Monocytes recruited to the lungs of mice during immune inflammation ingest apoptotic cells poorly

Abstract

Apoptotic cells must be cleared to resolve inflammation, but few resident alveolar macrophages (AMo) from normal lungs ingest apoptotic cells. We examined how Mo ingestion of apoptotic cells is altered during immune inflammation induced by intratracheal challenge of primed C57BL/6 mice using sheep red blood cells. Resident AMo were labeled in situ before challenge using intravenous PKH26 to distinguish them from recruited monocytes. Using flow cytometry, we identified phagocytosis of fluorescently-labeled apoptotic thymocytes by alveolar mononuclear phagocytes in vitro and in vivo, and measured surface molecule expression. Intratracheal challenge induced rapid recruitment of monocytes, peaking at Day 3 and decreasing thereafter, whereas numbers of resident AMo did not change significantly. At all times, the percentage of phagocytes ingesting apoptotic thymocytes in vitro was greater among resident AMo (28-45%) than among recruited monocytes (9-19%), but was low in both cell types relative to ingestion of immunoglobulin-opsonized targets. There was also a nonsignificant trend toward lower ingestion by monocytes in vivo. MerTK, a receptor tyrosine kinase crucial for apoptotic cell phagocytosis, was expressed by resident AMo, but not by recruited monocytes. Relative to resident AMo, monocytes recruited to the alveolus ingest apoptotic cells meagerly, possibly due to absence of MerTK expression.

Figure 1

PKH26 treatment permits distinction between resident AMø and recruited monocytes during immune lung inflammation. (A and B) Baseline BAL data. AMø from unimmunized C57BL/6 mice were analyzed by flow cytometry using identical cytometer instrument settings, either without PKH treatment (A) or 24 h after injection of PKH26 by tail vein (B). Note the near absence of overlap in the FL2 fluorescence intensity (vertical axis) between the two populations. (C and D) Peripheral blood data. Mononuclear cells purified from peripheral blood of primed C57BL/6 mice 3 d after intratracheal SRBC challenge were analyzed by flow cytometry. Note minimal difference in FL2 fluorescence (vertical axis) between control mice not receiving PKH26 (C) and PKH26-treated primed mice (D). (E–H) BAL data during inflammation. SRBC-primed C57BL/6 mice received PKH26 by tail vein-injection, and 24 h later were either killed as a Day 0 control (E and F) or were challenged with 5 × 10⁸ SRBC by the intratracheal route and assayed on various later days by flow cytometric analysis of BAL. Representative data are shown from a mouse 12 d after intratracheal challenge (G and H). (E and G) Light scatter data, as forward angle scatter (FSC) on the horizontal axis and side scatter (SSC) on the vertical axis. (F and H) Fluorescence data, as FL1 on the horizontal axis and FL2 (PKH26 staining) on the vertical axis. Note the appearance of PKH_neg cells in the lower left-hand quadrant following immunization (H), and the absence of FL1 + cells (F and H).

Figure 2

Kinetics of mononuclear cell recruitment during immune lung inflammation. SRBC-primed mice received PKH26 by tail vein-injection and were challenged by the intratracheal route 24 h later with 5 × 10⁸ SRBC. At the indicated days after intratracheal SRBC-challenge, mice underwent BAL, and absolute cell counts of PKH_pos Mø (black squares) and PKH_neg Mø (open circles) cells were calculated. Results are mean ± SEM of 3–10 mice per time point assayed individually in at least three separate experiments per time-point.

Figure 3

Characterization of mononuclear phagocyte subpopulations during immune lung inflammation. SRBC-primed mice received PKH26 by tail vein-injection and were challenged by the intratracheal route 24 h later with 5 × 10⁸ SRBC. At various later times, alveolar cells were harvested by BAL and cells within light scatter-defined gates were analyzed for the indicated antigens by flow cytometry. (A, C, E, G, and I) PKH_pos Mø. (B, D, F, H, and J) PKH_neg Mø. (A–H) Representative histograms from individual mice, 3 d after intratracheal challenge. Horizontal axis, log FL1 fluorescence; dotted line, isotype control; solid line, specific staining. (I and J) Representative two-color fluorescence plots from a single mouse, 12 d after intratracheal challenge. Horizontal axis, log FL1 fluorescence (CD11b); vertical axis, log FL3 fluorescence (CD11c). Quadrants indicate specific staining for each cell type.

