巨噬细胞吞噬凋亡中性粒细胞的实时动态观察及其转归的研究

1、Dynamic process of phagocytosis and fates of macrophagesafter their ingestion of apoptotic neutrophils1Wang Jiong, Huang Weilin, Wang Cheng, Liu RongyuDepartment of Pulmonology, Anhui Geriatrics Institute, the First Affiliated Hospital of AnhuiMedical University, Hefei, Anhui ( 230022)AbstractCleara

2、nce of apoptotic neutrophils by macrophages is important for the successful resolution of acute inflammation and homeostasis. In this study, the dynamic process of phagocytosis of apoptotic neutrophils was measured in real time and special attention was paid to the fates of macrophages after their p

3、hagocytosis in vitro, we staged the recognition and tethering, internalization, digestion and exocytosis stages of phagocytosis of apoptotic neutrophils. Furthermore we found that some macrophages underwent cell death after their ingestion of apoptotic cells. The dead macrophages consisted of autoph

4、agy, apoptosis and oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes. These results demonstrated that after ingestion of apoptotic neutrophils, macrophages might undergo autophagy and apoptosis. Autophagy of macrophage after ingestion of apop

5、totic cells may be one of the new mechanisms in immune response. Keywords: macrophage; cell death; phagocytosis; time-lapse imaging1. IntroductionRespiratory infectious disease is a common and leading cause of morbidity and mortality worldwide 1. The acute response to an infectious insult is usually

6、 a rapid recruitment and infiltration of neutrophils. The neutrophils can effectively bind and kill microorganisms that might cause damage to tissues, then they timely undergo a vigilantly controlled programmed cell death known as apoptosis 2. Efficient removal of these intact apoptotic neutrophils

7、by macrophages is believed to be critical for the successful resolution of acute inflammation and maintenance of tissue homeostasis 3, 4. Voluminous studies have been focused on the recognition and uptake of apoptotic neutrophils by macrophages, but the dynamic process of phagocytosis and the fates

8、of macrophages after their ingestion of apoptotic neutrophils have not been well documented 5.The murine macrophage-like RAW264.7 cells had been used to establish assays of phagocytosis of apoptotic Jurkat (human T-cell lymphoma) cells and CTLL-2 cells 6, 7. To gain a better understanding of the mac

9、rophage/apoptotic neutrophil interactions and the fates of macrophages after their ingestion, we measured the dynamic process of phagocytosis of human apoptotic neutrophils by RAW264.7 cells in real time and special attention was paid to the subsequent changes of macrophages after their ingestion of

10、 apoptotic neutrophils in cell culture condition.2. Material and Methods2.1 Neutrophil isolation and apoptosis induction10ml heparinized venous blood was obtained from healthy volunteers after informed written consent signed by each volunteer and approved by the local ethical committee. Neutrophils

11、were isolated under sterile conditions using the Percoll discontinuous density gradient centrifugation as previously described with some modification8, 9. Briefly, 10ml heparinized venous blood was incubated for 5 min in 50 ml red blood cell lysis buffer (170 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7

12、.3) to remove red blood cells. The remaining cells were layered on the top of Percoll discontinuous density gradient and centrifuged (1000g, 30min, room temperature) without braking. The isolated neutrophils 1 This study is supported by the Nature Science Foundation of China (30670936) and Doctoral

13、Fund of the Ministry of Education of China (20050366002).- 1 -were washed three times with PBS; cultured at a density of 1 ×106 cells/ml in Dulbeccos modified Eagles medium (DMEM) (Gibco) supplemented with 10%fetal calf serum (FCS), 2 mmol/L glutamine, 100 IU/ml penicillin and 100 mg/ml strepto

14、mycin. Viability and purity of neutrophils isolated using this method were 97% and 95% respectively. Spontaneous apoptosis was achieved by culturing for 20h at 37° C in a humidified incubator with 5% CO210. At this time, apoptosis assessed by Annexin V-FITC apoptosis detection kit (BD Bioscienc

15、e, USA) using flow cytometry was 2560% ，whereas necrosis was less than 3%.2.2 Live cell imaging of Raw264.7 cells engulfing apoptotic neutrophils2×104 Raw264.7 cells grew overnight at 37° C with 5% CO2 in feeding media consisting of DMEM and 10% FCS in a 35mm2 dish. The dish was then place

