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Life after corpse engulfment: phagocytosis of apoptotic cells leads to VEGF secretion and cell growth

HEIKO A. GOLPON^*,, VALERIE A. FADOK, LAIMA TARASEVICIENE-STEWART^*, ROBERTAS SCERBAVICIUS^*, CLEMENS SAUER, TOBIAS WELTE, PETER M. HENSON and NORBERT F. VOELKEL^*,1

^* Pulmonary Hypertension Center, University of Colorado, Health Sciences Center, Denver, Colorado, USA;
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado, USA; and
Research Center of Immunology, Otto-von-Guericke University, Magdeburg, Germany

¹Correspondence: Division of Pulmonary Sciences, and Critical Care Medicine, 4200 East Ninth Ave., C272, Denver, CO 80262, USA. E-mail: norbert.voelkel{at}uchsc.edu

SPECIFIC AIMS

Removal of apoptotic cells by neighboring viable cells or professional phagocytes is essential for the maintenance of tissue homeostasis. The present work addresses the question of whether in addition to the well-known secretion of anti-inflammatory cytokines, the phagocytosis of apoptotic cells by phagocytes leads to the production of factors that promote cell growth.

PRINCIPAL FINDINGS

1. Phagocytosis of apoptosed cells changes the expression of growth and survival factors
To study the relationship between programmed cell death and cell growth in integrated tissues, the mouse mammary epithelial cell line HC-11 was chosen as the nonprofessional phagocyte to engulf either apoptotic or necrotic cells. It has recently been shown that HC-11 epithelial cells are capable of engulfing apoptotic cells. Apoptosis was induced in Jurkat T cells (human lymphoma T cells) by UV irradiation, whereas necrosis was induced by repeated cycles of freezing and thawing. The number of viable, apoptotic, and necrotic cells was assessed by FACS analysis after staining with annexin-V and propidium iodide. Affymetrix microarray expression profiling was used to compare the response pattern of naive with that of HC-11 cells that had been exposed to apoptotic or necrotic cells. This technique was used to screen for growth factor- and apoptosis-related genes whose expression might be altered after engulfment of apoptotic or necrotic cells. An increased number of transcripts for transforming growth factor family members (TGF-, TGF-ß1, and TGF-ß2) was found after phagocytosis of apoptotic cells. The number of transcripts for hepatocyte growth factor (HGF) was increased when HC-11 had been exposed to apoptotic but not necrotic cells. Several genes related to angiogenesis processes were affected in their expression when HC-11 cells had been exposed to apoptotic cells. VEGF-A was up-regulated whereas VEGF-B, PlGF, angiopoietin 1 and 2, and endostatin were decreased.

Several genes encoding apoptosis-related genes were altered in their expression when HC-11 cells were exposed to apoptotic or necrotic cells. The apoptosis suppressor Bcl-2, which appears to function in a feedback loop system with caspases, was up-regulated when HC-11 cells had engulfed apoptotic bodies but was down-regulated when they were exposed to necrotic cells.

2. Phagocytosis of apoptotic cells induces VEGF protein secretion
A gene induced by HC-11 cells that had phagocytosed apoptotic, but not necrotic cells, was that encoding VEGF (VEGF-A). Accordingly, VEGF protein levels in the cell supernatants were measured using a specific ELISA. HC-11 cells that had ingested apoptotic cells released significant amounts of VEGF into the cell culture medium (Fig. 1 ). This was not seen in the naive HC-11 cells or in those that had interacted with the necrotic cells. We incubated unstimulated mouse peritoneal macrophages with viable or apoptotic HC-11 cells and measured VEGF protein levels. Consistent with the HC-11 cells, we noted an increase in VEGF protein levels in peritoneal macrophages exposed to apoptotic but not to viable cells. The VEGF protein level here was 50% of the level found in peritoneal macrophages stimulated with lipopolysaccharide (Fig. 1) . We have previously reported the induction of TGF-ß by a variety of cells after interaction with apoptotic cells and have suggested an important role for this in the anti-inflammatory response. As shown in Fig. 1 , increased levels of secreted TGF-ß protein were also observed in HC-11 cells when they had interacted with apoptotic but not necrotic Jurkat T cells.

View larger version (18K): [in this window] [in a new window]   Figure 1. Apoptotic Jurkat T cells induce A) VEGF and B) TGF-ß protein expression in HC-11 epithelial cells. VEGF and TGF-ß protein release into the medium were determined by ELISA. HC-11 cells were cultured with viable, apoptotic, or necrotic Jurkat T cells for 24 h in serum-free medium (X-Vivo 10). Data are displayed as mean cytokine production ± SE for 7 experiments. VEGF secretion was noted in peritoneal macrophages (C) cultured with apoptotic HC-11 cells for 24 h as well as in peritoneal macrophages treated with LPS at 1 ng/mL (n=3; ±SE).

