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. 2010 Jul 6;5(7):e11439.
doi: 10.1371/journal.pone.0011439.

Selective depletion of eosinophils or neutrophils in mice impacts the efficiency of apoptotic cell clearance in the thymus

Hye-Jung Kim  1 , Eric S Alonzo, Guillaume Dorothee, Jeffrey W Pollard, Derek B Sant'Angelo
Affiliations

Selective depletion of eosinophils or neutrophils in mice impacts the efficiency of apoptotic cell clearance in the thymus

Hye-Jung Kim et al. PLoS One. .

Abstract

Developing thymocytes undergo a rigorous selection process to ensure that the mature T cell population expresses a T cell receptor (TCR) repertoire that can functionally interact with major histocompatibility complexes (MHC). Over 90% of thymocytes fail this selection process and die. A small number of macrophages within the thymus are responsible for clearing the large number of dying thymocytes that must be continuously cleared. We studied the capacity of thymic macrophages to clear apoptotic cells under acute circumstances. This was done by synchronously inducing cell death in the thymus and then monitoring the clearance of apoptotic thymocytes. Interestingly, acute cell death was shown to recruit large numbers of CD11b(+) cells into the thymus. In the absence of a minor CSF-1 dependent population of macrophages, the recruitment of these CD11b(+) cells into the thymus was greatly reduced and the clearance of apoptotic cells was disrupted. To assess a possible role for the CD11b(+) cells in the clearance of apoptotic cells, we analyzed mice deficient for eosinophils and mice with defective trafficking of neutrophils. Failure to attract either eosinophils or neutrophils to the thymus resulted in the impaired clearance of apoptotic cells. These results suggested that there is crosstalk between cells of the innate immune system that is necessary for maximizing the efficiency of apoptotic cell removal.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Heterogeneous phenotype of thymic resident myeloid cells.
(A) Thymic stromal cells were enriched by collagenase/dispase digestion followed by percoll density gradient centrifugation. Cells were then stained with anti-CD11b and anti-F4/80 and analyzed by FACS. Red circles mark the three different cell populations discussed in the text. In this experiment, populations #1, #2 and #3 represented 0.02%, 0.43% and 0.61% of the analyzed cells, respectively. While the absolute cell number varied as a result of the preparation, the percentage of each cell type relative to each other was consistent over many experiments. (B) Size and surface marker expression (blue lines) of each population was determined by FACS. Populations were identified as in (A) and stained with the antibodies as indicated. Isotype controls (red lines) were used to correct differences in the autofluorescence of each cell population. (C) Individual cells from populations #2 and #3 were sorted onto glass slides followed by staining with H&E. Data are representative of more than ten experiments (a and b) and three experiments (c).
Figure 2
Figure 2. Time course analysis of apoptosis induced by irradiation.
Thymuses from wild type B6 mice were harvested and frozen post-irradiation at the indicated times (1 hour through 24 hours). Sections were stained by TUNEL (green) to detect apoptotic cells and with anti-DEC205 (red) to mark the thymic epithelium. Panel labeled “control' was a section stained with secondary antibodies only. Panel labeled “w/o irr.” Was a section from a nonirradiated mouse. Original magnification was 200X.
Figure 3
Figure 3. Time course analysis of F4/80 positive cells following irradiation.
Thymuses from wild type B6 mice were harvested and frozen post-irradiation at the indicated times (1 hour through 24 hours). Sections were stained with anti-F4/80 (red), which identifies macrophages and eosinophils. Panel labeled “control' was a section stained with secondary antibody only. Panel labeled “w/o irr.” was a section from a nonirradiated mouse. Original magnification was 200X.
Figure 4
Figure 4. No increase in eosinophils or neutrophils in spleens or lymph nodes post-irradiation.
Spleens and lymph nodes from wild type B6 mice were harvested and frozen either prior to irradiation or sixteen hours post-irradiation. Sections were stained by TUNEL to detect apoptotic cells, CD11b to detect neutrophils or SiglecF to detect eosinophils. Original magnification for each staining is listed in the figure. Data are representative of three independent experiments.
Figure 5
Figure 5. Migration of cells of the innate immune system into the thymic cortex following induction of thymocyte apoptosis.
