Mfge8 is critical for mammary gland remodeling during involution

Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling.     

Ultrastructural evidence for apoptosis of pavement cells, chloride cells, and hatching gland cells in the developing branchial area of the trout Salmo trutta

Death by apoptosis of branchial epithelial cells was studied in brown trout embryos by means of transmission electron microscopy. Superficial pavement cells are sloughed off for the renewal of the epithelium after an apoptotic degeneration with shrinkage of the cytoplasm and loss of desmosomal contacts. Chloride cells appear as immature, mature and degenerating cells. Degenerating chloride cells, which are separated from the ambient water by pavement cells, show condensation of the cytoplasm and structural alterations in the tubular system and the mitochondria. Hatching gland cells degenerate either into apoptotic bodies or into cellular debris, depending on the functional stage of the cell. There was no phagocytosis by macrophages or adjacent cells of the degenerating chloride and hatching gland cells, but an infiltration of leucocytes was always observed in the epithelium undergoing cellular degeneration. In some instances, secondary necrosis of apoptotic hatching gland cells was observed. Apoptosis occurs in the three types of cells since early stages of development. However, a massive wave of cellular death occurred in pavement and hatching gland cells during the hatching stage and in the chloride cells during post-hatching stages.     

Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.     

Poractant alfa (Curosurf?) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo

Background

Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf^®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.

Methods

Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.

Results

Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.

Conclusions

We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.     

Histologic changes in tissue components of the hyperplastic thyroid gland during its involution in the rat

Male Fischer rats were fed a low-iodine diet containing thiouracil for 21 days to produce hyperplastic thyroid glands, and then fed a high-iodine diet for various time intervals, from 5 hr to 180 days, in order to study the morphological changes that occur during involution. Thyroids were fixed by perfusion fixation and embedded in Epon. Sections were examined by light microscopy. Initially at 0 days of involution (at the time of the change to the high-iodine diet), follicular lumens were very narrow and capillary lumens were very wide. The capsule was thick and infiltrated with mononuclear leukocytes. No obvious changes occurred for 1 day after the change in diet, but shortly thereafter capillary lumens began to narrow. By 4 days, most capillary lumens were close to normal size; capillaries formed a more or less normal bed except that many were embedded in a relatively thick or wide interfollicular matrix. This matrix was largely gone by 21 days. Between 1 and 21 days, follicular lumens dilated progressively as colloid accumulated. The density of staining of the accumulated colloid varied from follicle to follicle, and this variation was also observed in older controls. Inflammatory cells gradually disappeared from the capsule and most were gone by 15 days. Starting at approximately 15 days and continuing to 180 days, one or more disintegrating cells were found in some lumen profiles. Colloid goiters were not observed in these rats even after several months of involution. Some lumens were rather large, however, and small fractions of the follicles, both small and large, were bounded by flat cells and resembled "cold" follicles morphologically.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

Background  

Mammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs) to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis.     

Phosphatidylserine- and integrin-mediated phagocytosis of apoptotic luteal cells by macrophages of the rat

Corpora lutea disappear from ovaries in the absence of conception. The present study was undertaken to examine the hypothesis that disappearance of corpora lutea is accomplished through apoptosis-dependent phagocytosis of luteal cells. When bone marrow cells expressing green fluorescence protein were transplanted into X-ray-irradiated mice, macrophages derived from donor mice were detected within corpora lutea, suggesting macrophage infiltration into the tissue. Dispersed rat luteal cells underwent spontaneous apoptosis during culture and were phagocytosed by luteal macrophages. Treatment with doxorubicin increased the extent of apoptosis in luteal cells, and those cells were more efficiently phagocytosed than cells left untreated. The phagocytosis was inhibited by liposomes containing phosphatidylserine or a peptide containing the integrin-targeted sequence, and was stimulated by milk fat globule epidermal growth factor 8. These results collectively indicate that apoptotic luteal cells are phagocytosed by macrophages in a manner mediated by phosphatidylserine and integrin.     

Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland

Following the cessation of lactation, the mammary gland undergoes a physiologic process of tissue remodeling called involution in which glandular structures are lost, leaving an adipose tissue compartment that takes up a much larger proportion of the tissue. A quantitative morphometric analysis was undertaken to determine the mechanisms for clearance of the epithelial cells during this process. The involution process was set in motion by removal of pups from 14-day lactating C57BL/6 mice. Within hours, milk-secreting epithelial cells were shed into the glandular lumen. These cells became apoptotic, exhibiting exposure of phosphatidylserine residues on their surfaces, activation of effector caspase-3, staining for caspase-cleaved keratin 18, loss of internal organellar structure, and nuclear breakdown, but minimal blebbing or generation of apoptotic bodies. Clearance of residual milk and the shed epithelial cells was rapid, with most of the removal occurring in the first 72 h. Intact apoptotic epithelial cells were engulfed in large numbers by residual viable epithelial cells into spacious efferosomes. This process led to essentially complete involution within 4 days, at which point estrous cycling recommenced. Macrophages and other inflammatory cells did not contribute to the clearance of either residual milk or apoptotic cells, which appeared to be due entirely to the epithelium itself.     

Appearance of apoptotic cells and granular cells in the silkworm midgut lumen during larval-pupal ecdysis

To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.     

Apoptosis contributes to vascular lumen formation and vascular branching in human placental vasculogenesis

Placental vasculogenesis consists of several stages, including appearance of hemangioblasts and angiogenic cell islands, setting up a primitive vascular network, and transition from vasculogenesis to sprouting and nonsprouting angiogenesis. In the present study, we hypothesized that placental vasculogenesis and angiogenesis require apoptosis during the formation of primitive vascular pattern, vessel elongation, and angiogenic branching. Vasculogenesis and apoptotic cells were identified using CD31 immunohistochemistry, hematoxylin-eosin (H-E) staining, CD31-TUNEL double-labeling, and transmission-electron microscopy (TEM). No TUNEL-positive cell was detected in angiogenic cell islands; however, several TUNEL-positive cells were observed during the primitive lumen formation. Interestingly, some of the stromal cells located between vasculogenic areas during the endothelial tube elongation and angiogenic branching also were TUNEL-positive. The presence of morphological aspects of apoptosis, such as nuclear shrinkage and nuclear bodies (apoptotic bodies), also was confirmed in H-E-stained and TEM-depicted sections. Quantitative analysis showed that higher ratios for apoptotic cells were found in the core stroma of villi among the vascular branching areas and in the primitive capillary lumen compared to angiogenic cell cords and vasculatures with advanced lumens (P < 0.05). In conclusion, our results suggest that apoptosis likely is involved in the physiologic mechanisms of placental vasculogenesis and angiogenesis, such as lumen formation and angiogenic branching.     

Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells.

One of the key features associated with programmed cell death in many tissues is the phagocytosis of apoptotic bodies by macrophages. Removal of apoptotic cells occurs before their lysis, indicating that these cells, during the development of apoptosis, express specific surface changes recognized by macrophages. We have compared the mechanisms by which four different macrophage populations recognize apoptotic cells. Murine macrophages elicited into the peritoneal cavity with either of two different phlogistic agents were able to phagocytose apoptotic cells. This phagocytosis was inhibited by phosphatidylserine (PS), regardless of the species (human or murine) or type (lymphocyte or neutrophil) of the apoptotic cell. In contrast, the murine bone marrow macrophage, like the human monocyte-derived macrophage, utilized the vitronectin receptor, an alpha v beta 3 integrin, for the removal of apoptotic cells, regardless of their species or type. That human macrophages are capable, under some circumstances, of recognizing PS on apoptotic cells was suggested by the observation that PS liposomes inhibited phagocytosis by phorbol ester-treated THP-1 cells. These results suggest that the mechanism by which apoptotic cells are recognized and phagocytosed by macrophages is determined by the subpopulation of macrophages studied.     