3. Phagocytosis of apoptotic cells leads to secretion of biologically active factors that induce proliferation of microvascular endothelial cells
To assess whether factors secreted by HC-11 cells were indeed biologically effective, cell supernatants were incubated for 18 h with rat pulmonary microvascular endothelial cells (RPMVEC) and cell proliferation was measured. Only the conditioned medium derived from HC-11 cells that had engulfed apoptotic cells stimulated endothelial cell growth (Fig. 2 ). No such stimulation was seen with medium derived from naive HC-11 cells or those incubated with necrotic Jurkat cells (nor from medium derived from cultured apoptotic Jurkat cells alone). To support the concept of involvement of the secreted VEGF in endothelial cell growth, the conditioned medium was added to RPMVEC and cells were exposed to neutralizing antibodies against VEGF or TGF-ß. Neutralizing VEGF antibodies blocked the growth of the RPMVEC induced by conditioned medium derived from the HC-11 cells after engulfment of apoptotic Jurkat cells (Fig. 2A ). In contrast, neutralizing TGF-ß antibodies had no effect on RPMVC growth induced by the supernatants (Fig. 2B ).

View larger version (17K): [in this window] [in a new window]   Figure 2. Conditioned medium from HC-11 cells that had been exposed to apoptotic Jurkat T cells (Apo CM) stimulated growth of rat lung microvascular endothelial cells. Cell proliferation was assessed 18 h after addition of the conditioned medium. Cell proliferation was inhibited by a neutralizing anti-VEGF antibody (Apo CM+anti-VEGF) (A), but not by a neutralizing anti-TGF-ß antibody (Apo CM+anti-TGF-ß) (B). Data points represent means of 3 independent experiments.

4. Phagocytosis of apoptosed Jurkat T cells by epithelial cells (HC-11) protects microvascular endothelial or naive HC-11 cells against apoptosis
By incubating HC-11 cells with conditioned medium (HC-11 cells that had phagocytosed apoptotic Jurkats), we saw a decrease in spontaneous apoptotic events compared with HC-11 cells incubated under standard conditions. We found that the conditioned medium from HC-11 cells that had phagocytosed apoptotic but not viable or necrotic Jurkat T cells protected both endothelial cells (RPMVEC) and naive HC-11 cells against UV irradiation-induced apoptosis. We induced apoptosis in endothelial cells by incubating cells with TNF- + cycloheximide. We observed that the conditioned medium also protected endothelial cells from apoptosis induced by TNF- + cycloheximide. Protection of the surrounding tissue after interaction with neighboring apoptosed cells may represent a mechanism for limiting the extent of the original injury and initiating repair.

CONCLUSIONS AND SIGNIFICANCE

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical for the restoration of normal tissue structure and function. Dead cells are rapidly cleared from the body but the physiological ghosts of the cells persist in the form of altered behavior of the phagocytes. It has been clearly demonstrated in the past that engulfment of apoptotic cells by professional and nonprofessional phagocytes sets up an anti-inflammatory milieu within the tissue that is mediated in part by the autocrine/paracrine action of TGF-ß. Here we show that phagocytes that had been exposed to apoptotic cells are "reprogrammed" in that they secrete growth and survival factors, which subsequently promote the growth of endothelial cells and inhibit their apoptosis.

Our microarray survey demonstrated that several genes related to growth factors were altered in their expression when apoptotic or necrotic cells had been engulfed. As expected, we noted an increase in the number of transcripts for genes of the TGF-ß family after engulfment of apoptotic bodies. Many of the genes are linked to angiogenesis, which raises the question whether phagocytosis of apoptotic cells is supportive or even critical for the formation of new vessels. Seguro et al. recently observed that during development of tubule-like structures, a fraction of endothelial cells became apoptotic. However, it is not known whether these apoptotic cells were also phagocytosed and whether angiogenesis factors were released in this process. Here we demonstrate that phagocytes that had engulfed apoptotic bodies secrete bioactive substances that lead to endothelial cell proliferation. It is highly likely that aspects of the observed biological properties of the conditioned medium were mediated in part by VEGF, since anti-VEGF antibodies inhibited the proliferation of endothelial cells elicited by the conditioned medium. VEGF regulates endothelial cell survival by up-regulation of the antiapoptotic protein Bcl-2 and via the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Based on our microarray analysis, we noted an increase of transcripts for Bcl-2 in HC-11 cells after exposure to apoptotic bodies.

Which molecule or set of molecules in the apoptotic cell body causes reprogramming of the phagocyte gene expression pattern and the receptors that participate in this response are unknown. It is likely that the increased expression of VEGF is related to externalized phosphatidylserine in the apoptotic cell membranes. The role of the phosphatidylserine receptor in this respect needs further investigation.

Our results presented here may explain aspects of mechanisms that regulate, in the context of integrated tissues, the relationship between programmed cell death and cell growth. The concept of "life after corpse removal" is of interest with respect to cell-cell interactions in remodeling, inflammation, cell injury, and tumorigenesis. This concept leads to the recognition that the act of apoptotic body removal represents a new form of life-promoting cell-cell interaction.

View larger version (53K): [in this window] [in a new window]   Figure 3. Sequence of events that leads from phagocytosis of apoptosed cells to proliferation and apoptosis resistance of neighboring cells. VEGF secreted by mouse mammary gland epithelial HC-11 cells after engulfment of Jurkat cells stimulates the growth of coincubated rat lung microvascular endothelial cells and improves epithelial wound closure.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1853fje;

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