBM)-derived eosinophil culture was determined at the indicated time points (a). In (b) Pearson correlation between PIR-A and Bim expression is shown (r=0.943, P = 0.0004). LDBM cells from wild type (WT) and Pirb^−/− mice were obtained and eosinophils were grown untreated (UT), in the presence of anti-PIR-A/B, or isotype control antibodies (Iso). Representative dot plots (c) and a summary of the flow cytometric analysis of anti-Siglec-F and annexin-V stained LDBM cells is shown (d, e). Cells were stained with modified Wright Giemsa stain and cellular morphology was assessed (f). Viable (i.e. Siglec-F⁺Annexin-V⁻) Pirb^−/− cells were sorted and PIR-A immunoprecipitated. Thereafter, antibody coated membranes were treated with anti-phospho-tyrosine antibodies conjugated to horseradish peroxidase and PIR-A:protein interactions were assessed (g). Representative flow cytometric histogram plot (h) and quantitative analysis of phopsho-Erk 1/2 expression in Siglec-F⁺Annexin-V⁻ and Siglec-F⁺Annexin-V⁺ cells from WT and Pirb^−/− cells is shown (i). Expression of PIR-A and PIR-B on the surface of eosinophils from various in vivo sources is depicted (j); *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative dot-plots and mean and s.e.m of five experiments conducted in duplicates (a-f); representative blots of one out of three independent experiments (g); representative histogram plot and pooled mean and s.e.m from five mice; and mean and s.e.m of at least five mice.

Figure 7. Pirb^−/− mice display increased bone marrow eosinophil apoptosis as well as decreased PB and tissue eosinophilia following mucosal aeroallergen challenge

Wild-type (WT) and Pirb^−/− mice were challenged with saline or Aspergillus fumigatus (Asp). Following six challenges, mice were bled and assessed for total serum IgE (a). Bone marrow was obtained and stained with anti-Siglec-F, anti-CCR3 antibodies and annexin-V. Siglec-F⁺CCR3^int and Siglec-F⁺CCR3^hi cells were gated and assessed for the frequencies of annexin-V positive cells (b, c). Saline- and Asp-challenged WT and Pirb^−/− mice were assessed for allergen-induced PB eosinophils (d), bronchoalveolar lavage fluid (BALF) eosinophil percentages (e) and total BAL eosinophils (f). Eosinophils from saline and Asp-challenged BALF were assessed for annexin-V positivity (g). *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of five experiments each containing six to eight mice per group (a-g).