(A) FACS analysis of thymic stromal cells sixteen hours after irradiation collected as described in Figure 1. Microscopic analysis of sorted cells showed the morphological differences of eosinophils (top panel) and the myeloid type DCs (bottom panel). (B) Thymic stromal cells were stained for CD11b and Ly-6G before and after irradiation. The percentage of CD11b+Ly-6G+ cells is shown. (C) Thymuses were harvested from control (top panels) or irradiated (bottom panels) mice. Frozen sections were stained with CD11b+ (red), Ly-6G+ (green) and Siglec-F+ (red). Sections are representative of multiple samples from more than six experiments.
Figure 6
Figure 6. Influx of innate cells rapidly declines twenty-four hours after irradiation.
Thymuses were harvested from mice at the indicated times after irradiation. Stromal cells were enriched as described and stained with anti-CD11b, -F4/80, SiglecF and -Ly-6G. Numbers in the plots are the percentage of cells within the electronic gate. N = 6 and the data show the results from two independent experiments. The mean is shown by a horizontal bar and statistics were calculated with 2-tailed, nonpaired Mann-Whitney test. *  =  <0.01; **  =  <0.001; ***  =  <0.0001.
Figure 7
Figure 7. Nearly all apoptotic cells appear to be associated with CD68+ cells.
(A) Thymic macrophages (CD11bhi and CD11blo) from non-irradiated mice were stained for surface CD68 expression (blue line). Red line is isotype control staining. (B) Intracellular staining for CD68 (blue line) in the indicated cell populations 16 hours post irradiation. Red line is isotype control staining. (C) Frozen sections of thymuses harvested 16 hours post irradiation were stained with DAPI and an antibody against CD68. Only thymic cortical areas are shown, since CD68+ cells were mainly found in the cortex (original magnification: 100X). (D) Clearance of apoptotic cells (top panels) was analyzed by staining thymic sections by TUNEL (red) and CD68 (green). Original magnification: 400X. Enlargement of a single CD68+ cell (bottom panels) shows multiple engulfed thymocytes, each ringed with CD68 (original magnification: 1000X). All sections are representative of multiple samples from more than six experiments.
Figure 8
Figure 8. FACS analyses of the innate cells in the spleen and bone marrow from WT and Csf op/op mice.
Cells from the spleen and bone marrow from WT and Csfop/op mice were stained for CD11b and F4/80. Eosinophils and neutrophils were identified by staining cells for CD11b and SiglecF or CD11b and Gr-1, respectively. Percent of cells in each area is indicated in the Figure.
Figure 9
Figure 9. Impaired clearance of apoptotic thymocytes in Csf1 op/op mice.
(A) Thymic stromal cell subpopulations from WT (Csf1 op/+) and osteopetrosis (Csf1 op/op) mice were stained with the indicated antibodies and analyzed by FACS. Numbers indicate the percentage of cells within the circles. The red circle marks population #1, which is not present in Csf1 op/op mice. (B) Comparison of thymic stromal cell subpopulations between WT (Csf1 op/+) and osteopetrosis (Csf1 op/op) mice 16 hours after irradiation. Thymic stromal cells were enriched and stained for CD11b and F4/80. (C) Representative images showing the frequency of apoptotic cells in WT (Csf1 op/+) and Csf1 op/op mice in nonirradiated mice in mice 24 hrs receiving 1Gy irradiation. Apoptotic cells were detected by TUNEL (green) and their cortical localization was visualized by counterstaining of thymic sections with an antibody against DEC205 (red). Original magnification was 100X. Sections are representative of multiple samples from more than ten experiments.
Figure 10
Figure 10. Eosinophils and neutrophils are essential for efficient clearance of apoptotic thymocytes.
(A) FACS analyses of thymic stromal cells from wild type (C57Bl/6) mice, CSF1 deficient (CSF1op/op) mice, eosinophil deficient mice (ΔdblGATA), mice deficient for neutrophil migration (CXCR2−/−) 16 hours after apoptosis induction. Cells were stained with the indicated antibodies and analyzed for FACS. The percentage of CD11b+F4/80lo (eosinophils and myeloid DCs) (top), eosinophils (middle) and neutrophils (bottom) is shown. (B) Frozen thymic sections were prepared 16 hrs after irradiation from wild type (B6), ΔdblGATA and CXCR2−/− mice were stained either by TUNEL (green) or TUNEL and with an antibody against DEC205 (red). Top panels are at low magnification (original magnification: 50X) to show an “overview” of TUNEL positive cells in the thymus. Bottom panels (original magnification: 100X) shows apoptotic cells (TUNEL, green) and thymic cortical epithelial cells (DEC205+, red). Note the formation of cell clusters composed of non-engulfed apoptotic cells in CXCR2−/− mice.
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