16、d in a chamber on the stage of an Olympus IX-70 inverted microscope equipped with a CCD camera controlled by SPOT RT software (Diagnostic Instruments, Inc., Sterling Heights, MI). Live cell imaging of phagocytosis assays were done as previously described10, 11. About 2×103 apoptotic neutrophils

17、 were added to the dish and allowed them to interact with the macrophages. For time-lapse sequences, images were obtained automatically every 30 seconds for a period of 512h. The series of images were compiled into digital movies by MetaMorphTM7.0 Image Processing software (Universal Imaging Corpora

18、tion).2.3 Transmission electron microscopyMacrophages (2× 105 cells) were transferred to a 35mm dish in 1000µL DMEM with 10%FCS and allowed to grow overnight before co-incubation with apoptotic neutrophils (4 × 105cells) at 37°C for 60 minutes. Then the non-ingested neutrophils w

19、ere removed by vigorous washing. After that RAW264.7 cells were fixed in 2.5% glutaraldehyde, scraped and pelleted by centrifugation. Ultrathin sections were cut using an ultramicrotome(LKB-4, Sweden). Sections stained with uranyl acetate and lead citrate were observed under a transmission electron

20、microscope (JEM-1230, Japan).2.4 Confocal microscopyAfter interacting with cultured neutrophils labeled with 7-AAD for 60minutes, washing away the non-ingested ones, the macrophages were stained in 0.05mM MDC/100µg/ml AO, 100µg/ml AO, 100µg/ml AO /100µg/ml EB respectively for 15m

21、in12, 13. Then the cells were washed three times with PBS and scanned using Zeiss LSM510 confocal microscopy within 1 hour. Monodansylcadaverine( MDC) is a specific dye for autophagosome, acridine orange is a cell-permeable dye that intercalates into DNA and results a green color while ethidium brom

22、ide enters cells with disrupted membrane integrity and intercalates into RNA and double-stranded DNA to appear orange. Thus, differential uptake and binding of these dyes allow us to identify macrophages which had engulfed apoptotic neutrophils in autophagy, apoptosis and oncosis. In each experiment

23、, at least 100 macrophages which had engulfed 7-AAD labeled apoptotic neutrophils were counted, and the proportion of macrophages with positive granules stained in MDC, AO and EB was expressed as a percentage respectively. Macrophages without interaction with apoptotic neutrophils stained in AO/MDC,

24、 AO and AO/EB were used as corresponding controls.2.5 StatisticsData were expressed as the means ±S.E and were analyzed for significant differences by Independent-Samples T Test and One-Way ANOVA with SPSS 10.0. Differences were considered statistically significant if P value <0.05.3. Result

25、s3.1 Live cell imaging of the phagocytosis of apoptotic neutrophils by macrophage - 2 -and macrophage cell death after phagocytosisIn time-lapse sequences, we found that most neutrophils appeared to interact actively with the ramified macrophages. The macrophages could recognize and tether the apopt

26、otic neutrophils, and then internalization ensued. Analysis of 30 macrophages in 20 independent experiments showed that most macrophages engulfed intact apoptotic neutrophils within a few minutes to 1 hour. As shown in the movie (see supplementary material) and Fig.1, we recorded the steps of phagoc

27、ytosis and the macrophage cell death after ingestion of apoptotic neutrophils. The stages of phagocytosis were distinguishable: I: recognition and tethering: the apoptotic neutrophil was recognized and finally tethered to the macrophage. This stage lasted about 5 minutes (Fig.1 image 13). II: intern

28、alization: the macrophage initially protruded pseudopodia to prey the neutrophil and the two cells membrane fused. This lasted about 3 minutes (Fig.1 image 4 6). III: digestion: the engulfed neutrophil was processed within the macrophage. This stage kept on about 2 minutes (Fig.1 image7, 8). IV: exo

29、cytosis: the neutrophil became smaller and was excreted out of the macrophage. This stage lasted about 2 minutes (Fig.1 image9, 10). During the process of phagocytosis, the macrophage was highly active and underwent dramatic structural changes from ramified cell to round one.As time lapsed (1 hour l