Role of macrophages and multinucleate giant cells in the resorption of corpora amylacea in the involuting bovine mammary gland

Summary Microscopic examination of involuting bovine mammary tissue revealed elevated concentrations of corpora amylacea in alveolar lumina. Morphologic relationships between amyloid bodies, macrophages, and multinucleate giant cells (MGCs) suggested phagocytosis and degradation of the deposits by the phagocytic cells. Resorption of amyloid material by macrophages and MGCs during the process of mammary involution may be instrumental in preventing accumulation of corpora amylacea in secretory tissue which may interfere with mechanisms of milk synthesis and secretion.     

Immunogenicity of apoptotic cells in vivo: role of antigen load, antigen-presenting cells, and cytokines.

Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least 20-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of IL-10 and IL-1beta cytokines, and is enhanced in IL-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mphi), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mphi and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.     

Degradation of chromosomal DNA during apoptosis

Apoptosis is often accompanied by degradation of chromosomal DNA. CAD, caspase-activated DNase, was identified in 1998 as a DNase that is responsible for this process. In the last several years, mice deficient in the CAD system have been generated. Studies with these mice indicated that apoptotic DNA degradation occurs in two different systems. In one, the DNA fragmentation is carried out by CAD in the dying cells and in the other, by lysosomal DNase II after the dying cells are phagocytosed. Several other endonucleases have also been suggested as candidate effectors for the apoptotic degradation of chromosomal DNA. In this review, we will discuss the mechanism and role of DNA degradation during apoptosis.     

Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells

Chronic obstructive pulmonary disease is a highly prevalent, complex disease, usually caused by cigarette smoke. It causes serious morbidity and mortality and costs the global community billions of dollars per year. While chronic inflammation, extracellular matrix destruction and increased airway epithelial cell apoptosis are reported in chronic obstructive pulmonary disease, the understanding of the basic pathogenesis of the disease is limited and there are no effective treatments. We hypothesized that the accumulation of apoptotic airway epithelial cells chronic obstructive pulmonary disease in could be due to defective phagocytic clearance by alveolar macrophages. There have been no previous studies of the phagocytic capacity of alveolar macrophages in chronic obstructive pulmonary disease using physiologically relevant apoptotic airway epithelial cells as phagocytic targets. We developed a phagocytosis assay whereby cultured 16HBE airway epithelial cells were induced to apoptosis with ultraviolet radiation and stained with mitotracker green. Alveolar macrophages from bronchoalveolar lavage from eight control and six chronic obstructive pulmonary disease subjects were analysed following 1.5 h incubation with apoptotic airway epithelial cells, then staining with macrophage marker anti CD33. CD33+/mitotracker green + events (i.e., alveolar macrophages which had phagocytosed apoptotic airway epithelial cells) were analysed using flow cytometry. Phagocytosis of polystyrene microbeads was investigated in parallel. A significantly reduced proportion of alveolar macrophages from chronic obstructive pulmonary disease subjects ingested apoptotic airway epithelial cells compared with controls (11.6 +/- 4.1% for chronic obstructive pulmonary disease versus 25.6 +/- 9.2% for control group). Importantly, the deficiency was not observed using polystyrene beads, suggesting that the failure to resolve epithelial damage in chronic obstructive pulmonary disease may result, at least partially, from specific defects in phagocytic ability of alveolar macrophages to ingest apoptotic airway epithelial cells.     

