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. 2009 Oct;297(4):L677-86.
doi: 10.1152/ajplung.00030.2009. Epub 2009 Jul 24.

Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences

R William Vandivier  1 , Tiffany R Richens, Sarah A Horstmann, Aimee M deCathelineau, Moumita Ghosh, Susan D Reynolds, Yi-Qun Xiao, David W Riches, Jonathan Plumb, Eric Vachon, Gregory P Downey, Peter M Henson
Affiliations

Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences

R William Vandivier et al. Am J Physiol Lung Cell Mol Physiol. 2009 Oct.

Abstract

Cystic fibrosis (CF) is caused by mutated CF transmembrane conductance regulator (CFTR) and is characterized by robust airway inflammation and accumulation of apoptotic cells. Phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, because it prevents postapoptotic necrosis and actively suppresses release of a variety of proinflammatory mediators, including IL-8. Because CF is associated with accumulation of apoptotic cells, inappropriate levels of IL-8, and robust inflammation, we sought to determine whether CFTR deficiency specifically impairs efferocytosis and its regulation of inflammatory mediator release. Here we show that CFTR deficiency directly interferes with efferocytosis by airway epithelium, an effect that is not due to altered binding of apoptotic cells to epithelial cells or altered expression of efferocytosis receptors. In contrast, expression of RhoA, a known negative regulator of efferocytosis, is substantially increased in CFTR-deficient cells, and inhibitors of RhoA or its downstream effector Rho kinase normalize efferocytosis in these cells. Impaired efferocytosis appears to be mediated through an amiloride-sensitive ion channel, because amiloride restores phagocytic competency in CFTR-deficient cells. Finally, ineffective efferocytosis in CFTR-deficient cells appears to have proinflammatory consequences, because apoptotic cells enhance IL-8 release by these cells, but not by wild-type controls. Therefore, in CF, dysregulated efferocytosis may lead to accumulation of apoptotic cells and impaired regulation of the inflammatory response and, ultimately, may suggest a new therapeutic target.