Figure 4

Phagocytosis of apoptotic thymocytes by recruited and resident mononuclear phagocytes in vitro. At various days after intratracheal SRBC challenge of primed PKH-labeled mice, cells were recovered by BAL, washed, and allowed to adhere to plastic plates. CMFDA-labeled apoptotic thymocytes (A–E) or CMFDA-labeled apoptotic thymocytes opsonized by IgG (F) were added to the adherent Mø at a ratio of 10:1 and incubated for 90 min. Slides were vigorously washed to remove external thymocytes and Mø were released into suspension using trypsin/EDTA, and analyzed by fluorescence microscopy (A and B) or flow cytometry (C–F). (A and B) Photomicrographs of Mø containing internalized apoptotic thymocytes; epifluorescence illumination (A); phase contrast (B). Note the distinction between the uningested green thymocyte not associated with Mø (white arrow), and a PKH_pos Mø containing two ingested yellow thymocytes (yellow arrowheads). (C and D) Representative dot plots from individual mice incubated either without (C) or with (D) CMFDA+ apoptotic thymocytes. Note appearance of FL1+ cells in both right-hand quadrants of D, indicating Mø that have ingested apoptotic thymocytes. (E) Kinetics of Mø phagocytosis of apoptotic thymocytes in vitro. Black squares, PKH_pos Mø; open circles, PKH_neg Mø. (F) Kinetics of phagocytosis of opsonized apoptotic thymocytes in vitro. Dark bars, PKH_pos Mø; light bars, PKH_neg Mø; ND = not done. Results in E and F are mean ± SEM of 3–9 mice assayed individually in at least three independent experiments; *P < 0.05, unpaired t test compared with PKH_neg Mø at the same time-point.

Figure 5

Kinetics of phagocytosis of apoptotic thymocytes in vivo. SRBC-primed mice received PKH26 by tail vein-injection and were challenged by the intratracheal route 24 h later with 5 × 10⁸ SRBC to induce lung inflammation. At the indicated days after intratracheal challenge, mice each received 20 × 10⁶ CMFDA-labeled apoptotic thymocytes by the intratracheal route, and were allowed to recover from anesthesia. Two hours later, mice were killed, alveolar cells were recovered by BAL, and the percentage of phagocytic Mø in the PKH_pos and PKH_neg subsets was determined by two-color flow cytometry. Black bars, PKH_pos Mø; white bars, PKH_neg Mø. Results are mean ± SEM of 3–8 mice in at least two independent experiments per time-point. Day 0 indicates SRBC-primed C57BL/6 mice that had received PKH26 and labeled apoptotic thymocytes, but no intratracheal SRBC. n.d., not determined.

Figure 6

Expression of receptors important for apoptotic cell clearance. SRBC-primed mice received PKH26 by tail vein-injection, were challenged by the intratracheal route 24 h later with 5 × 10⁸ SRBC, and were assayed on various later days for expression of CD51 (left-hand column), PSR (middle column), and MerTK (right-hand column) by flow cytometric analysis of BAL. (A and B) Representative flow cytometry data from individual mice 6 d after intratracheal challenge, gated on (A) PKH_pos Mø and (B) PKH_neg Mø. (C) Kinetics of receptor expression. Black bars, PKH_pos Mø; white bars, PKH_neg Mø. Results are expressed as change in mean fluorescence channel number (Δ MCF) from isotype control, as mean ± SEM of 3–5 mice per time-point, assayed individually. *Significantly different from other Mø population, P < 0.05, unpaired t test. Note difference in scale of Δ MCF between different receptors.