An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response

Abstract

Cells undergo programmed cell death/apoptosis throughout the lifespan of an organism. The subsequent immunologically silent removal of apoptotic cells plays a role in the maintenance of tolerance; defects in corpse clearance have been associated with autoimmune disease. A number of receptors and signaling molecules involved in this process have been identified, but intracellular signaling downstream of corpse recognition is only now being defined. Calcium plays a key role as a second messenger in many cell types, leading to the activation of downstream molecules and eventual transcription of effector genes; however, the role of calcium signaling during apoptotic cell removal is unclear. Here, using studies in cell lines and in the context of a whole organism, we show that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that the genes required for calcium flux are essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium through CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in Caenorhabditis elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes seemed to affect early signaling events, leading to a decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the secretion of TGF-beta by the phagocytes during the engulfment of apoptotic cells. Taken together, these data point to an earlier unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: the formation of the phagocytic cup and the internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.

Figure 1. Functional requirement of calcium flux during engulfment of apoptotic cells

a) Apoptotic Jurkat cells were added (time 0) to Fluo-4 labeled LR73 phagocytic cells and cytoplasmic calcium levels were assessed over time using a fluorescent microplate reader. BAPTA and EGTA were added to LR73 cells 5 min prior to addition of apoptotic cells. b) Labeled apoptotic thymocytes were incubated with LR73 cells in the presence or absence of BAPTA or EGTA, as well as in medium with or without calcium, and engulfment was assessed by a flow cytometry-based phagocytosis assay. c) Labeled apoptotic thymocytes were incubated with LR73 cells in complete fluorimetry medium, medium without Ca²⁺, medium without Mg²⁺, or medium without Ca²⁺ but with 2-fold higher Mg²⁺ concentration so that the total divalent cation concentration in the medium was as in complete medium and engulfment was assessed by flow cytometry. Significance is indicated as follows: p = 0.01−0.05 (*), 0.001−0.01 (**), <0.001 (***). All of these experiments were repeated a minimum of three times.

Figure 2. Calcium is important for actin polymerization and phagocytic cup formation

a) Representative photographs of actin polymerization during apoptotic cell engulfment. Quantitation of the percentage of apoptotic cells that displayed phagocytic cup formation. Apoptotic cells were labeled with TAMRA and incubated with NIH/3T3 cells and cells treated with nothing (WT), vehicle (DMSO), BAPTA, or the actin polymerization inhibitor cytochalasin D. Actin was stained with Alexa 488-phalloidin. Significance is indicated as follows: p>0.05 – (ns), 0.01–0.05 – (*), 0.001–0.01 – (**), <0.001 – (***). n = 10.

Figure 3. Both internal calcium stores and cell membrane calcium channels like the CRAC channels are important for the engulfment of apoptotic cells

a) LR73 cells were incubated with labeled apoptotic thymocytes in the presence or absence of the indicated drugs and scored in the engulfment assay. Drugs shown were added at the same time as apoptotic thymocytes to minimize gross effects on the phagocytes. This is a representative of three independent experiments. b) ORAI 1, 2 and 3 transcript levels in NIH3T3 cells transfected with siRNA specific for the genes indicated on the x-axis as measured by real-time PCR. c) NIH3T3 that were transfected with the indicated siRNA were incubated with labeled apoptotic thymocytes and analyzed for engulfment. b&c are representative of two independent experiments. Significance is indicated as follows: p>0.05 – (ns), 0.01–0.05 – (*), 0.001–0.01 – (**), <0.001 – (***).

Figure 3. Both internal calcium stores and cell membrane calcium channels like the CRAC channels are important for the engulfment of apoptotic cells

a) LR73 cells were incubated with labeled apoptotic thymocytes in the presence or absence of the indicated drugs and scored in the engulfment assay. Drugs shown were added at the same time as apoptotic thymocytes to minimize gross effects on the phagocytes. This is a representative of three independent experiments. b) ORAI 1, 2 and 3 transcript levels in NIH3T3 cells transfected with siRNA specific for the genes indicated on the x-axis as measured by real-time PCR. c) NIH3T3 that were transfected with the indicated siRNA were incubated with labeled apoptotic thymocytes and analyzed for engulfment. b&c are representative of two independent experiments. Significance is indicated as follows: p>0.05 – (ns), 0.01–0.05 – (*), 0.001–0.01 – (**), <0.001 – (***).

Figure 4. jph-1 and stim-1 are important for corpse clearance in C. elegans

a) Quantitation of the number of undegraded refractile corpses in the gonad of worms after RNAi knockdown of the indicated genes. Control worms were maintained on bacteria transformed with an RNAi vector containing no insert. b) Defective corpse internalization in jph-1(RNAi) and stim1(RNAi) worms was determined by scoring the number of actin halos in worms with RNAi knockdown of the indicated genes. Lack of halos in ced-1(RNAi) worms served as the positive control for defects in corpse internalization. Significance is indicated as follows: p>0.05 – (ns), 0.01–0.05 – (*), 0.001–0.01 – (**), <0.001 – (***). c) DIC micrographs showing the C. elegans adult hermaphrodite gonad in control (left) or the increased number of corpses in jph-1 deficient animals. Arrows point to refractile cell corpses.

Figure 5. Both intracellular and extracellular Ca²⁺ is crucial for TGF-β secretion by phagocytes encountering apoptotic cells

a) J774 phagocytes cells were incubated with apoptotic thymocytes in the presence of absence of calcium in the medium, or in the presence of calcium but with calcium inhibitor drugs. The supernatants were collected and analyzed for TGF-β levels by ELISA. Drugs were added at the same time as the apoptotic cells. b) NIH3T3 cells transfected with control or orai1, orai2 and orai3 specific siRNA were incubated with apoptotic thymocytes and secretion of TGF-β was measured in the medium 24 hrs later. No apopt cells shows the amount of TGF-β secreted by NIH3T3 cells without addition of apoptotic thymocytes, while apopt cells alone is the amount of TGF-β in wells with apoptotic thymocytes without any phagocytes. Each figure is representative of at least three independent experiments. Significance is indicated as follows: p>0.05 – (ns), 0.01–0.05 – (*), 0.001–0.01 – (**), <0.001 – (***).