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Figures

Fig. 1.
Fig. 1.
CFTR deficiency is associated with fewer spontaneous efferocytosis events in vitro. Representative photomicrographs show spontaneous ingestion of apoptotic cells into spacious phagosomes by neighboring primary human airway epithelial cells (A and B) and human alveolar macrophages (C and D). Arrows indicate ingested apoptotic cells. E: quantitation of spontaneously ingested apoptotic cells by CFTR+/+ and CFTR−/− primary human airway epithelial cells [phagocytic index (PI)]. Values are means ± SE for 4 replicates per group. *Significantly different from CFTR+/+ cells (P < 0.05).
Fig. 2.
Fig. 2.
CFTR deficiency decreases efferocytosis. A and B: representative photomicrographs showing ingestion of apoptotic Jurkat T cells by primary human airway epithelial cells. Arrows indicate ingested apoptotic cells. C and D: CFTR+/+ and CFTR−/− primary airway epithelial cells were cocultured with no cells or with apoptotic Jurkat T cells and analyzed for PI or binding index (BI). Values are means ± SE for 5–7 replicates per group. *Significantly different from identically treated CFTR+/+ cells (P < 0.05). E and F: C38 and IB3-1 epithelial cells were cocultured with no cells or apoptotic Jurkat T cells for 4 h and analyzed for PI or BI. Values are means ± SE for 3 replicates per group. *Significantly different from identically treated C38 cells (P < 0.05). G: PI in CFTR+/+ and CFTR−/− mice instilled intratracheally with apoptotic polymorphonuclear leukocytes (neutrophils). After 30 min, whole lung lavage was performed, and alveolar macrophage PI was determined. Values are means ± SE for 5 animals per group.
Fig. 3.
Fig. 3.
CFTR ion channel function is not required for regulation of efferocytosis. A: to determine whether CFTR's ion channel function was required for regulation of efferocytosis by epithelial cells, C38 epithelial cells were cocultured with no cells or apoptotic Jurkat T cells for 4 h in the presence of DMSO or the specific CFTR channel inhibitor CFTRinh 172 (5 and 10 μM). Values are means ± SE for 4 replicates per group. *Significantly different from untreated C38 cells cocultured with apoptotic cells (P < 0.05). B: culture of epithelial cells at low temperature increases translocation of the ΔF508 CFTR mutation to membrane, resulting in increased function in primary epithelial cells and IB3-1 cells (10, 12, 47). Efferocytosis was studied in IB3-1 cells cultured at 37°C or 26°C for 18 h and then cocultured with Jurkat T cells for 4 h at 37°C. Culture at low temperature tended to increase efferocytosis by IB3-1 cells. Values are means ± SE for 5 replicates per group. *Significantly different from 37°C (P = 0.08).
Fig. 4.
Fig. 4.
CFTR deficiency inhibits phagocytosis mediated by the phosphatidylserine (PS) receptor and the Fcγ receptor IIa (FcγRIIa) but does not significantly affect latex bead ingestion. A and B: representative photomicrographs showing ingestion (uptake) and binding of E(PS)-αCD59 by C38 epithelial cells. C and D: PI (after hypotonic lysis) and BI (before hypotonic lysis) of various erythrocyte (E) targets cocultured with C38 and IB3-1 epithelial cells. Values are means ± SE for 5–6 replicates per group. *Significantly different from identically treated C38 cells (P < 0.05). E and F: PI and BI of IgG-coated erythrocytes cocultured with C38 or IB3-1 epithelial cells that had been transfected with vehicle, empty vector, or FcγRIIa. Values are means ± SE for 3 replicates per group. *Significantly different from identically treated C38 cells (P < 0.05).
Fig. 5.
Fig. 5.
CFTR deficiency increases RhoA activity. A: Western blot analysis of total RhoA protein and baseline and GTPγS-loaded RhoA activity in C38 and IB3-1 epithelial cells. Statistical analysis was performed on densitometry readings of total or active RhoA relative to its β-actin control for C38 and IB3–1 cells (n = 3). B: Western blot analysis of total RhoA protein and baseline and GTPγS-loaded RhoA activity in CFTR+/+ and CFTR−/− primary airway epithelial cells. Representative blots of 2 replicates are shown for CFTR+/+ and CFTR−/− cells. C: actin polymerization (green) and stress fiber formation in C38 and IB3-1 cells exposed to complete medium (media control), serum starvation for 6 h, or lysophosphatidic acid (LPA) for 10 min. Serum starvation and LPA enhanced actin stress fibers (white arrow) more in IB3-1 than in C38 cells. Fluorescence images are representative of 4 independent experiments.
Fig. 6.
Fig. 6.
Inhibitors of RhoA, Rho kinase, and amiloride-sensitive ion channels restore phagocytic competency in CFTR−/− cells. CFTR+/+ and CFTR−/− airway epithelial cells were treated with or without C3 transferase (A and B), Y-27632 (C–F), or amiloride (G and H) and then cocultured with no cells (NC) or apoptotic Jurkat T cells (ApoJkts). Values are means ± SE for 3–6 replicates per group. C3 transferase, Y-27632, and amiloride corrected defective efferocytosis in untreated IB3-1 and CFTR−/− primary epithelial cells without affecting binding. *Significantly different from identically treated CFTR+/+ cells (P < 0.05) **Significantly different from untreated CFTR−/− cells (P < 0.05). †Significantly different from untreated C38 cells (P < 0.05).
Fig. 7.
Fig. 7.
Apoptotic cells enhance IL-8 release from CFTR−/− epithelial cells in a Rho kinase-independent manner. A: IL-8 release in C38 and IB3-1 epithelial cells cocultured with no cells (NC), viable cells (V), or apoptotic Jurkat T cells (Apo) in the presence of no stimulus (No Stim), TNFα, or IL-1β. Values are means ± SE for 4–6 replicates per group. *Significantly different from IB3-1 cells without coculture (P < 0.05). **Significantly different from identically treated C38 cells (P < 0.05). B: IL-8 release in CFTR+/+ and CFTR−/− primary epithelial cells treated as described in A. Values are means ± SE for 5 replicates per group. *Significantly different from CFTR−/− cells without coculture (P < 0.05). C: test for involvement of the RhoA/Rho kinase pathway in apoptotic cell-enhanced IL-8 release from CFTR−/− cells. C38 and IB3-1 epithelial cells were cocultured with no cells or apoptotic Jurkat T cells in the presence or absence of IL-1β and in the presence or absence of the Rho kinase inhibitor Y-27632 (1 and 5 μM), and supernatants were collected and analyzed for IL-8. Values are means ± SE for 3 replicates per group. *Significantly different from identically treated C38 cells (P < 0.05). **Significantly different from identically treated IB3-1 cells without coculture (P < 0.05).
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