30、ater), the macrophage which ingested the apoptotic neutrophil underwent a characteristic sequence of morphological changes, beginning with cell shrinkage, followed by membrane blebbing, and ultimately concluding in cell membrane rupture (Fig.1 image1115). Subsequently the neighboring macrophage also

31、 underwent cell shrinkage, the membrane blebbing and rupture, just as the preceding one (Fig.1 mage1620).3.2 Form of macrophage cell death observed by transmission electron microscopyAs shown in Fig.2, cells characterized by depletion of organelles with intact non-pyknotic nuclei, autophagosomes wer

32、e identified as autophagy. Apoptotic cells characterized by loss of cytoplasm, pyknotic nuclei assumed the appearance of crescent shape or irregularly shaped nuclei along with marginated chromatin were also found. In addition, we observed that some macrophages exhibited cell and organelle swelling w

33、hich are characteristics of oncosis. So, after interaction with apoptotic neutrophils, the form of macrophage cell death was a mixture of autophagy, apoptosis and oncosis.3.3 Confocal microscope analysis of macrophages after ingestion of apoptotic neutrophilsAs shown in Fig.3, the macrophages contai

34、ned MDC, AO and EB positive granules with 7-AAD labeled apoptotic neutrophils. Data from three independent experiments showed that MDC positive macrophages representing autophagic ones comprised about 8.00±2.00% of the numbered macrophages, while macrophages with AO positive granules and EB pos

35、itive cells indicating apoptotic and oncotic macrophages comprised 12.33±2.08% and 3.66±1.50%, respectively. The predominant form of macrophage cell death was apoptosis and autophagy.4. DiscussionClearance of apoptotic cells by phagocytic process was demonstrated in mammals and nematodes i

36、n the early 1980s 14, 15. But the process is still incompletely understood. Cvetanovic16 identified specific recognition step during macrophages engulfing apoptotic cells. Hoffmann et al17 developed a novel system to distinguish tethering from ingestion during human monocytes differentiated macropha

37、ges engulfing apoptotic erythrocytes. DeCathelineau and Henson18 and Gardai et al19 coined the unique term efferocytosis to describe the process of clearance of apoptotic cells. In their efferocytosis model, they proposed three steps of clearance of apoptotic cellstethering, tickling and internaliza

38、tion. Here, we reported for the first time the dynamic process of phagocytosis of human - 3 -apoptotic neutrophils by RAW264.7 cells. We found that the phagocytosis of apoptotic neutrophils by macrophages could also be divided into the recognition and tethering, internalization, digestion and exocyt

39、osis stages. These observations confirmed and extended previous studies.Autophagy, apoptosis and oncosis have been well described among other forms of cell death while necrosis is defined as the sum of changes occurring in the cells after they have died regardless of the prelethal process 20, 21. Ph

40、agocytosis-induced apoptosis had been well elucidated 22. MDC, a well-known specific fluorescent dye that accumulates in acidic vacuoles, was often used as a valid marker of autophagosomes23. In the present study, we used it to determine whether autophagy was an alternative mode of macrophage cell d

41、eath after ingestion of apoptotic neutrophil. We found that MDC decorated the engulfed apoptotic neutrophils and transmission electron microscope confirmed the phenomena. So we concluded that after ingestion of apoptotic neutrophils, the macrophages, besides underwent apoptosis, and also underwent a

42、utophagy.Macrophages play a central role in the control of inflammatory diseases such as asthma, pneumonia. They are professional scavenger cells for clearance of apoptotic cells. Our results shown that after ingestion of apoptotic cells, a portion of macrophages might undergo different forms of cel

43、l death. Limited macrophage cell death may be beneficial for macrophage turnover and the process of inflammation. But massive macrophage cell death may be an important factor in the pathogenesis and progression of severe asthma, pneumonia.Taken together, our study measured the dynamic process of pha

44、gocytosis of apoptotic neutrophils by macrophages and showed that after ingestion of apoptotic neutrophils, macrophages might undergo autophagy and apoptosis. Autophagy of macrophage after ingestion of apoptotic cells may be one of the new mechanisms in immune response.AcknowledgmentsMuch gratitude

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