Degeneration and death,by apoptosis and necrosis,of the pavement and chloride cells in the gills of the teleost Oreochromis mossambicus

Summary Degeneration and death of branchial epithelial cells were studied in an African cichlid fish. In both freshwater and seawater fish the superficially located pavement cells are sloughed off at the end of their lifecycle. This process is preceded by degeneration via a process of cytoplasmic shrinkage and condensation related to apoptotic (physiologically controlled) cell death. The chloride cells are pleomorphic, i.e., accessory, mature, and degenerating cells. Degeneration of chloride cells mainly occurs by apoptosis. Degenerating cells show shrinkage and densification of cytoplasm and nuclei, and swelling of the tubular system; these cells are then separated from the ambient water by pavement cells. They are finally phagocytosed and digested by macrophages. Apoptosis of chloride cells, but not of pavement cells, is greatly stimulated when the fish are in seawater; this reflects an increase in cellular turnover of the chloride cells. Accidental cell death (necrosis) of pavement cells or chloride cells is rarely observed in fully adapted freshwater and seawater fish. Its incidence increases in the first few days following transfer of fish from fresh water to seawater.     

Fonction catabolique de l'épithélium mammaire

Summary The histochemical (iron, lipopigments, acid phosphatase, leucine aminopeptidase) and cytologic (lysosomes) changes occuring during pregnancy, lactation and involution of mouse, rat, rabbit, guinea-pig mammary glands are studied by light microscopy and electron microscopy.In all the animals examined, the mammary epithelium has an intracellular digestive system which is adapted to subserve two functions. The first one is the segregation of cytoplasmic components which often precedes cellular involution. The second one is the regulation of secretory processes in the non lactating glands. This digestion of endogenous materials results in the formation of various lytic bodies: dense bodies sometimes containing ferritin, vacuolated dense bodies with membranous residues, autophagic vacuoles. The lysosomes can give large complex dense bodies like lipofuscin pigments with or without ferritin.Leucine aminopeptidase which always disappears in the mouse mammary epithelium during lactation is not present in rat, rabbit, guinea-pig mammary epithelium. In these species only the vascular tissue contains the enzyme. This observation indicates that leucine aminopeptidase does not take care of the overproduction of secretory products in the non-lactating glands.Acid phosphatase is concentrated in secretory granules and in lytic bodies: multivesicular bodies, dense bodies with ferritin, vacuolated dense bodies, lipopigments. This enzyme constitutes probably a mechanism for controlling and triggering the destruction of the secretory material with no active elimination.The iron of the mammary epithelium appears in virgin mice older than 30 weeks and in mice, rats, rabbits, guinea-pigs during glandular cells involution. This is a catabolic iron located in lysosomes. Its amount depends upon the iron content of the milk and upon the competitive secretory and catabolic activities of the glandular cells. An explanation of iron disappearance during a second pregnancy and lactation is discussed.     

鸡胚巨噬细胞AcP酶变化及AcP酶与凋亡细胞的关系

本研究采用电镜及酶细胞化学的方法观察了鸡胚脾脏不同胚龄组巨噬细胞溶酶体酸性磷酸酶（AcP酶）的变化、凋亡实验组巨噬细胞及其AcP酶与凋亡细胞的关系。取10天、13天和17天鸡胚脾脏,按Gomori法显示AcP酶,各胚龄脾脏巨噬细胞AcP酶细胞化学反应阳性,按AcP酶染色阳性做溶酶体计数,结果显示随着胚龄的增加溶酶体数随之增加,尤以第17天组溶酶体数增加最为明显,所得数据经统计分析表明各胚龄组间溶酶体数的差异有统计学意义。凋亡实验组采用放线菌酮诱导15天鸡胚脾脏细胞凋亡,结果显示凋亡细胞为各类幼稚血细胞,以幼稚淋巴细胞为主。巨噬细胞未见凋亡,而是吞噬了大量的凋亡细胞和凋亡小体,AcP酶反应颗粒不仅出现在巨噬细胞的溶酶体、吞噬体,还见于高尔基复合体、内质网等。细胞AcP酶反应强度数字化结果表明：凋亡组酶活性显著高于对照组,差别有统计学意义,提示胚胎巨噬细胞在凋亡细胞出现时AcP酶活性增强,说明巨噬细胞吞噬和消化凋亡细胞或凋亡小体是通过AcP酶等活性物质来实现的。